The nature of immunoglobulin species in passive immunity of neonatal mice
by Richard Adam Wilson
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Microbiology
Montana State University
© Copyright by Richard Adam Wilson (1970)
Abstract:
Passive immunity in neonates was studied in conventionally reared and germfree mice, using three
assay methods to determine the immunoglobulin concentrations in neonatal and dam sera and in
colostrum. Female mice were antigenized with one or two injections of DNP-BGG in complete
Freunds adjuvant given by intradermal injection near the mammary chain. The female mice were mated
10 days after the last injection.
At day one and day five post-partum, blood samples were obtained from the females and one half of
their litters were sacrificed. Colostrum was flushed from the neonatal stomachs and neonatal serum was
collected. All specimens were stored in the frozen state until assayed. Mammary glands were excised
from the females after they had been sacrificed on day five post-partum.
Hemolysin and hemmaglutinin titres were performed on sera and some of the polostral samples. A
modification of the radialimmunodiffusion technique of Masseyeff and Zisswiller was employed to
measure the four immunoglobulins, IgA, IgM, IgG1 and IgG2, and the specific antibody concentrations
in colostrum and serum.
The data indicates that neonates acquired IgG2 and IgG1 passively by both placental and lacteal
transmission and IgA and IgM primarily by lacteal transmission. The concentration of the
immunoglobulins and specific antibodies in serum or colostrum of the dams was not an apparent factor
in determining transmission of the immunoglobulins to the neonate. A hierarchy directly related to the
species of immunoglobulin and how well they traverse the placenta or mammary gland barrier was
observed. IgA was associated with "local" gut immunity while IgG1 and IgG2 were found in greatest
concentration in the sera of fhe neonates. S ta te m e n t o f P e r m is s io n to Copy
In p r e s e n t in g t h i s t h e s i s in p a r t i a l f u l f i l l m e n t o f th e r e q u ir e ­
m ents f o r an advanced d e g r e e a t Montana S t a t e U n i v e r s i t y , I a g ree t h a t
th e L ib r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r i n s p e c t i o n .
I fu r th e r
a g r e e t h a t p e r m is s io n f o r e x t e n s i v e . c o p y in g o f t h i s t h e s i s f o r
s c h o la r l y p u r p o se s may b e g r a n ted by my m ajor p r o f e s s o r , o r , in h i s
a b s e n c e , by th e D ir e c t o r o f L i b r a r ie s .
I t i s u n d er sto o d t h a t any
c o p y in g o r publ i c a t i o n c o f t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l n o t be
a llo w e d w ith o u t my w r i t t e n p e r m is s io n .
S ig n a tu r e
D ate
-De^C .
4 ,/W P
THE NATURE OF IMMUNOGLOBULIN SPECIES IN
PASSIVE IMMUNITY OF NEONATAL MICE
by
RICHARD ADAM WILSON
A t h e s i s su b m itte d to th e G raduate F a c u lty in p a r t i a l
• f u l f i l l m e n t o f th e r e q u ir e m e n ts f o r th e d e g r e e
of
MASTER OF SCIENCE
in
M ic r o b io lo g y
Approved:
H ead, M ajor D epartm ent
I t f i - <k)
, Exami
MONTANA STATE UNIVERSITY
Bozeman, Montana
Decem ber, 1970
iii
ACKNOWLEDGEMENT
The a u th o r w is h e s t o e x p r e s s a p p r e c ia t io n to Dr. John W. J u t i l a
f o r th e t im e ly d i r e c t i v e s and s c i e n t i f i c . i n s p i r a t i o n he gave th rou gh ­
ou t th is in v e s tig a tio n .
The a s s i s t a n c e an,d s u g g e s t io n s o f h i s
com m ittee m em bers, Dr. Wil1Iiam H i l l and Dr. Frank Newman a r e g r a t e f u l l y
ack n ow led ged .
The s u p p o r t, encouragem ent and s a c r i f i c e s m ade-by h i s
w i f e , B e v e r ly , and fo u r so n s i s humbly a c c e p te d .
The a u th o r i s g r a t e f u l f o r th e A l l i e d P r o f e s s i o n a l H e a lth G rant,
a d m in is te r e d by Dr. R ich ard H. McBee f o r f i n a n c i a l s u p p o r t.
IV
TABLE 0F CONTENTS
Page
v it a :
.................................................................................
ACKNOWLEDGEMENT . . . .............................
ii
............................. ................................. i i i
TABLE OF CONTENTS .......................................
iv
LIST OF T A B L E S ..............................' .........................
v ii
LIST OF FIGURES....................................................................■ . . . . - .......................
ix
ABSTRACT.................................
xi
INTRODUCTION
....................................................................................................................
I
I n t r o d u c t io n t o th e t h e s i s .........................................................................
10
MATERIALS AND METHODS .................................................................................................
14
A n i m a l s .............................
A n tig e n p r e p a r a tio n
. ..................................................... .
.......................................................................................
P r e p a r a tio n o f a n t i s e r a and c o lo s tr u m
............................................
T is s u e sp ecim en s . . . . . . . . . . . .
F lu o r e s c e n t s t u d i e s
14
. . . . . . .
.............................
. .
.............................................
14
16
18
.
19
Q u a n t it a t iv e im m unoglobulin a s s a y . . . . . . . . . . . .
20
P a s s iv e h e m a g g lu tin a tio n
23
Mammary g la n d p la q u e form ing c e l l a s s a y
RESULTS .............................................................................
.......................................
. . . . .
.........................
Im m unoglobulin l e v e l s in CR dams f o flo w in g a s i n g l e
i n j e c t i o n o f 2 3 0 p g s DNP^-BGG in C F A ..................................................
fgA .
25
27
27
27
V
Page
IgM
XgG1
31
........................
31
XgGg
^X
Inmaunog l o b u l in l e v e l s . . ip. n e o n a te s born to dams g iv e n
a s i n g l e i n j e c t i o n o f DNP^^-BGG . = ............................. «
IgA . . . . . . . .
,
,
32
, ,
32
IgM . .........................- v .......................................................................
•
32
IgG1
........................
37
IgG2
x. ........................
37
Im m unoglobulin l e v e l s in th e sorym o f CR fe m a le s
f o ll o w i n g two i n j e c t i o n s o f 2 3 0 ngs DNP^y-BGG . . . . . . .
IgA .............................
. .
. ..........................................................
IgM . . . . . . . . . . . . . . . . .
.................................
IgG^ and IgGg ......................................................................................
37
40
40
•
40
Im m unoglobulin in n e o n a te s born to dams g iv e n two
i n j e c t i o n s o f BNPgy^BGG . . . . . . .
............................................
40
XgM .
.
.
.
.
.
.
.
I gG^
.
.
.
.
.
.
.
.
* * * * * * * %
Ig
IgM
IgG1
^^
A*1
Im m unoglobulin l e v e l s in .germ free m ice a f t e r
r e c e iv in g , a s i n g l e a n t ig e n a t io n o f D N P g y -B G G ............................... '
IgA
41
........................ .... ......................................
. . . . . . .
. . . . . . .
42
42
. . . . . .
42
. ® . ® . .
”n 42
vi
Page
IgG2
............................. .... ................................. '..........................................
Inpjm noglobulin l e v e l s in th e n e o n a te s born to GF
dams g iv e n a s i n g l e i n j e c t i o n o f DNPgy-BGG w ith GFA
. . .
IgA , . . ............................
IgM . . . . . . . . . . . .
46
46
46
.................................................
46
IgGl
................................................ ......................................■.......................
46
IgG2
......................
47
A s y n o p s is o f th e s p e c i f i c a n tib o d y r e sp o n s e . . . . . . . .
47
P a s s iv e h e m a g g lu tin a tio n t i t r a t i o n s
. . . . . . . . . . .
52
D e t e c t io n o f im m unoglobulin s p e c i e s in th e c o lo s tr u m . . .
54
DISCUSSION
61
SUMMARY........................................................................
68
APPENDIX
. . . . . . . . . . . . . . . . .
. . .
70
A ppendix A - T a b le on d ia m e te r o f p r e c i p i t i n r in g s . . . .
71
A ppendix B - F lu o r e s c in g c e l l s
. .
72
. . . . . . . . . . . . . .
73
. . . . .
A p pendix C - ( a ) Equipm ent la y o u t
.............................
.............................
(b‘) P r e c i p i t i n r in g s in agar d i s c s .
LITERATURE C f T E D ......................................
. . . . .
73
74
v ii
LIST GF TABLES
Page
T a b le
T a b ls
I
II
T a b ls ' H I
T a b le
T a b le
Tab 3,e
T ab le
IV
V
VI
V II
T a b le V III
Im m unoglobulin l e v e l s a t day I and day
5 p o s t-p a r tu m in dams a n t ig e n iz s d w ith
a s i n g l e i n j e c t i o n o f DNP^-BGG . . . . . . . .
28
Im m unoglobulip l e v e l s in th e serum o f
n e o n a te s born to dams a n t ig e n iz e d w ith
a s i n g l e i n j e c t i o n o f D N P ^-B G G ..................................
36
Im m unoglobulin l e v e l s in dams a n t ir
g s n iz e d w ith two i n j e c t i o n s o f DNP^y-BGG
. . .
38
. . .
39
Im m unoglobulin l e v e l s in germ free p i c e
a n t ig e n iz e d w ith a s i n g l e i n j e c t i o n o f
DNP6 7 -BGG , . . .
...............................................................
43
Im m unoglobulin l e v e l s in s e turn o f n s o n a t e s
b orn to germ free fe m a le s a n t ig e n iz e d w ith
a s i n g l e i n j e c t i o n o f DNP67-BGG .............................
45
Im m unoglobulin l e v e l s in th e s e r a o f th e
■peonates b orn to dams a n t ig s p iz e d w ith
two i n j e c t i o n s o f DNPc ^-B G G ........................
o/
.
H e m a g g lu tin a tin g apd h e m o ly sin t i t r e s ip
serum o b ta in e d from fe m a le s i n j s c t s d one
o r two tim e s in tr a d e r m a lly w ith DNP67-BGG . . . .
53
The number o f c e l l s p e r 50 m ic r o s c o p ic
f i e l d s ( o i l im m ersion ) e x h i b it i n g
f lu o r e s c e n c e in mammary t i s s u e s e c r e t i o n s
t r e p t e d w ith f l u o r e s c e n t a n t i- h e a v y c h a in
s e r a (Ig A , IgM, IgG ^, IgG2 ) .............................................
57
(a )
(b )
CR fe m a le s t h a t r e c e iv e d a s i n g l e
a n t ig e n a t f o n . . ........................ ....
57
CR fe m a le s t h a t r e c e iv e d two
i n j e c t i o n s ..................................................... ....
57
v iii
Page
(c)
T a b le
IX
GF fe m a le s t h a t r e c e iv e d a s i n g l e
i n j e c t i o n o f D N P .................................. ....
57
Im m unoglobulin l e v e l s in c o lo str u m
h a r v e s te d from stom ech s o f n e o n a te s . . . . . .
(a )
(b )
Im m unoglobulin l e v e l s i n c o lo str u m
h a r v e s te d from stom achs o f n e o n a te s
b o m to CR dams g iv e n a s i n g l e d o se
o f DNP6 7 -BGG . . , ................................................
. ,
59
Im m unoglobulin l e v e l s in c o lo str u m
h a r v e s te d from stom achs o f n e o n a te s
, born to CR dams g iv e n two i n j e c t i o n s
o f DNP, ..-B G G ....................................................................
59
6/
(c)
59,
Im m unoglobulin l e v e l s in c o lo str u m
h a r v e s te d from stom achs o f n e o n a te s
b orn to g erm free dams g iv e n a s i n g l e
a n t ig e n a t io n w ith DNP67-BGG
59
LIST OF FIGURES
F ig u r e I
A h y p o t h e t ic a l m odel f o r th e t r a n s p o r t
q f gamma A and SP a c r o s s m u cosal membrane
e p it h e liu m
*
* *
«
0
0
0
0
0
F ig u r e 2
P roposed m odel o f th e c o l o s t r a l fgA
F ig u r e 3
Stan d ard c u r y e s d e v e lo p e d from r a d ia l
im m u n o d iffu sio n r e a c t io n s w it h v a r io u s
s p e c i f i c g o a t a n ti-m o u s e im m unoglobulin
serum and d i l u t i o n o f a p o o l o f norm al
S w iss serum
*
*
* *
* **
0
F ig u r e 4
0
0
0
0
0
. « . «
0
(a )
L e v e ls o f im m unoglobulin i n th e serum
o f fem a le e x p e r im e n ta l a n im a ls
* * * *
(b )
Im m unoglobulin l e v e l s in c o n tro l,
anim al s
F ig u r e 5
Im m unoglobulin l e v e l s in th e sepum o f CR
n e o n a te s (Dams - s i n g l e i n j e c t i o n ) * * * * *
F ig u r e 6
Im m unoglobulin l e v e l s in th e serum o f CR
n e o n a te s (Dams
two i n j e c t i o n s ) 0 . * * * *
F ig u r e 7
Im m unoglobulin l e v e l s i n th e serum o f GF
n e o n a te s (Dams - s i n g l e i n j e c t i o n ) « . „ * * *
F ig u r e 8
F req u en cy o f s p e c i f i c a n tib o d y i n th e s e r a
o f CR fem a le and n e o n a te s f o llo w in g a s i n g l e
i n j e c t i o n o f DNP^,,-BGG * * . , . * * , * « * = *
F ig u r e 9
F req u en cy o f s p e c i f i c a n tib o d y in ttie s e r a
o f CR fe m a le s and n e o n a te s f o llo w in g two
i n j e c t i o n s o f DNP^^^BGG « * « . * * « . * »
F ig u r e 10
F req u en cy o f s p e c i f i c a n tib o d y in th e s e r a
o f GF fe m a le s and n e o n a te s f o llo w in g a
s i n g l e i n j e c t i o n o f DNU^-BGG , .......................*
X
Pagq
F ig u r e I1I
Number o f p e l l s in mammary g la n d w ith
f lu o r e s c e l n a t e d a n t i- h e a v y c h a in s e r a . .
And, L e v e ls o f im m u n oglob u lin s ii%
c o s t r a l sam p les from n e o n a t a l stom achs . . . .
56
xi
ABSTRACT
P a s s iv e im m unity in n e o n a te s was s t u d ie d i # c o n v e n t io n a lly
r e a r e d and germ free m ic e , u s in g t h r e e a s s a y m ethods to d e ter m in e th e
im m unoglobulin c o n c e n t r a t io n s in n e o n a t a l and dam s e r a and in
c o lo s tr u m . Fem ale m ice w ere aq.tigeni% ed w ith one o r two i n j e c t i o n s
o f DNP-BGG in c o m p le te Freunds a d ju v a n t g iv e n by in tr a d e r m a l in j e c t io n ,
n e a r th e mammary c h a in . The fem a le m ice w ere m ated 10 days a f t e r th e
la s t in je c tio n .
At day one and day f i v e p o s t-p a r tu m , b lo o d sam p les w ere o b t a in ­
ed from th e fe m a le s and pne h a l f o f t h e i r l i t t e r s w ere s a c r i f i c e d .
C olostrum was f lu s h e d fpom th e n e o n a t a l stom achs and n e o n a t a l serum
was c o l l e c t e d . A l l sp ecim en s w ere s to r e d in t h e fr o z e n s t a t e u n t i l
a s s a y e d . Mammary g la n d s w ere e x c is e d from th e fe m a le s a f t e r th ey
had b een s a c r i f i c e d on day f i v e post-vpartum .
H em olysin and hem m aglutitifn t i t r e s w ere perform ed on s e r a and
some o f th e p o l o s t r a l sa m p le s. A m o d if ic a t io n o f t h e radialimmuno-rd i f f u s io n te c h n iq u e o f M a s s e y e ff and Z i s s w i l l e r was em ployed to
m easure th e fo u r im m u n o g lo b u lin s, IgA , IgM, IgG^ and IgG g, and th e
s p e c i f i c a n tib o d y c o n c e n t r a t io n s in c o lo s tr u m and serum .
■The d a ta i n d i c a t e s t h a t n e o n a te s a c q u ir e d IgGg and IgG^ p a s s i v e l y
by b o th p l a c e n t a l and l a p t e a l t r a n s m is s io n and IgA anh IgM p r im a r ily
by l a c t e a l t r a n s m is s io n . The c o n c e n t r a t io n o f th e im m unoglobulin s
and s p e c i f i c a n t ib o d ie s in serum o r c o lo s tr u m o f th e dams was n o t an
a p p a ren t f a c t o r in d e te r m in in g t r a n s m is s io n o f th e im m unoglobulin s
to t h e n e o n a t e . A h ie r a r c h y d i r e c t l y r e l a t e d to th e s p e c i e s o f
im m unoglobulin and how w e l l th e y t r a v e r s e th e p la c e n t a o r mammary
g la n d b a r r ie r was o b s e r v e d . IgA was a s s o c i a t e d w ith " lo c a l" g u t
im m unity w h ile IgG^ and IgGg were found in g r e a t e s t c o n c e n tr a t io n in
th e s e r a o f fh e n e o n a t e s .
INTRODUCTION
An abundant lit e r a t u r e ; c l e a r l y i n d i c a t e s t h a t p a s s iv e immunity
a f f o r d s e a r ly p r o t e c t i o n a g a i n s t i n f e c t i o n f o r many mammalian n e o n a te s
(B r a m b e ll, 1 9 6 8 ) .
Im m unoglobu lins e n t e r th e f e t p l b lo o d s tr e a m by
p l a c e n t a l p a s s a g e a n d /o r th e n e p p a ta l b lo o d str e a m v i a a b s o r p tio n o f
c o lo s tr u m th ro u g h th e i n t e s t i n e .
R a b b its , g u in e a p i g s and human
n e o n a te s a q u ir e a m a jo r ity o f t h e i r p a s s iv e a n t ib o d ie s a lm o st e x c lu ­
s i v e l y in th e c o lo s tr u m .
D o g s, m ice and r a t s o b t a in im m unoglobulin s
in u te r o and a q u ir e a n t ib o d ie s from c o lo s tr u m .
The p l a c e n t a l
s t r u c t u r e a s w e l l a s i t s p e r m e a b ilit y p r o p e r t i e s appear to d i c t a t e
f e t a l a c q u i s i t i o n o f p a s s iv e a n t i b o d i e s . •
T here have b e e n e x t e n s i v e and v a r ie d s t u d ie s o f c o l o s t r a ! t r a n s ­
f e r o f immune gamma g l o b u l i n s .
In t h i s r eg a rd th e v a s t l i t e r a t u r e
d e a lin g w ith c o l o s t r a ! im m u n oglob u lin 8 in b o v in e , o v in e and p o r c in e
d e s e r v e s some a t t e n t i o n b u t o n ly t h o s e i n v e s t i g a t i o n s w h ich w i l l
em p h asize a p a r t i c u l a r p o i n t are in c lu d e d in t h i s d i s c u s s i o n .
A g e n e r a l s tu d y o f th e im m unoglobulin c o n te n t o f th e serum o f
c a lv e s in England was r e p o r te h by Sm ith e t a l .
(1 9 6 7 ).
As ope m ight
e x p e c t t h e r e were g r e a t v a r i a t i o n s in immpnog lo b u lin le v e l s - w h ic h d id
n o t r e l a t e to th e serum o r c o l o s t r a l l e v e l s o f th e dam.
T h ese i n v e s t i ­
g a t o r s o b se r v e d no r e l a t i o n s h i p s b etw een im m unoglobulin c o n te n t o f
serum and when c a lv in g o c c u r r e d .
T h is l a t t e r o b s e r v a t io n i s n o t in
a c c o rd w it h th e f in d in g o f G ray, F is c h e r and McEwen (1 9 6 5 ) in a su rv ey
2
o f c a t t l e in S c o t la n d .
Sm ith e t a l .
(1 9 6 7 ) a t t r i b u t e th e v a r i a t i o n In im m unoglobulin
l e v e l in c a lv e s to m ethods o f husbandry in th e c o u n tr y and s u g g e s te d
a g e n e t i c v a r i a t i o n am ongst o f f s p r i n g i s in v o lv e d , e s p e c i a l l y th a t
w h ich i n f l u e n c e s th e a b s o r p t iv e c a p a b i l i t i e s o f th e i n t e s t i n e .
The
•im m unoglobulin c o n c e n t r a t io n was a s s a y e d by ah s o r p t io m e t r ic t e c h n ic s .
C o lo s t r a l t r a n s f e r o f R in d e r p e s t n e u t r a l i z i n g a n tib o d y compares
f a v o r a b ly b etw een F r e is e n cows and w a ter b u f f a lo ( SIghn e t a l . , 19670»
In t h e i r s t u d ie s F r e is e n dams w ere v a c c in a t e d w ith a I a p in iz e d s t r a i n
and w a ter b u f f a lo , w ere v a c c in a t e d w ith a v ia n iz e d and c a p r ia n iz e d
s t r a i n s o f R in d e r p e s t v i r u s 6 -7 m onths p r e -p a rtu m .
C a lv e s su c k le d
2 - 4 h o u rs p o st-p a rtu rn and s e q u e n t ia l a n tib o d y a s s a y s b egan 4 -5 hours
la te r .
Peak t i t r e s w ere seen , a t 8 h o u rs a f t e r s u c k lin g and p e r s i s t e d
f o r 21 d a y s .
The t i t r e s s lo w ly d e c r e a s e d to zero a t 6 - 7 m onths o f a g e .
The h a l f - r l f f e was c a l c u l a t e d to b e 33 days and 29 days f o r th e
w a ter b u f f a lo and F r e ls e n c a l v e s , r e s p e c t i v e l y .
The h a l f - l i f e
compared fa v o r a b ly w ith th e r e s u l t s r e p o r te d by Brown (1 9 5 8 ) who
exam ined R in d e r p e s t p e u t r a l i z i n g a n tib o d y in b o v in e .
An i n t e r e s t i n g
o b s e r v a t io n was t h a t th e h a l f - l i f e e x c e e d e d t h a t p f m a te r n a lly
d e r iv e d f o o t and mouth d i s e a s e a n tib o d y a s d eterm in ed by G raves ( 1 9 6 3 ) .
The p o s s i b i l i t y o f d i s s i m i l a r e x p e r im e n ta l p r o t o c o l m ust b e e n t e r t a i n ­
e d , y e t , t h i s sp ea k s t o o t h e r unknown f a c t o r s , sueh a s , t h o s e
3
i n f l u e n c i n g th e a b s o r p tio n and p a s s a g e o f im m u n oglob u lin s in th e g u t.
Sm ith (1 9 6 2 ) r e p o r te d t h a t E s c h e r ic h ia COI i s e p t ic e m ia commonly
o c c u r s in c o lo str p m -'d e p r iv e d c a l v e s .
In o t h e r s t u d i e s , hypogamma­
g lo b u lin e m ia was d e t e c t e d in c o l o s t r a l - f e d c a lv e s r e s u l t i n g in c o l i b a c i l l o s i s (F ey and M argaran t, 1 9 6 1 ) .
T h ese w orkers have r e p o r te d an
in c id e n c e o f hypogam m aglobulinem ia in 10% o f "normal c a lv e s " th ey
have s t u d ie d .
I t seem s p o s s i b l e t h a t hypogam m aglobulinem ia p r e c e d e s
th e e s t a b lis h m e n t o f g u t f l o r a v f a i n g e s t i o n o f f e c a l m a t e r ia l from
u d d ers o r e n v ir o n m e n ta l f o m i t e s .
In a d d i t io n , t h o s e s m a ll q u a n t i t i e s
o f im m u n oglob u lin s t h a t a r e p a s s e d from dam to o f f s p r i n g may be
ab sorb ed by o rg a n ism s in o r o f th e i n t e s t i n e and n e v e r c o n t r ib u t e to
p a s s iv e im m unity o f th e c a l f .
D ata p e r t i n e n t to t h i s th e o r y a r e found in a r e p o r t b y K la u s s ,
B en n et and J o n e s ( 1 9 6 9 ) .
Three c a lv e s from a group o f 1 0 , demon­
s t r a t e d low IgM and IgG l e v e l s in serum b e g in n in g day I and l a s t i n g
u n t i l day 7 .
One o f th e dams o f t h e s e c a lv e s had norm al serum and
c o l o s t r a l l e v e l s o f IgM and IgG.
Two q f th e t h r e e c a lv e s w ere born
.
o f dams w it h an i n f e c t i o u s p r o c e s s ; on e a m a s t i t i s and t h e o t h e r ? a
case o f e n t e r i t i s .
The m a s t i t i s cow had an in c r e a s e d c o n c e n tr a t io n
o f IgM i n h e r serum w h ich i s i n d i c a t i v e o f an a c u te r e s p o n s e .
The
e t i o l o g i c a l a g e n ts w ere n o t in d ic a t e d n or were th e s u b c l a s s e s o f IgG
r e p o r te d .
T here w as, h ow ever, a c o n t r ib u t io n to serum im m unoglobulin
4
l e v e l s o f th e c a l f presu m ab ly th rou gh th e c o lo str u m o f th e dam in
every c a se .
The p r e s e n c e o f IgG , IgM, and IgA in serum and c o lo s tr u m o f .
th e dam and i t s g r a d u a l a c c u m u la tio n in th e i n t e s t i p a l c o n t e n ts
and n e o n a t a l serum i n d i c a t e a t r a n s f e r f u n c t io n o f th e mammary
g la n d and th e i n t e s t i n e .
Z u ffa (1 9 6 4 ) v a c c in a t e d b rood sows w ith m o d ifie d v i r u s o f
A u je sky* s d i s e a s e in a stu d y o f p l a c e n t a l t r a n s f e r o f v i r u s and
c o l o s t r a l t r a n s m is s io n o f a n tib o d y .
w ere t r a n s m itt e d d i a p l a c e n t n l l y .
N e ith e r v i r u s n or a n tib o d y
H ow ever, 24 h ou rs p o st-p ^ rtu m th e
p i g l e t serum t i t r e was e q u a lly h ig h a s in m a te r n a l serum .
The h a l f -
l i f e o f t h e s e a n t ib o d ie s was c a lc u l a t e d to b e 1 0 .8 d a y s .
P a s s iv e im m unity t o A s c a r js suum was t r a n s f e r r e d in c o lo str u m
from sows t o t h e i r o f f s p r i n g ( K e lle y and N ayah, 1 9 6 5 ) .
The humdral
a n t ib o d ie s d i r e c t e d a g a in s t a s c a r j s la r v a e c o n fe r r e d im m unity by
im peding t h e m ig r a t io n o f th e la r v a q to th e l u n g s .
S p r in t (1 9 4 9 )
found t h a t m ig r a tio n o f A s c a r is la r v a e was sto p p ed in th e l i v e r o f
young immune m ic e .
S o u ls b y ( 1 9 6 1 ) ,o b se r v e d a c e l l u l a r r e a c t io n
ab ou t th e 3rd s t a g e la r v a e g iv e n i n t r a p e r i t o n e a l l y to immune a n im a lsi
The r e a c t io n in th e l i v e r and i n t e s t i n e in c lu d e d a l e u c o c y t e r e sp o n se
w h ich c o u ld p o s s i b l y have b lo c k e d m ig r a t io n .
5
The p r e s e n c e o f ab sorb ed c o l o s t r a l a n tib o d y w i l l e f f e c t a
r e sp o n s e t o ■E s c h e r ic h ia c o l i (6 s p e c i e s ) fn p o r c in e n e o n a t e s .
Sharpe (1 9 6 6 ) compared a n tib o d y r e s p o n s e in n o r m a lly r e a r e d p i g l e t s
p a r t i a l l y d e p r iv e d o f c o lo s tr u m and n o te d t h a t th e d e p r iv e d p i g l e t s
p roduced a n tib o d y a t an e a r l i e r age and w ere s t i l l a b le to absorb:,
b u t to a l e s s e r d e g r e e , a n t ib o d ie s when r e tu r n e d to a sow 26 hours
p o s t-p a r tu m .
b o th a n t i- " 0 " and an ti-" K " a n tib o d y t i t r e s v a r ie d in a
s im i la r f a s h io n .
T i t r e s w ere n e g a t iv e a t b i r t h as d e term in ed by
th e t e c h n iq u e s em p loyed.
P i e r c e and Sm ith (1 9 6 7 ) i n v e s t i g a t e d hom ologous and h e ter o ^
lo g o n s c o I o s t r q l im m unoglobulin t r a n s f e r in p o r c in e n e o n a t e s .
The
c o l o s t r a l im m u n oglob u lin s were ab sorb ed s i m i l a r i t y i f fe d s e p a r a t e ly
in h ig h c o n c e n t r a t io n s
I n t e s t i n a l a b s o r p tio n in p i g l e t s fe d h e t e r o ­
lo g o u s c o lo s tr u m o f b o th w ere ab sorb ed r e a d i l y w ith o u t ap p a ren t
c o m p e t itio n .
I n t e r n a l d e g r a d a tio n o f im m u n o g lo b u lin s, h o w ev er,
made d e f i n i t i v e i n t e r p r e t a t i o n d i f f i c u l t .
P o r t e r ’ s (1 9 6 9 ) work
su p p o r ts t h e f in d in g s o f P ie r c e and F e i n s t e i n (1 9 6 5 ) and e a r l i e r
w ork ers t h a t th e i n t e s t i n a l t r a c t o f n e o n a t a l b o v in e w i l l absorb
p r o te in s in d is c r im in a te ly .
On the- o t h e r hand, in v i t r o s t u d ie s
( P ie r c e and S m ith , 1967b ) i n d i c a t e a s e l e c t i v e m echanism w ith th e
m ost pronoun ced im m unoglobulin p a s s a g e in th e m id d le t h ir d (ile u m )
o f th e p o r c in e s m a ll i n t e s t i n e .
A l s o , . H a llid a y and K eck w ith ( 1 9 6 0 ) ,
6
Locke e t a l .
(1 9 6 4 ) have showp. t h a t IgG immune g l o b u l in s a r e absorbed
more e f f e c t i v e l y th an IgM In th e new bqrn r a t apd p i g .
A s i e v e - d i k e f u n c t io n has b e e n p o s t u la t e d f o r thq mammary glan d
o f th e r a t (L o w e ll and M organ, 1 9 6 5 ) .
The dom inant p r o t e i n s demon-,
s t r a t e d i n r a t m ilk a r e serum alb u m in , t r a n s f e r r i n ,and a lp h a - g lo b u lin ^
L ip o - p r o t e in s w ere n o t found in t h e m ilk w hich a t t e s t s bo th e s e l e c t ­
i v i t y o f t h i s g la n d .
A ls o , th e h e a v ie r m o le c u la r w e ig h t f r a c t io n o f
a lp h a g lo b u l in s w ere n o t t r a n s f e r r e d .
D ix o n , e t a l .
(1 9 6 1 ) have shown la r g e amounts o f im m unoglobulin
a r e t r a n s f e r r e d from serum to c o lo s tr u m in p r o c in e due t o a co n cen t
t r a t i n g m echanism in a l y e p la r c e l l s o f th e mammary g la n d .
Immuno-
f l o u r e s c e n t s t u d ie s o f th e udder showed a few s p e c i f i c a l l y s t a i n i n g
c e lls .
P i e r c e apd F e i n s t e i n ( 1 9 6 5 ) , Lom asi e t a l .
(1 9 6 9 ) have shown
IgG^ " f a s t m oving f r a c t io n " p r e d o m in a te s in b o v in e c o lo s tr u m .
I t is
d i s t i n c t from th e I lS s e c r e t o r y IgA r e p o r te d in r a b b it s (Cebra' and
S m a ll, 1 964) and humans ( T o u r v ilI e e t a l , , 1969) and in m ice
( A s o f sky and H y lto n , 1 9 6 8 ) .
Mach, Pahud and I s l i k e r (1 9 6 9 ) r e p o r t f in d in g IgA w ith " se cr e t,
t o r y p ie c e " in b o v in e c o lo str u m ^nd s a l i n a .
F ree "S" p i e c e was demop^
s t r a t e d in b o v in e c o lo s tr u m apd m ilk b u t n o t in serum .
7
A s tu d y £p d e t e c t p o r c in e c o l o s t r a l a n tib o d y to E s c h e r ic h ia
c o l i 08 and E s c h e r ic h ia c o l i 0141 was con d u cted by P ark er ( i 9 6 0 ) .
He was u n a b le to d e t e c t IgA in sow serum o r p o s t - c o l o s t r a l p i g l e t
serum; IgM was found t o b e th e m ain component w ith a c t i v i t y in th e
sow serum o r p o s t - c o l o s t r a l p f g l e t serum; IgM was found to b e th e
m ain com ponent w it h a c t i v i t y in th e sow serum .
On th e o t h e r hand,
th e a g g l u t i n a t in g a c t i v i t y found in c o l o s t r a l sam p les was low ered
when t r e a t e d w ith r a b b it a n t i^ p o r c ip e IgA a n tib o d y .
The c o n c lu s io n
was made th e IgA d o e s n o t p a r t i c i p a t e in p a s s iv e im m unity o f th e
p i g l e t b e c a u s e i t was n o t found in th e serum .
Sephadex f r a c t i o n ­
a t i o n , h e x o s e .a ssa y (10%) and im m unochem ical .d e te r m in a tio n s i n d i c a t e
th e Im m unoglobulin found by Mach and co-,w orkers (1 9 6 9 ) may b e IgA .
T h ese w ork ers p o s t u la t e d t h a t s y n t h e s i s o f "T p ie c e d t a k e s p la c e in
s e c r e t o r y e p i t h e l i a l c e l l s and IgA s y n t h e s i s in l o c a l i z e d plasmorc y te s.
A so fsk y and S m all (1 9 6 7 ) in c o r p o r a te d r a d i o - l a b e l l e d amino
a c id s in " in v it r o "
s t u d i e s and i n d ic a t e d "T p ie c e " i s s y n t h e s iz e d
i n th e r a b b it mammary g la n d s..
T h ese s t u d ie s s t r o n g ly su p p o rt th e e a r l i e r p r o p o s a l by Tomasi
I
e t a l . (1 9 5 8 ) t h a t "T p ie c e " i s s y n t h e s iz e d by a c e l l c lo n e d i s t i n c t
from th e IgA c lo n e , m o reo v er, i t h as b e e n s u g g e s te d b o th may be
s tim u la t e d ' E f f e c t i v e l y by l o c a l
a n t ig e n a t io n .
8
A h y p o t h e t ic a l m odel f o r th e t r a n s p o r t o f . IgA and "T p ie c e "
a c r o s s mucous membranes h a s b een p o s t u la t e d by T o u r v i l l e , A d le r ,
B ie n e n s to c k and Tom asi ( 1 9 6 9 ) , F ig u r e I .
The s t r u c t u r e f o r s e c r e t o r y XgA h a s b e e p p ro p o sed by Cebra
( 1 9 6 9 ) , F ig p r e 2 .
S e c r e t o r y IgA i s n o t e f f e c t e d in t h e u s u a l manner by th e p r o ­
t e o l y t i c en zy m es, p a p a in o r p e p s in , and i s more r e f r a c t i v e to
i n t e s t i n a l p r o t e o l y t i c a c t i v i t i e s th an IgG and IgM.
T h is i n d i c a t e s a
p r o t e c t i v e r o l e f o r "T p ie c e " w h ich may in c r e a s e th e e f f e c t i v e h a l f l i f e o f t h e s e a n t ib o d ie s in th e i n t e s t i n e o r o t h e r s i t e s on th e mucouA
membrane, ( Tomasi , G a lv a n ic © , 1 9 6 8 ) .
o r IgM.
"T p ie c e " w i l l n o t b in d to IgG
T here a r e no known a n t ig e n i c s i m i l a r i t i e s b etw een "T p ie c e "
and l i g h t o r h eavy c h a in s d e te r m in a n ts .
T here i s e v id e n c e , (B pand t-
z a e g , 1 9 6 9 ) t h a t "T p ie c e " o b ta in e d by r e d u c t iv e tr e a tm e n t o f
s e c r e t o r y IgA -has a n t ig e n i c d e te r m in a n ts t h a t a r e n o t r e c o g n iz e d by
a n t i s e r a r a is e d to "T p ie c e " s tim u la t e d by th e H S s e c r e t o r y IgA
m o le c u le .
Complement f i x a t i o n d o e s n o t o c c u r b e c a u s e s e c r e t o r y IgA i s
u n a b le to a c t i v a t e C^.
R ecen t s t u d i e s by Vaerman and Heremans (1 9 6 9 )
have shown t h a t s c i a l i c a c id removed b y n eu a m in id a se from th e s e c r e ­
t o r y IgA m o le c u le , d o e s n o t a l l o ^ com plem ent a c t i v a t i o n .
In th e
p r e s e n c e o f .ly s o z y m e and com plem ent ( A d i h o l f i , 1966) b a c t e r i o l y t i c
L umen
Apical
mucin
layer
• IfA -SP '
F ig u r e I .
A h y p o t h e t ic a l m odel f o ^ th e tr a n s p o r t o f gamma A and
SP a c r o s s m u cosal membrane e p it h e liu m .
(From D.
T o u r v i l l e , R. A d le r , J . B ie n e n s to c k and T .B . T om asi,
J.E .M .
1 2 9 , 2 , 1 9 6 9 .)
V
3
] \
\L
F ig u r e 2 .
N
YZ
XZ
\
P rop osed m odel o f th e c o l o s t r a l IgA . T c h a in i s r e p r e ­
s e n te d by a l i n e o f lo n g d a s h e s .
(As ad ap ted from John
J . C eb ra, B a c t. R eview s V o l. 3 3 , Number 2 , June 1 9 6 9 .)
1-0
r e a c t io n s w ith E s c h e r ic h ia c o l i have b een d e m o n str a ted .
T h is a c t i v i t y
h a s n o t b e e n o b s e r v e d in o t h e r sy ste m s n or con firm ed b y o t h e r w o rk ers.
A lth o u g h th e e x a c t m echanism o f "T piece*' has n o t b e e n f u l l y
e lu c i d a t e d and H S s e c r e t o r y IgA was n o t s p e c i f i c a l l y a s s a y e d fo r in ■
many o f t h e a f o r e m en tio n ed s t u d ie s i t i s e v id e n t t h a t s e c r e t o r y IgA
sh o u ld n o t .b e c o n s id e r e d i n hum oral im m unity b u t p r im a r ily in l o c a l
i n t e s t i n a l im m u n ity .• "T p ie c e " p erform s th r e e b a s i c f u n c t io n s in th e
i n t e s t i n e ; ( I ) a tta c h m e n t and a b s o r p t iv e d is c r im in a t io n a t th e
e p i t h e l i a l membrane l e v e l ,
( 2 ) im m o b iliz a tio n a g a in s t f lu s h i n g a c t io n s
in th e g u t and ( 3 ) to p r o t e c t th e im m unoglobulin from p r o t e o l y t i c
enzym es in th e i n t e s t i n e .
■ In trod u ction to th e T h e s is
S e c r e t o r y IgA h as b e e n s tu d ie d i n r a b b it s by C eb ra, R ob b in s,
S m a ll, F e i n s t e i n and o t h e r s ? in man by P a tto n and Pigm an, T om asi,
Z eiglem an e t a l - , Lehner e t ^ l . ,■ C rabbe, H erem ans, G e lz a y d , Rosen e t
a l . , B r a n d tz a e g , J e f f r i e s , C h ord irk er and o t h e r s and in m ice by Mandel
and A so fsk y and o t h e r s .
I t i s p o s s i b l e t h a t IgA h as b e e n s tu d ie d o r
d is c o v e r e d in o t h e r s p e c ie s b u t n o t e n c o u n te r e d d u r in g t h i s l i t e r a t u r e
search .
The e x i s t e n c e o f s e c r e t o r y IgA and i t s r e le v a n c e in l o c a l i z e d
im m unity and in p a s s i v e i n t e s t i n a l im m unity in n e o n a te s makes i t a
11
p e r t i n e n t s tu d y b o th o f econom ic a s w e l l a s academ ic im p o r ta n c e .
R e a liz in g f u r t h e r , t h a t la r g e a n im a ls and humans are n o t am enable
f o r i n t e n s i v e r e s e a r c h , th e mouse becom es th e m odel o f c h o ic e *
C o n v e n tio n a l r e a r e d (CR) and germ free (CF) m fce were s e l e c t e d f o r
t h i s s t u d y , e s p e c i a l l y s in c e IgA h a s n o t b een (u n e q p iv o c a b ly )
d em o n stra ted i n th e g e r m fr ee m ou se, h e n c e , i t s e r v e ? a u s e f u l c o n t r o l
sy ste m .
T h ere are o b v io u s sh o r tc o m in g s in i n t e r p o l a t i n g and e x t r a ­
p o l a t i n g d a ta from one mammalian sy ste m in t o a n o th e r , h e n c e , t h i s
stu d y i s u n d erta k en to " d em o n stra te th e c l a s s e s o f im m unoglobulin s
s y n t h e s iz e d in CR and CF m ice s tim u la t e d by a w e ll d e f in e d a n t ig e n ."
Em phasis w i l l b e d i r e c t e d tow ard th e e x a m in a tio n o f th e s e c r e t o r y
immune sy ste m and to r e l a t e f in d in g s t o t h e s t u d ie s o f o t h e r mammalian
s y s te m s .
The "modus op eran d !" o f e x p e r im e n ts d e a lin g w ith s e c r e t o r y IgA
g r e a t l y I n f lu e n c e s th e f i n d i n g s .
T h u s, Genco and Taubmann (1 9 6 9 )
e l i c i t e d a s e c r e t o r y IgA r e sp o n se by l o c a l l y a n t ig e n i z ip g r a b b it
mammary g la n d s w h ile Ogra e t a l .
(1 9 6 0 ) d em o n stra ted a s i g n i f i c a n t
d i f f e r e n c e in s e c r e t o r y IgA t i t r e s i f human s u b j e c t s w ere a n t ig e n iz e d
w ith l i v e p o l i o v i r u s r a th e r th an dead v i r u s ; th e r o u te o f i n j e c t i o n
and n a tu r e o f th e a n t ig e n w ere deemed to b e o f s ig n a l im p o r ta n c e .
P an se and D u tta (1 9 6 4 ) o b se r v e d ho p r o t e c t io n o f n e o n a t a l r a b b it s i f
12
t h e m other had b een a n t ig e n iz e d w ith f o r m a lin iz e d o r i n a c t i v a t e d
v a c c in e ev en th ough m a te r n a l serum t i t r e s in d ic a t e d a good r e s p o n s e .
S i g n i f i c a n t p r o t e c t i o n was c o n fe r r e d i f th e m other was a n t ig e n iz e d
w ith l i v e v i b r i o v a c c in e .
P an se e t a l ,
(1 9 6 9 ) d em o n stra ted a s im ila r
phenomenon w ith p a r e n t e r a l a d m in is t r a t io n o f p a s s iv e a n t ib o d ie s from
serum o f a n t ig e n iz e d f e m a le s i
Intramammary i n s t i l l a t i o n o f myxp-
v i r u s and a d e n o v ir u s e l i c i t e d n e u t r a l i z i n g a n t ib o d ie s in g o a t s (immuno­
g lo b u l in s n o t s p e c ia t e d ) (G u e r in , M it c h e ll and P a s ie k a , 1 9 6 9 ) .
In a
s im i la r ex p e r im en t p r o p a g a tio n o,f H erpes s im p le x f a i l e d when i n s t i l ­
le d in g o a t mammary g la n d in 2 o f 3 t e s t a n im a ls , t h e r e f o r e , a
s i g n i f i c a n t t i t r e was n o t e l i c i t e d .
S t u d ie s c o n c e r n in g th e e f f e c t o f th e serum c o n c e n t r a t io n o f IgA
c i r c u l a t i n g o r in f u s e d , on th e s e c r e t o r y IgA c o n c e n tr a t io n in secrett o r y s u b s ta n c e s a r e n o t c o n c lu s i v e .
The d a ta a v a i l a b l e i s lim it e d
and d o e s n o t c o n c l u s i v e l y in d i c a t e a p o s i t i v e e f f e c t .
To r e l e g a t e s e c r e t o r y im m u n oglob u lin s t o th e IgA c l a s s i s
u n d e r s ta n d a b le , e s p e c i a l l y jjith th e f in d in g o f "T p i e c e " .
In mammals
w h ich have n o t shown s e c r e t o r y IgA i n s a l i v a , c o lo str u m and i n t e s t i n a l
w a s h in g s , a d i f f e r e n t l o c a l immune m echanism r e q u ir e s a t t e n t i o n s o r ,
p erh a p s a more i n t e n s e s e a r c h f o r "T p ie c e " u t i l i z i n g more s e n s i t i v e
m ethods and g r e a t e r c o n c e rn f o r t h e te c h n iq u e s em ployed f o r a s s a y in g
IgA a c t i v i t y .
E s p e c i a l l y im p o r ta n t i s th e a n t ig e n a t io n p r o t o c o l w hich
13
i s o b v ia te d in th e in tr o d u c t o r y rem arks.
L o c a liz e d (ju x ta p o s e d to
th e mammary g la n d s ) a p t ig e n a t io n sh o u ld e l i c i t C d lo s t r a l IgA in th e
c o n v e n t io n a l and g erm free m opse,
IgA p r o d u c tio n in some m urine
s t r a i n s d is p la y s d i s p a r i t y under v a r io u s e n v iro n m e n ta l c o n d i t i o n s ,
i . e . , th e a b se n c e o f IgA in th e qerum and c o lo str u m o f germ free m ice
in c o n t r a s t to t h e p r e s e n c e o f IgA in th e serum and
qoI o strum
of
c o n v e n t io n a lly r e a r e d , ( A s o f sky and H y lto n , 1 9 6 8 ).
The g erm free m ice
in t h i s s tu d y p roduced a p r o t e i n m a t e r ia l t h a t c o p r e c i p i t a t e d w ith
IgA apd had a s e d im e n ta tio n c o e f f i c i e n t o f 4S^
There was good e v id e n c e
t h i s m a t e r ia l was "T" p i e c e s i n c e , i n th e p r e s e n c e o f IgA myeloma
p r o t e i n and r a d i o - l a b e l l e d amino a c id s vyhich were added t o a t i s s u e
c u lt u r e o f germ free mammary g la n d , and I lS r a d i o - l a b e l l e d p r o t e in
was form ed, (M urine s p e c i e s n o t s p e c i f i e d ) .
Four im m unoglobulin s p e c i e s , IgG ^, IgG ^, IgM and IgA have been
shown to in c r e a s e i n th e serum n n d /o r c o lo s tr u m o f many s t r a i n s o f
m ice f o ll o w i n g a n t ig e n a t f o n w ith a v a r i e t y o f a n t ig e n s .
T h e ir
a b s o lu t e r o l e in p a s s i v e im m unity and o r i g i n in th e serum o f th e
n e o n a te i s s t r o n g ly d e b a te d .
T h u s, t h e s e s t u d ie s so u g h t to i d e n t i f y
s p e c i f i c immunoiglobulin s p e c ie s in t h e dam and n e o n a te s u s in g immun­
i z a t i o n p r o c e d u r e s , s e r o l o g i c a s s a y s , and, an im a ls r e a r e d under
a n t ig e n f r e e e n v iro n m en ts i n o r d e r to t r a c e th e o r i g i n and a c t i v i t y o f
p a s s i v e l y a c q u ir e d im m u n oglob u lin s in n e o n a te s*
MATERIALS AND METHODS
A n im a ls:
Randomly b red c o n v e n t io n a lly r e a r e d (CR) and g erm free (GE)
m
Sw is s Manor m ic e , o b t a in e d in 1964 from Manor Farms ( S t a a t s b u r g , New
York) w ere u se d th r o u g h o u t t h e s tu d y .
The CR m ice w ere m a in ta in e d •
on P u r in a L a b o ra to ry Chow and w a ter ad lib it u n i w ith o c c a s i o n a l f e e d ­
in g s o f Quaker R o lle d O a ts .
The GF m ice w ere h o u sed in T r e x le r germ-
f r e e u n i t s , fe d s t e r i l e P u r in a L a b o ra to ry Chow # 5 QlOC and s t e r i l e
w a te r ad_ lib it u m w ith o c c a s i o n a l f e e d in g s o f s t e r i l e Quaker R o lle d
O a ts.
The g erm free te c h n iq u e s u se d in th e stu d y w ere t h o s e adapted in
p r e v io u s work in g n o t o b i o t i c sy ste m s (R eed , 1 9 6 6 ).
The g erm free
fe e d was d r ie d f o r 30 m in u te s , a u to c la v e d f o r 35 m in u te s a t 120°C and
18 pounds p r e s s u r e , th e n d r ie d a g a in f o r 30 m in u te s .
The s t e r i l i z e d
f e e d s w ere c u ltu r e d in T h i o g l y c o l l a t e b r o th and B r a in H ea rt L iv e r
b r o th to in s u r e s t e r i l i t y b e f o r e commitment to th e g erm free e n v ir o n ­
m en t.
F e c a l sam p les from germ free m ice w ere c u ltu r e d a t m onth ly
in te r v a ls .
A n tig e n P r e p a r a tio n ^ ■■
D in itr o p h e n y la t e d b o v in e gamma g l o b u l in ( DNP-BGG) was p rep ared
a c c o r d in g t o th e te c h n iq u e d e s c r ib e d by E is e n ( 1 9 6 4 ) .
The sodium
s a l t o f 2 ,4 - d in it r o b e n z e n e s u l f o n a t e (2,4-D N B S) was c r y s t a l l i z e d by
15
ad d in g 7 ,5 gms o f 2,4-DNBS to 500 ml o f e t h y l a lc o h o l (95% ).
T h is
s o l u t i o n was h e a te d t o 7 0 °C in a d o u b le b o i l e r and m a in ta in e d a t
t h i s tem p e r a tu re u n t i l th e m a te r ia l was d i s s o l v e d .
One and f i v e
t e n t h s grams o f N o r it powder was th e n added and th e s o l u t i o n was
s t i r r e d f o r two m in u te s .
N o r it and i m p u r it ie s w ere removed fro m . s u s p e n s io n by p a s s in g
th rou gh a d o u b le l a y e r o f Whatman #3 f i l t e r p ap er in a D -3 Buchner
f u n n e l.
S u c tio n was a p p lie d to a id f i l t r a t i o n flo w -
The f i l t r a t e
»
was r e f r i g e r a t e d to h a s t e n c r y s t a l fo r m a tio n .
The c r y s t a l s were
a g a in d i s s o l v e d in h o t a lc o h o l (95%) and th e p r o ced u re r e p e a te d .
F i n a l l y , c r y s t a l s w ere form ed in a la r g e e v a p o r a tin g d is h and th en
s t o r e d in a dark b o t t l e u n t i l u se d in t h e s u b s t i t u t i o n p r o c e d u r e .
To 50 ml o f d i s t i l l e d w a ter was added 500 mg o f th e sodium
s a l t o f DNBS ( c r y s t a l l i z e d ) ' , . 500 t)g o f b o v in e gamma g l o b u l in (BGG)
( F r a c t io n I I , Armour L a b o r a t o r ie s , K ankakee, I l l i n o i s ) and 500 mg
anhydrous p o ta s siu m c a r b o n a te .
The s o l u t i o n was s t i r r e d f o r 24 h o u r s ,
th e n d ia ly z e d a g a i n s t 16 ch an ges o^ d i s t i l l e d w a ter o y e r t h e n e x t 8
d ays.
The d i a l y s a t e was k e p t r e f r i g e r a t e d th ro u g h o u t t h e d i a l y s i s .
A l l o f t h e g la s s w a r e was co v e re d w ith aluminum f o i l to p r o t e c t th e
l i g h t s e n s i t i v e 2,4-D N P .
To d e ter m in e th e s u b s t i t u t i n g e f f i c i e n c y o f t h e p r o c e d u r e ,
th e d i a l y s a t e was d i l u t e d I : 100 w ith d i s t i l l e d w a ter and th e o p t i c a l
16
d e n s i t y m easured a t 360 n an om eters (pm) and 278 ntn in a Perk i n Elmer m odel 124 S p e c tr o p h o to m e te r .
The M olar E x t in c t io n C o e f f i c i e n t
f o r 2,4-DNP u se d in a l l c a l c u l a t i o n s was 1 7 ,4 0 0 and th e
was 1 4 .8 ( a s d eterm in ed in ou r l a b o r a t o r y ) .
17
f o r BGG
C a lc u la t io n s o f th e
amount o f h a p t e p c a r r ie r s y n t h e s iz e d in d ic a t e d t h a t 65 r e s id u e s o f
DffP r e a c t e d w ith ea ch BGG m o le c u le . , H en ce , th e a n t ig e n u se d in t h i s
stu d y i s r e f e r r e d to a s :DNP^^-BGG.
P r e p a r a tio n o f a n t i s e r a and c o lo s tr u m :
S ix t e e n fem a le CR S w iss Manor m ice w ere i n j e c t e d in tr a d e r m a lly
( l . D . ) in 2 s i t e s ; ( I ) th e m id lin e b etw een th e m ost p o s t e r i o r mammary
g la n d s and ( 2 ) th e m id lin e b etw een th e secon d m ost a n t e r io r mammary
g la n d s .
The a n t ig e n i n j e c t e d c o n s is t e d o f 2 3 0 pg, DNP^-BGG e m u ls if ie d as
a s a l i n e s o l u t i o n ip an e q u a l volum e o f Freunds co m p le te a d ju v e n t
(CFA).
P r io r t o i n j e c t i o n th e DNP^^-BGG was p a s s e d th rou gh a 0 .4 5 p
m i l l i p o r e membrane c o n ta in e d ip a Sw inney a d a p te r .
h a n d led a s c e p f i c a l l y th r o u g h o u t th e s tu d y .
The a n t ig e n was
C o n tr o l a n im a ls r e c e iv e d
0 .2 ml o f s t e r i l e s a l i n e in CFA a d m in is te r e d in th e same f a s h io n .
Ten days l a t e r , e i g h t e x p e r im e n ta l a n im a ls r e c e iv e d a se co n d i n j e c t i o n
o f th e t e s t a n t ig e n by th e in tr a d e r m a l r o u t e .
t e n d ays a f t e r th e l a s t i n j e c t i o n .
A l l m ice w ere mated
■17
At p a r t u r i t i o n and a t 5 d ays p o s t-p a r tu m th e fe m a le s w ere b le d
from t h e r e t r o - o r b i t a l p le x u s and a t t h e same tim e , one h a l f o f th e
l i t t e r was p r o c e s s e d in th e f o llo w in g m anner: th e n e o n a te s w ere b le d
v i a t h o r a c ic i n c i s i o n , th e b lo o d c o l l e c t e d in n o n -h e p a r in ig e d hem ato­
c r i t tu b e s (G lay-A dam s) and th e serum h a r v e s te d a f t e r c e n t r i f u g a t i o n
in a Clay^Adams .m ic r o h e m a to c r it c e n t r i f u g e .
The s e r a w ere s to r e d in
th e f r o z e n s t a t e u n t i l a s s a y e d .
C olostru m yras. r in s e d from e x t i r p a t e d n e o n a ta l stom ach s w it h in
24 h ou rs o f b i r t h and m ature m ilk Vfas h a r v e s te d a t 5 d ays p o s t ­
partum -
The stom achs w ere r in s e d w ith a sp ra y o f s t e r i l o w a te r to
remove r e s i d u a l b lo o d b e f o r e b e in g opened a lo n g th e g r e e t e d c u r v a tu r e .
The c o lo s tr u m o r m ilk o f one n e o n a te was e x p r e s s e d th fo u g h th e i n c i s i o n
and susp en d ed in I ml s t e r i l e w a te r .
The s u s p e n s io n Was washed back
and f o r t h in a 5 ml s y r in g e u n t i l a sm ooth s u s p e n s io n was o b t a in e d .
The a d d it io n o f 0 .1 M HCl p r e c i p i t a t e d t h e c a s e in a t pH 4 .8 w hich was
s u b s e q u e n tly c le a r e d b y c e n t r i f u g a t i o n in a r e f r i g e r a t e d I n t e r n a t io n a l
HR-2 c e n t r i f u g e a t a sp eed o f 1 0 ,0 0 0 fpm and a tem p era tu re o f $°C.
The c le a r f l u i d b etw een th e upp er l i p i d la y e r and t h e sed im en ted
m a t e r ia l was c a r e f u l l y removed and th e pH a d ju s te d t o 7 .2 w ith 0 .1 M
KOH.
C l a r i f i e d c o lo s tr u m o r m ilk was k e p t fr o z e n u n t i l a s s a y e d .
The
pH o f th e c o lo s tr u m a t th e tim e o f c o l l e c t i o n ranged b etw een 6 .7 and
6 .9 .
A t th e tim e o f a s s a y th e c o l o s t r a l sam p les were c o n c e n tr a te d
18
a g a in s t d i a l y s i s tu b in g f i l l e d w ith CARBOWAX ( F is h e r ) to a f i n a l
volum e o f 0 . 2 - 0 . 3 m l.
G erm free a n im a ls w ere g iv e n a s i n g l e i n j e c t i o n o f 260 ]ig o f
DNPgJ--BGG in CFA and m ated 10 days l a t e r .
G ravid fe m a le s w ere r e ­
moved from th e g erm free en viron m en t z e r o to f i v e days p re-p a rtu m and
m a in ta in e d in s t e r i l e i s o l e t s ( C arw erth F arm s).
h a l f o f th e l i t t e r was removed and p r o c e s s e d .
At p a r t u i t i o n o n e The fe m a le s w ere n o t
b le d on day z e ro in o r d e r t o re d u c e t h e p o s s i b i l i t y o f c o n ta m in a tin g
th e r e m a in in g l i t t e r m a t e s .
C o n tr o l a n im a ls - CR and GF w ere i n j e c t e d
w ith a s a lin e - a d j u v a n t s u s p e n s io n in a manner s im ila r t o th e DNPg,.-BGG
a d ju v a n t a n t ig e n a t io n p r o c e d u r e .
R e s id u a l a d ju v a n t a t th e i n j e c t i o n
s i t e s was o b se r v e d p o st-m o rtem in a l l m ic e ,
T is s u e s p e c im e n s :
A d u lt fe m a le
5
w ere b le d from th e r e t r o ^ -o rb ita l p le x u s and th en
s a c r i f i c e d b y c e r v i c a l d i s l o c a t i o n f i v e days p o s t-p a r tu m .
The s k in
was opened v e n t r a l l y b y a Y shaped i n c i s i o n and th e s k in f l a p s were
r e f l e c t e d u s in g b lu n t d i s s e c t i o n .
Numbering th e fo u r p a ir s o f mammpry g la n d s , eep h a l'id t o c a u d a l,
th e r i g h t fo u r th and t h e l e f t secon d mammary g la n d s w ere e x c is e d ,
and f i x e d in 10% n e u t r a l i z e d f o r m a lin .
A 4 cm p o r t io n o f th e ile u m ,
5 cm p r o x im a l to th e cecum , was e x c is e d and f ix e d in n e u t r a li z e d
f o r m a lin .
V
19
The t i s s u e s w ere s e c t io n e d a t 5 - 7 jn icron s on an I n t e r n a t io n a l
C r y o s ta t a t 2 0 ° C and f l o a t e d on a s l i d e w ith a few drop s o f c p ld
a b s o lu t e e t h y l a l c o h o l .
The s l i d e s w ere s to r e d a t 4 ° C u n t i l s tu d ie d
by f l u o r e s c e n t a n tib o d y t e c h n iq u e s .
F lu o r e s c e n t s t u d i e s :
G oat a n ti-m o u se im m u n oglob u lin s ( a n t i - G ^ A-,
,-M ) (MELPAR) was
c o n ju g a te d w it h f l u o r e s c e i n is o t h i o c y a n a t e on c e l i t e (C a lb ie c h e m .) by
p r e p a r in g th e f o llo w in g s o lu t i o n :
Goat a n ti-m o u s e IgG , IgG^, IgA , o r IgM . . . .
C arbonate b u f f e r , pH 1 0 .3
F l u o r e s c e in e i s o t h i o c y a n a t e
D i s t i l l e d w a ter
0 .5 ml
............................................0 .5 ml
. . . . . . . . .
. . . . . . . . . . . . . . .
1 0 .0 mg
1 .2 5 ml
The s o l u t i o n iyas h e ld a t 5°C f o r 24 h ou rs b e f o r e chrom atograph­
in g in a 2 .0 x 33 cm g l a s s column packed w it h Sephadex G-50 ( F in e )
w h ich had b een a d ju s te d t o pH 7 .2 w ith p h o sp h a te b u f f e r e d s a l i n e .
The f i r s t peak o b t a in e d , a t th e v o id v o lu m e, was u se d in th e s tu d y .
F iv e ml o f f l u o r e s c e i n - l a b e l l e d a n t i s e r a w ere ab sorb ed w it h 500 mg
o f d r ie d m ouse l i v e r p ow d er, c e n t r if u g e d and d i lu t e d I : 100 b e fo r e
u se.
A n t is e r a were s t o r e d in th e fr o z e n s t a t e .
T is s u e s e c t i o n s w ere f lo o d e d w ith th e s p e c i f i c a n t i s e r a and
in c u b a te d in a m o is t chamber 4 -6 h o u rs a t room te m p e r a tu r e .
The
20
were r in s e d f o r a t o t a l o f 8 h ou rs w ith a change o f PBS a t fo u r hours
and th e n m ounted on 1 x 3
and 22 x 40 mm c o v e r s l i p s .
in c h s l i d e s u s in g G ly cero l-P B S ( 9 : 1 ) pH 7 .2
The s l i d e s were o b se r v e d f o r f lu o r e s c e n t
c e l l s w ith a L e i t z f l u o r e s c e n t m ic r o sc o p e equ ip p ed w ith th e f o llo w in g
f i l t e r s : UG-38 h e a t f i l t e r , UG-12 e x c i t e r f i l t e r and a #530 b p r r ie r
f i l t e r . ■ The in c id e n t l i g h t so u r c e was s u p p lie d by an 0sram -200
m ercury vap or lam p.
The number o f f lu o r e s c i n g c e l l s
(A ppend ix B)
o b s e r v e d in 50 o i l im m ersion f i e l d s w ere r e c o r d e d .
Q u a n t it a t iv e Im m unoglobulin A s s a y :
(E quipm ent, A ppendix C)
A r e c e n t m o d if i c a t i o n (M a s s e y e ff and Z i s s w i l l e r , 1 969) o f th e
c l a s s i c M a n c in i, C arbon ara, Heremans (1 9 6 3 ) p r o c e d u r e f o r s i n g l e
r a d i a l iitimunod i f f u s i o n was u sed to q u a n t it a t e th e l e v e l o f s p e c i f i c
im m u n oglob u lin s in s e r a and c o lo s tr u m .
Ten m il li m e t e r d i s c s w ere c u t from 1.07° S p e c ia l N o b le Agar p r e ­
p ared in 0 .2 M V ern a l B u f f e r , pH 8 . 6 , and poured 1 .5 mm in d ep th in
H yland im m u n o d iffu sio n p l a t e s .
The d i s c s were exp osed to a d r y in g
en viron m en t o f 3 7 °C f o r 30 m in u te s .
F ifty
p i o f a n t i s e r a (M elpar,
F a l l s Church, V i r g in ia ) d i lu t e d 1 :5 0 w it h V e m o l b u f f e r , 0 .0 2 M, pH
8 . 6 , was a p p lie d to th e d i s c s .
A f t e r 18 hou rs a t 2 2 -2 5 °C , th e a n t i ­
s e r a had a p p a r e n tly d i f f u s e d in t o th e d i s c s and were s u i t a b l e fo r
a d d it io n o f a n t ig e n .
A 2 mm w e ll was c u t in th e c e n t e r o f ea ch d i s c
w ith a b lu n t e d 13 gauge hypoderm ic n e e d l e .
21
Stan d ard c u r v e s ( F i g . 3 ) f o r ea ch im m unoglobulin w ere p rep ared
u s in g t w o - f o ld d i l u t i o n s o f p o o le d norm al mouse s e r a o b ta in e d from
r e t r o - o r b i t a l b l e e d in g s o f t e n 3 -6 month o ld m ale and fe m a le CR S w iss
Manor m ic e .
S e r i a l d i l u t i o n s w ere made in 17= BSA and in DNPgy-BSA.
F iv e p i o f a 1:2 and 1 :1 6 d i l u t i o n w ere added to th e w e l l s o f th e
d i s c s c o n t a in in g , r e s p e c t i v e l y , a n t i- I g A and a n ti-Ig M ; 5 p i o f a 1:32
and 1 :6 4 d i l u t i o n w ere added to w e l l s o f d i s c s c o n t a in in g a n ti-Ig G ^
and a n t i - IgG g.
The a n t ig e n (m ouse serum ) was added to t h e w e l l s w ith
a f i n e l y drawn c a p i l l a r y . tu b e c o n n e c te d to an o i l f i l l e d
1 .7 5 mm (GD) d ia m e te r , a t ta c h e d to an o i l f i l l e d
p Beckman S p in co m i c r o t i t r a t o r .
ty g o n t u b in g ,
s y r in g e , ad apted to
The p r e c i p i t i n r e a c t io n p r o g r e s s e d
f o r 3 6 -4 8 h ou rs and t h e u n p r e c ip it a t e d p r o t e i n s w ere removed by wash­
in g w it h 2 ch an ges p f 5 ml norm al s a l i n e f o r 2 4 h o u r s .
The d i s c s were
s t a ip e d w it h *0.6% B u ff a lo B la c k in 77» a c e t i c a c id f o r 20 m in u tes and
th e n d e c o lo r iz e d w it h c o p iu s amounts o f 107= a c e t i c a c id i n m eth a n o l.
* The s t a i n was p rep a red in th e f o llo w in g manner:
B u ff a lo B la c k NBR
M ethanol
.
.
.
.
.
.
.
.
. . . . . . . . . . . . . . . .
6 gm
405 ml .
D i s t i l l e d HgO ................................................
405 ml
A c e t ic A c i d ........................ ....................... ....
90 ml
The s o l u t i o n was f i l t e r e d th rou gh 2 l a y e r s o f gau ze
and s t o r e d a t room te m p e r a tu r e .
Reciprocal
chIutron o f Atorrn+)Serum
22
WU/ C/ uo/j_z>v&y
F ig u r e 3
/o
Jay.auj'ty /(J
Stan d ard c u r v e s d e v e lo p e d from r a d i a l im m u n od iffu sion
r e a c t io n s w ith v a r io u s s p e c i f i c g o a t a n ti-m o u se immuno­
g l o b u l in serum and d i l u t i o n o f a p o o l o f norm al S w iss
serum .
2:3.
The d ia m e te r s o f th e p r e c i p i t i n r in g s (A ppendix C) w ere m easured
w ith a G a ev ert m ea su rin g m ic r o s c o p e .
The d ia m e te r , in m i l l i m e t e r s ,
was p l o t t e d on s e m i- lo g r it h m ic p ap er as a f u n c tio n o f th e r e c ip r o c a l
d i l u t i o n o f th e a n t ig e n .
To d e te r m in e t h e q u a n tity o f immune g l o b u lin r e a c t in g s p e c i f i c ­
a l l y w ith th e DNP d e te r m in a n t in each p r e p a r a tio n a n o th e r s e t o f
a n t ig e n ( serum o r c o lo s tr u m ) w h ich had b een absorbed w ith h e a t d e­
n a tu r e d D N P ^y-B S A w ere a p p lie d to a d u p li c a t e s e t o f d i s c s .
The
d i f f e r e n c e in th e d ia m e te r o f th e r a d i a l d i f f u s i o n p a t t e r n betw een
nonabsorb ed and ab sorb ed serum o f c o lo str u m was e s t a b l i s h e d to be
a n tib o d y s p e c i f i c f o r t h e DNP d e te r m in a n t.
The d i s c s , c o n ta in e d in H yland immune d i f f u s i o n p l a t e s and s e a le d
w ith v a s e l i n e ■, w ere in c u b a te d in a m o is t en viron m en t a t room tem per­
a tu r e f o r 24 h o u r s .
The d ia m e te r o f t h e p r e c i p i t i n r in g s t h a t formed
vferq m easured in th e same manner a s th e sta n d a rd p r e c i p i t i n r in g s
form ed by norm al mouse serum and w ere r e p o r te d as a p e r c e n t o f th e
norm al m o u se■serum .
P a s s iv e H e m a g g lu tin a tio n :
A m o d if ic a t io n o f th e m ethod o f B u llo c k and K a n to r , (1 9 6 5 ) wqs
f o llo w e d u s in g 2 ,4 - d in it r o f lu o p o b e n z e n e (DNFB) (Sigm a C hem ical
Company, S t . L o u is , M is s o u r i) .
To a s o l u t i o n o f 20 ml o f e t h y I e n e -
d i a m i n t e t r a a c e t i c a c id (EDTA) pH 8 . 4 , p lu s 0 .3 ml o f 2% DNFB in
24
a c e to n e was added 10 ml o f 10% s u s p e n s io n o f r a b b it red b lo o d c e l l s
w h ich had b e e n c o l l e c t e d in s t e r i l e A l s e v e r *s s o l u t i o n and washed
t h r e e tim e s w ith p h y s i o l o g i c a l s a l i n e b e f o r e u s e .
The h a p te n -KRBC m ix tu r e was in c u b a te d f o r 15 m in u te s a t 37°C.
F o llo w in g i n c u b a t io n ? th e m ix tu r e was d i l u t e d in 180 ml EDTA b u f f e r ,
pH 7 . 5 , c o n t a in in g 1% g lu c o s e .
The c e l l s were c e n t r if u g e d a t a low
s p e e d , a p p r o x im a te ly 1800 rpm (500G ) f o r 10 m in u te s .
The c e l l s were
washed t h r e e tim e s w ith EDTA-17» D e x tr o se b u f f e r , pH 7 .5 , and d i lu t e d
to a f i n a l c o n c e n t r a t io n o f 10%.
Mouse s e r a o b ta in e d from -DNPf. ^-BGG a n t ig e n iz e d S w iss Manor M ice
(CR) was u se d to t e s t th e hapten-KRBC sy ste m in a p a s s iv e h em agglu t­
i n a t i o n p r o c e d u r e em p loyin g a d o u b lin g d i l u t i o n o f a n t i s e r a i n 0 ,1
ml to w h ich was added 0 .0 5 ml o f a 27» s u s p e n s io n o f h a p t e n e te d - r a b b it
red b lo o d c e l l s .
The r e a c t io n p r o c e ed ed a t 37°C f o r 3 -5 h ou rs a f t e r
w h ich t h e tu b e s w ere c e n t r if u g e d f o r I m in u te and o b s e r v e d f o r
a g g lu tin a tio n .
Guinea p i g com plem ent d i l u t e d 1 :1 0 was added to th e
sy ste m and th e tu b e s w ere returnee), to th e w a ter b a th (37°C ) f o r an
a d d i t io n a l h o u r .
The l a s t tu b e show ing a g g l u t i n a t io n o r h e m o ly s is
was c o n s id e r e d th e end p o i n t f o r d e te r m in in g th e t i t r e o f h em a g g lu ti n a t i n g and h e m o ly tic a n t ib o d ie s , r e s p e c t i v e l y .
Mammary g la n d p la q u e form in g c e l l a s s a y ;
Many m o d if i c a t i o n s o f th e h e m o ly tic p l a q u e - i n - g e l te c h n iq u e
d e v e lo p e d by J e r n e and N ord in (1 9 6 3 ) a r e r e p o r te d .
The p r o t o c o l
b e in g d e v e lo p e d f o r th e a s s a y o f mammary g la n d - d e r iv e d p la q u e form ing
c e l l s f o ll o w s m ethods d e s c r ib e d by Ingraham and B u ssard (1 9 7 0 ) and
in c lu d e s m a t e r ia ls qnd m ethods d e s c r ib e d by D a n ie ls and W e ig le (1 9 6 8 )
and d em o n stra ted i n our la b o r a to r y by C h i l l e r ( 1 9 7 0 ) .
Two e x c is e d mammary g la n d s xyere fo rc q d th rou gh 8 0 -9 0 mesh s t a i n ­
l e s s s t e e l o r b r a s s s c r e e n t o e f f e c t a c e l l u l a r s u s p e n s io n in W e ig le 1s
b a la n c e d s a l t s o l u t i o n (WBSS).
L ip id s and strom a w ere s e p a r a te d from
th e t i s s u e fra g m en ts and c e l l s a f t e r a 15 m in u te in c u b a t io n in an
ic e b a t h .
The c e l l s su sp en d ed b etw een th e l i p i d l a y e r and th e
se d im e n ted strom a w ere c o l l e c t e d and washed t h r e e tim e s w ith WBSS.
F in a f ", c e l l s u s p e n s io n s w ere a d ju s te d to 10^ to 10^ c e l l s p e r ml
and d e term in ed by hemoc y to m e te r c o u n t s .
Mammary g la n d c e l l s u s p e n s io n s w ere m ixed w ith 0 .0 5 ml o f a 1:15
s u s p e n s io n o f D N P ^ y -B S A -G o a t RBC in 0 ,5 ml o f 1% a g a r o se in WBSS.
The
m ix tu r e was e v e n ly d i s t r i b u t e d o v e r a g l a s s m ic r o sc o p e s l i d e p r e v io u s ­
l y c o a te d w it h 0.1% a g a r o s e .
The s l i d e s w ere in c u b a te d 3 - 4 hours a t
3 7 °C in a m o is t a tm o sp h ere.
D ir e c t p la q u e s were d e v e lo p e d by adding
g u in e a p i g com plem ent (a b so rb ed w it h g o a t r b c ' s ) d i l u t e d 1 : 1 0 .
2.6
I n d i r e c t p la q u e s w ere d e v e lo p e d by a d d it io n o f g o a t a n ti-m o u se
a n tise r u m a t a f i n a l c o n c e n tr a t io n o f I : 100 in com plem ent ( 1 : 1 0 )
ab sorb ed w ith g o a t red b lo o d c e l l s .
P la q u e s were o b s e r v e d w ith a 40X
m a g n ify in g g l a s s and i n d i r e c t i l l u m in a t io n .
RESULTS
Im m unoglobulin l e v e l s in CR dams f o llo w in g a s i n g l e i n j e c t i o n o f
230 u gs DNlc 7-BGG i n CFA.
Fem ale m ice g iv e n a s i n g l e dcpse o f DNP-BGG were b le d I and 5
days p o st-p a rtu rn f o r serum a s s a y s o f im m unoglobulin c o n c e n tr a t io n and
s p e c i f i c a n tib o d y t i t r e s .
T h ese d a ta a r c p r e s e n te d in T a b le I .
Data
o b t a in e d from fe m a le e x p e r im e n ta l a n im a ls ap p ears in g r a p h ic form in
th e b ar g r a p h , in F ig u r e & a .
4 b.
C o n tr o l d a ta a r e r e p r e s e n te d in, F ig u r e
The v a lu e s a r e a v e r a g e s o f day I and day 5 p o st-p a rtu rn s e r a
l e v e l s , h ow ever, n o t in c lu d e d i n t h e s e v a lu e s are n e g a t iv e serum
l e v e l s w h ich may have o c c u r r e d a s a r e s u l t o f t e c h n ic a l e r r o r s .
For
exam ple: a n e g a t iv e f in d in g in a dam s e r a o b ta in e d on e day p o s t ­
partum i s n o t in c lu d e d in th e c o m p u ta tio n o f th e a v e r a g e i f a sam ple
from th e same a n im a l, ta k e n f i v e d a y s l a t e r d em o n stra ted d e t e c t a b le
l e v e l s o f immune g l o b u l i n s .
IgA .
Two o f 5 m ice t e s t e d I day p o st-p a r tu m had c o n c e n tr a t io n s
o f IgA e q u a l to 171% (a v e r a g e ) o f c o n t r o l serum v a lu e s', w h ile 5 o f th e
7 a n im a ls t e s t e d 5 d ays p o st-p a r tu m (T a b le I ) showed IgA l e v e l s eq u a l
to 117 (a v e r a g e ) o f serum c o n t r o l l e v e l s .
A lth ou gh b o th ( 2 ) I day
p o s t-p a r tu m m ice had s p e c i f i c a n tib o d y l e v e l s w h ich e x c e e d e d 15% o f
:
th e im m unoglobulin c o n c e n tr a t io n ,, the. serum o f o n ly I o f 5 show ing
IgA a t day 5 p o s t-p a r tu m c o n ta in e d d e t e c t a b l e l e v e l s o f s p e c i f i c
T a b le I .
Day from
la s t
in je c tio n
■39 -
In m u n o g lo b u lin l e v e l s a t day I and day 5 p o st-p a r tu m in dams a n t i g e n iz e d w ith a s i n g l e i n j e c t i o n o f DNP^^-BGG.
IgA
IgM
1SG1
IgGg
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
0
( )-/
0
40'
41 .
0 (0 )
1 1 9 (2 4 )
7 1 (2 )
1 0 0 (2 5 )
7 6 (1 6 )
----
1 1 2 (1 0 )
----
1 1 9 (2 4 )
1 3 8 (3 4 )
1 9 2 (3 1 ) 1 3 2 (0 )
1 0 4 (2 2 )
—
1 2 5 (2 5 )
2 0 0 (1 7 ).
1 3 3 (0 )
9 0 (3 0 )
1 5 6 (6 9 )
1 1 2 (1 2 )
1 0 7 (2 2 )
9 0 (1 0 )
1 2 5 (2 5 )
1 6 8 (1 8 )
1 6 1 (6 1 )
1 3 8 (4 1 )
9 1 (1 7 )
9 7 (1 3 )
1 3 8 (1 8 )
1 0 5 (0 )
1 5 0 (1 5 ) 1 2 5 (0 )
1 0 0 (1 5 )
1 0 0 (2 5 )
6 0 (0 )
1 5 0 (1 5 )
1 5 0 (1 5 )
1 7 3 (5 )
1 7 1 (2 3 ) 1 1 7 (3 )
1 2 4 (2 5 )
1 1 2 (2 0 )
8 9 (1 6 )
1 1 6 (2 5 )
1 2 6 (1 8 )
1 4 1 (1 3 )
0 (0 )
0 (0 )
44
8 (0 )
1 3 3 (0 )
1 2 3 (4 )
60
0 (0 )
1 2 3 (0 )
83
Mean
.a/
1 0 0 (0 )
9 1 (9 )
————
44
———™
0 (0 )
—— —
---- - ”
1 1 9 (5 )
F ig u r e s in p a r e n t h e s is i n d i c a t e t h e p e r c e n t o f th e im m unoglobulin t h a t r e a c te d
s p e c i f i c a l l y w ith DNP.
W The im m unoglobulin d a ta from one dam i s r e c o r d e d f o r each tim e p e r io d .
M
Od
29
U Single injection CR
I l I l Two injections CR
140
Single injection GF
Specific antibody level
-
o_
I
2
O 120
8$
3
IOO
CO
to
O
Z
z>
60
-
O
UJ
^
z
<
Ul
2
40
20
-
Ig A
F ig u r e 4
(a )
Ig M
Ig G|
L e v e ls o f im m unoglobulin in th e serum o f fem ale
e x p e r im e n ta l a n im a ls .
Ig G g
30
Ig A
F ig u r e 4
(b)
Ig M
Ig G|
Ig G g
Im m unoglobulin l e v e l s in c o n t r o l a n im a ls .
'3i
a n tib o d y .
IgM.
A l l o f th e m ice t e s t e d f o r IgM had norm al o r s l i g h t l y
e le v a t e d l e v e l s o f t h i s im m unoglobulin c l a s s a v e r a g in g 104 and 967=
on days I and 5 r e s p e c t i v e l y .
E ig h t o f 11 a n im a ls (T a b le I ) demon­
s t r a t e d s p e c i f i c a n tib o d y in e x c e s s o f 127. o f th e t o t a l IgM con cen ­
tr a tio n .
There appeared to b e no d e c li n e in th e l e v e l s o f s p e c i f i c
a n tib o d y o v e r an 83 day p e r io d .
IgG^.
m ice t e s t e d .
The IgG^ l e v e l was norm al in a group o f 9 o f th e 10
S p e c i f i c a n tib o d y l e v e l s w ere 10 and 717, ( a v e r a g e ) o f
t o t a l IgG^ in dam serum I and 5 days p o st-p a r tu m and p e r s i s t e d a t
h ig h l e v e l s th r o u g h o u t th e 83 day s tu d y .
IgG^.
Serum IgG^ l e v e l s o f th e 12 e x p e r im e n ta l a n im a ls w ere,
on th e a v e r a g e , s l i g h t l y h ig h e r th an in th e c o n t r o l s e r a .
E ig h t
o f th e 12 dams had d e v e lo p e d s p e c i f i c a n tib o d y t i t r e s o f 107. or
more o f t h e t o t a l IgGg im m unoglobulin l e v e l s ,
A d e c l i n e in a n t i -
DNP a n tib o d y was n o t e v id e n t a t 83 days p o s t im m u n iza tio n .
32
Im m unoglobulin l e v e l s in n e o n a te s born to dams g iv e n a s i n g l e
i n j e c t i o n o f DNP^r-BGG.
D ata o b ta in e d from serum o f n e o n a te s ap p ears in g r a p h ic form in
F ig u r e s 5 , 6- and ITY-
S p e c i f i c a n tib o d y l e v e l s (a v e r a g e ) and l e v e l s
o f im m u n oglob u lin s (a v e r a g e ) found in n e o n a t a l serum a t day I and
day 5 p o st-p a r tu m a r e shown.
IgA .
Serum l e v e l s o f IgA f a i l e d to r e f l e c t th e serum IgA
c o n c e n t r a t io n o f th e dams (T a b le I I ) .
T h u s, 2 o f th e 7 n e o n a te s had
p a r t i a l l y a d u lt l e v e l s , one o f w h ich p o s s e s s e d s p e c i f i c a n tib o d y 5
days p o s t-p a r tu m .
T h ese d a ta s u g g e s t t h a t IgA d o e s n o t c o n t r ib u t e
to c i r c u l a t i n g sepum a n tib o d y l e v e l s o r , i f i t d o e s , o n ly in m inim al
am ounts.
The s p e c i f i c a n tib o d y in th e serum o f 5 day o ld n e o n a te s
born o f fe m a le s v a c c in a t e d 41 days p r e v io u s ly may have b e e n t r a n s ­
m it te d from s i t e s o f s y n t h e s i s in th e mammary g la n d and to th e n eo ­
n a t e v i a l a c t e a l s e c r e t i o n s s in c e IgA in th e serum in th e . dam were
u n d e t e c t a b le .
IgM.
I t was o b s e r v e d t h a t w h ereas th e dams showed h ig h IgM
l e v e l s and s p e c i f i c a n tib o d y t i t r e s th e n e o n a te s e x h i b it e d low l e v e l s
o f IgM in serum o f 6 o f th e 7 a n im a ls t e s t e d a t I day p o st-p a r tu m .
T h is p r o b a b ly ■i n d i c a t e s poor p l a c e n t a l t r a n s m is s io n o f IgM t o .the
n e o n a te s.
S in c e 3 o f 7 n e o n a t a l s e r a had a d u lt l e v e l s o f IgM w ith
s p e c i f i c a n tib o d y a t day 5 p o s t-p a ttu m (55% o f a d u lt l e v e l s w ith 11%
33
MEAN LEVEL OF IMMUNOGLOBULIN ( % of NSP)
140
120
IOO
80
60
40
20
□a
I
5
La_da
I
5
I
19 M
Figure 5
Im m unoglobulin l e v e l s in th e serum o f CR n e o n a te s
(Dams - s i n g l e i n j e c t i o n ) .
5
34
140
CL
CO
Z
%
120
35
100
3
CD
9
O
0
Z
I
80
3
Z
1
60
SKt
O
#0 I
UJ
>
UJ
40-
z
<
UJ
S
I##
S tt
1
I
i; ^
®
ttttgttS
1I .
Ig A
Figure 6
r
sit
Ig M
igG,
Im m unoglobulin l e v e l s in th e serum o f CR n e o n a te s
(Dams - two i n j e c t i o n s ) .
Ig G 2
F ig u r e 7
Im m unoglobulin l e v e l s in th e serum o f GF n e o n a te s
(Dams - s i n g l e i n j e c t i o n ) .
Table I I .
la s t
in je c tio n
39 -
Im m unoglobulin l e v e l s in th e serum o f n e o n a te s b o r n to dams a n t i g e n iz e d w ith a s i n g l e i n j e c t i o n o f DNP 7-BGG.
Ig A
Ig M
Ig G 1
Day p ost-p artu rn
I
5
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
1 0 0 (1 3 ) 1 6 1 (3 3 )
9 1 (9 )
1 9 0 (3 0 )
— ™ — —
1 0 0 (2 0 )
0 (0 )
0 (0 )
1 3 2 (3 0 )
1 4 5 (1 6 )
—™——
41
0(0)
44
68(1)
0(0)
0 (0 )
44
9 0 (0 )
0 (0 )
0 (0 )
1 0 7 (3 6 )
0 (0 )
60
0 (0 )
0 (0 )
1 1 1 (2 4 )
0 (0 )
0 (0 )
83
0 (0 )
0(0)
0 (0 )
0 (0 )
29(1)
28(4)
1 8 (4 )
5 5 (1 1 )
. Mean
b/
0(0)
0 (0 )
40
a/
0 (0 )
202(29)
0(0)'
0(0)
83(0)
0(0)
Ig G 2
0(0)
1 0 6 (6 )
1 4 7 (2 9 )
9 5 (2 0 )
1 2 6 (4 )
————
1 2 0 (0 )
1 0 0 (0 )
1 4 3 (4 3 )
1 1 6 (1 6 )
80(20)
1 0 4 (2 ) •
5 0 (0 )
83(0)
1 0 0 (2 3 )
9 5 (2 0 )
4 0 (2 )
8 6 (1 6 )
9 3 (1 4 )
1 1 1 (1 7 )
The im m unoglobulin d a ta f o r each tim e p e r io d was o b ta in e d from n e o n a ta l p o o le d
s e r a from one h a l f o f th e l i t e r .
A verage l i t t e r s i z e i s 8 .
F ig u r e s in p a r e n t h e s is i n d i c a t e t h e p e r c e n t o f th e im m unoglobulin t h a t r e a c te d
s p e c i f i c a l l y w ith DNP.
37
s p e c i f i c a n tib o d y ) m a te r n a l IgM was p a s s e d in i'.la c t e a l s e c r e t i o n s and
in n e a r ly 507= o f t h e c a s e s was t r a n s m itt e d th rou gh t h e i n t e s t i n a l
m ucosa i n t o th e b lo o d stream o f th e n e o n a t e .
IgG^ .
As w ith IgM, IgG^ was a l s o tr a n s m itt e d to th e n e o n a te
p r im a r ily th rou gh l a c t e a l s e c r e t i o n s .
T here w ere a d u lt l e v e l s o f
IgG^ in th e s e r a o f a l l b u t one n e o n a te a t day 5 .
fpom 5 day o l d
The IgG^ f r a c t io n
n e o n a te s a l s o c o n ta in e d s p e c i f i c a n tib o d y a v e r a g in g
137= (0-337=) o f th e IgG^ l e v e l s in c o n t r a s t to an a v e r a g e o f 27=
s p e c i f i c a n tib o d y in th e serum o f I day o ld n e o n a te s (T a b le I I ) .
IgG^.
The d a ta show t h a t IgGg was a c q u ir e d b y t h e n e o n a te b o th
t r a n s p l a c e n t a l l y and to come e x t e n t th rou gh l a c t e a l s e c r e t i o n s .
A ll
o f '.the n e o n a te s s t u d ie d had a d u lt l e v e l s o f IgGg and s p e c i f i c a n t i ­
body in th e serum a t th e o n e -d a y a s s a y p e r io d .
have a c q u ir e d t h i s IgG^ from th e dam.
H en ce , th e y m ust
Im m unoglobulin IgGg l e v e l s
w ere r e l a t i v e l y unchanged and th e s p e c i f i c a n tib o d y t i t r e s rem ained
s t a b l e th r o u g h o u t th e stu d y p e r io d .
Im m unoglobulin l e v e l s in th e serum o f CR fe m a le s f o llo w in g two
.
—
' ■,.V- !
i n j e c t i o n s o f 230 pgs DNP^.-,-BGG.,
The r e s u l t s o f s t u d ie s on im m unoglobulin l e v e l s f o llo w in g 2
i n j e c t i o n s o f ENfP^-BGG a r e p r e s e n te d in T a b le s I I I and IV .
Table I I I .
Im m unoglobulin l e v e l s in dams a n t ig e n iz e d w ith two i n j e c t i o n s o f
DNP -BGG.
6 7
Day from
la s t
in je c tio n
IgA
IgM
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
‘
5
1 0 0 (0 )
47
—-----
1 0 0 (3 5 )
9 7 (0 )
8 9 (0 )
60
1 5 3 (1 0 )
1 8 3 (1 6 )
1 2 5 (5 )
1 1 0 (1 7 )
1 6 6 (3 2 )
228(0)
80
1 2 7 (9 )
1 5 0 (4 0 )
9 0 (0 )
1 4 1 (1 3 )
1 5 0 (1 0 )
7 4 (9 )
115
1 6 0 (2 0 )
145
8 3 (1 4 )
1 1 9 (1 2 )
150
Mean
1 3 0 (1 5 )
1 3 8 (2 3 )
0 (0 )
54
a/
—
W
IgGl
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
0 (0 )
1 0 8 (8 ) '
1 3 7 (0 )
8 0 (2 0 )
1 0 4 (1 7 )
1 0 5 (1 0 )
~— -
1 5 0 (2 0 )
0 (0 )
————
1 2 2 (1 2 )
d'(o)
8 3 (1 4 )
67(0)
7 6 (1 6 )
9 0 (0 )
1 4 1 (3 1 )
1 0 6 (6 )
1 2 3 (2 4 )
87(0)
1 3 3 (3 3 )
1 1 9 (4 )
93(26)
N o n - s p e c if i c a l Iy immune m ice
-----1 6 9 (3 )
90(8)
—™——
61
1 0 8 (8 )
9 7 (0 )
99
2 4 0 (4 0 )
1 1 2 (1 2 )
Mean
1 7 4 (2 4 )
0 (0 )
IgG2
1 0 4 (6 )
1 2 9 (1 5 )
79(0)
- ---1 6 9 (3 )
-------
1 6 7 (0 )
1 1 8 (1 3 )
1 1 6 (0 )
—-----
0 (0 )
9 0 (8 )
7 9 (0 )
1 1 6 (0 )
'i
The im m unoglobulin d a ta from one dam i s r e co r d e d f o r each tim e p e r io d .
F ig u r e s in p a r e n t h e s is i n d i c a t e th e p e r c e n t o f th e im m unoglobulin t h a t r e a c te d
s p e c i f i c a l l y w ith DNP»
Table IV.
Day from
la s t
in je c tio n
Immunog l o b u lin l e v e l s in th e s e r a o f th e n e o n a te s born to dams a n t i ­
g e n ! zed w ith two i n j e c t i o n s o f DNP^y-BGG.
IgA
Day p o st-p a r tu m
I
5
47 - /
IgM
Day p o st-p a r tu m
I '
5
—™——
—— —
Day p o st-p a r tu m
I
5
—= ——
IgG2
Day p o st-p a rtu m
I
5
—™——
-----
60
0 (0 )
0 (0 )
0 (0 )
8 5 (1 0 )
1 3 3 (0 )
0 (0 )
. 1 0 0 (0 )
1 0 0 (0 )
80
0 (0 )
0 (0 )
0 (0 )
0 (0 )
9 7 (0 )
8 9 (0 )
0 (0 )
1 0 8 (8 )
115
0 (0 )
____
0 (0 )
145
7 2 (0 )
0 (0 )
Mean
1 8 (0 )
1 2 2 (1 2 )
3 0 (3 )
9 0 (1 5 )
G ---
1 1 3 (2 6 )
0 (0 )
7 0 (2 )
9 5 (1 5 )
6 7 (0 )
1 1 3 (2 6 )
2 8 (3 )
9 7 (4 )
6 1 (5 )
7 0 (6 )
1 0 7 (1 1 )
—
N o n - s p e c if i c a l l y immune m ic e .
5.4
61
. . . .
1 0 0 (0 )
8 3 (0 )
0 (0 )
0 (0 )
---- -
99
s J The im m unoglobulin d a ta from one dam i s r e co r d e d f o r ea ch tim e p e r io d .
b/ '
F ig u r e s in p a r e n t h e s is i n d i c a t e th e p e r c e n t o f th e im m unoglobulin t h a t r e a c te d
s p e c i f i c a l l y w ith DNP.
'4'0
I rA.
An i n t e r e s t i n g f in d in g was t h a t IgA l e v e l s w ere h ig h e r a t
I and 5 days p o s t - p a r t u r i t i o n , f o llo w in g 2 a n t ig e n a t i o n s , th an th o s e
found a f t e r a s i n g l e i n j e c t i o n o f DNP^^-BGG.
M oreover, 7 o f th e 8
fe m a le s e r a a s s a y e d d em o n stra ted s p e c i f i c a n tib o d y a t s i g n i f i c a n t
l e v e l s , th a t i s ,
IgM.
above 8% o f th e t o t a l im m unoglobulin c o n c e n t r a t io n .
A l l b u t 7 o f th e a n im a ls d e v e lo p e d norm al o r n e a r norm al
l e v e l s o f IgM r e g a r d le s s o f th e la p s e o f tim e s in c e a n tig e n ,a tio n .
More th an one h a l f (557=) o f t h e s e s e r a c o n ta in e d s i g n i f i c a n t l e v e l s
o f s p e c i f i c ^ h tib o d y o v e r a 1 5 0 'day p e r io d .
IgG^ and IgG^.
tit r e d .
The IgG^ was found in th e s e r a o f a l l a n im als
F i f t y - f i v e p e r c e n t o f t h e group had l e v e l s o f s p e c i f i c a n t i ­
body t h a t e x c e e d e d 87=7,o f th e IgG^ c o n c e n t r a t io n .
U n e x p e c te d ly , th e
r e sp o n s e o f IgG^ was l e s s d ra m a tic th a n t h a t o b se r v e d f o llo w in g s i n g l e
a n t ig e n a t ib n .
S p e c i f i c a n tib o d y t i t r e s were n o t a s pred om in an t in .
t h e IgGg im m u noglobulin s p e c i e s (T a b le I I I ) w h ich i s i n c o n t r a s t t o
t h e t i t r e s f o llo w in g a s i n g l e e x p o su r e to th e a n t ig e n (T a b le I ) .
Im m unoglobu lins i n n e o n a te s b orn to dams g iv e n two i n j e c t i o n o f UNP^-,_
■
T-T-?
:
:
0/
BGG.
In s p i t e o f t h e e le v a t e d l e v e l s o f IgA in t h e dams, t h e r e was
n o t a com parable e l e v a t i o n o f t h i s im m unoglobulin in n e o n a t a l serum
a t I day p o s t-p a r tu m , (T a b le s I I I and IV ) . . M oreover, l i t t l e o r no
41
IgA was d e t e c t e d in n e o n a t a l s e r a 5 d ays p o st-p a r tu m (T a b le IV ) .
IgM.
A d u lt l e v e l s o f IgM w ere found in 2 o f 7 n e o n a te s a t I day
and 5 d ays p o s t - i n j e c t i o n .
The s p e c i f i c a n tib o d y t i t r e s w ere p r e s e n t
a t low l e v e l s in b o th n e o n a t e s .
IgG^.
T h r e e - fo u r t h s o f th e n e o n a te s had a d u lt l e v e l s o f IgG^
b u t had low t i t r e s o f s p e c i f i c a n tib o d y .
Four o f 5 n e o n a te s had IgG^
im m u n oglob u lin s in t h e i r s e r a a t I day p o st-p a r tu m w h ich i n d i c a t e s
s u b s t a n t i a l p l a c e n t a l p a s s a g e o f IgG^ (T a b le I V ).
little
T here was v e r y
IgG^ c o n t r ib u t e d to th e n e o n a t a l serum p o o l v i a l a t e a l s e c r e ­
t i o n d u r in g th e n e x t 5 d a y s .
IgGp.
IgGg was a t a d u lt l e v e l s in 6 o f 7 o f th e n e o n a te s s e r a
a s s a y e d a t I day and 5 day p o s t-p a r tu m .
W hile 42% o f t h e s e s e r a
d em o n stra ted s p e c i f i c a n tib o d y t h e r e was no ap p aren t in c r e a s e o f IgGg
a f t e r i n g e s t i o n o f c o lo s tr u m o r m ature m ilk;'
H ence, a s w ith an im als
r e c e i v i n g s i n g l e a n t ig e n a t io n s s u f f i c i e n t IgGg was a c q u ir e d p a s s i v e l y
by p l a c e n t a l t r a n s m is s io n .
P a s s iv e a n tib o d y a c q u ir e d th rou gh l a c t e a l and p l a c e n t a l t r a n s ­
m is s io n o f b o th IgG^ and IgGg s p e c i f i c a n tib o d y appeared to be r e ­
l a t e d to t h e r e s p e c t i v e c o n c e n tr a t io n o f IgG^ and IgGg found in th e
dam.
42
Im m unoglobulin l e v e l s in germ free m ice a f t e r r e c e i v in g a s i n g l e a n t i g e n a t io n o f DNP^-,-BGG.
G erm free m ice g iv e n a s i n g l e d o se o f s t e r i l e DNP^y-BGG were b le d
I and 5 days p o st-p a rtu rn f o r t o t a l and s p e c i f i c im m unoglobulin
d e t e r m in a t io n s .
The d a ta from t h e s e s t u d i e s are p r e s e n te d in T a b le s
V and V I .
IgA .
i
Ten s e r a from fe m a le germ free m ice w ere a s s a y e d f o r IgA
im m u n oglob u lin .
S i x o f te n c o n ta in e d im m unoglobulin, l e v e l s (4 0 and
837» r e s p e c t i v e l y a t I and 5 days p o st-p a r tu m ) s im i la r to t h e i r
c o n v e n t io n a l c o u n te r p a r ts (T a b le s I and V ),
d e t e c t e d in 3 o f th e 10 s e r a t e s t e d .
S p e c i f i c a n tib o d y was
None o f th e c o n t r o l a n im als
d is p la y e d a IgA l e v e l s a t day one o r day 5 p o s t-p a r tu m .
IgM.
The l e v e l s o f IgM in serum o f m ost immune GF m ice were
com parable to t h a t i n immune CR q iic e .
Four o f th e 14 a n im a ls d i s ­
p la y e d IgM s p e c i f i c a n tib o d y b u t m ost t i t r e s , on th e a v era g e. (87»),
w ere lo w e r th an in th e s e r a o f CR m ic e .
S u r p r i s in g l y , c o n t r o l m ice
(GF) had norm al serum l e v e l s o f IgM.
IgG .
F iv e o f 8 e x p e r im e n ta l a n im a ls had c o n c e n t r a t io n s o f
IgG^ s im i la r t o immune CR m ic e .
A l s o , th e s e r a o f 3 o f . t h e resp on d ­
in g m ice c o n ta in e d s p e c i f i c a n tib o d y .
Table V
Day from
la s t
in je c tio n
34 —
Im m unoglobu lin l e v e l s in g erm free m ice a n t'ig e n iz e d w ith a s i n g l e
i n j e c t i o n o f DNP^y-BGG.
IgA
Day p ost-p artu rn
I
5
0 ( 0 ) —^
47
8 6 (9 )
54
6 4 (0 )
——
-
——
— -
IgM
Day p o st-p a rtu rn
I
5
1 0 6 (4 )
1 2 0 (2 0 )
—— — —
IgG2
Day post-p artu rn
I
5
5 0 (0 )
9 0 (0 )
————
5 4 (0 )
----
“ “
“
0 (0 )
8 0 (1 6 )
1 4 3 (2 4 )
1 0 0 (0 )
58
IgG
Day p o s t -partum
I
5
————
1 0 7 (7 )
1 0 0 (0 )
—
1 3 7 (2 )
————
1 6 6 (0 )
8 6 (0 )
7 5 (0 )
. 7 5 (1 2 )
9 1 (2 5 )
7 3 (0 )
0 (0 )
0 (0 )
T T ------
5 1 CO)
1 1 7 (5 5 )
.6 5 ( 2 ).
6 0 (1 0 )
0 (0 )
—
—
0 (0 )
6 1 (0 )
1 1 6 (0 )
8 2 (2 )
90
0 (0 )
—
—
7 1 (4 )
—
106
5 0 (0 )
Mean
6 6 (1 1 )
33
0 (0 )
92
0 (0 )
106
0 (0 )
77
5 8 (0 )
78
7 5 (1 3 )
82
1 6 6 (5 6 )
— —
1 1 4 (1 4 )
— ——
1 6 6 (5 6 )
1 1 7 (2 9 )
8 1 (0 )
—
—
1 0 4 (1 4 )
7 5 (8 )
N o n - s p e c i f i c a l l y immune m ice
1 0 5 (0 )
——
0 (0 )
—
—
-
1 0 7 (0 )
—— — —
—
1 4 2 (2 )
0 (0 )
1 0 0 (0 )
1 0 2 (2 0 )
De= - -
1 0 0 (0 )
1 1 4 (0 )
—
1 0 0 (0 )
—
9 8 (2 3 )
7 7 (3 )
72(0)
————
0 (0 )
————
67(0)
Table V.
Day from
la s t
in je c tio n
Mean
a/
—
—^
Continued.
IgA
Day p ost-p artu rn
' I
5
0 (0 )
————
IgM
Day p ost-p artu rn
I
5
1 1 8 (I )
————
IgG^
Day p o st-p a r tu m
I
5
0 (0 )
— ——
IgG2
Day p o st-p a rtu m
I
5
7 0 (0 )
------
The im m unoglobulin d a ta from on e dam was r e c o r d e d f o r ea ch tim e p e r io d .
F ig u r e s in p a r e n t h e s is i n d i c a t e th e p e r c e n t o f th e im m unoglobulin t h a t r e a c te d
s p e c i f i c a l l y w ith th e DNP.
£
Table VI
Day from
la s t
in je c tio n
Im m unoglobulin l e v e l s in serum o f n e o n a te s b o r n to germ free fe m a le s
a n t ig e n iz e d w it h a s i n g l e i n j e c t i o n o f DNP^^-BGG.
IgA
IgM
Day p o st-p a r tu m
I
5
Day p o st-p a r tu m
I
5
IgG^
Day p o st-p a r tu m
I
5
34
0 (0 )
——
0 (0 )
47
0 (0 )
——
0 (0 )
58
1 0 0 (1 8 )
77
0 (0 )
——
0 (0 )
0 (0 )
7 5 (0 )
78
0 (0 )
——
0 (0 )
—
0 (0 )
82
0 (0 )
——
1 3 2 (7 )
1 1 2 (0 )
90
0 (0 )
——
0 (0 )
0 (0 )
98
0 (0 )
—
0 (0 )
106
0 (0 )
0 (0 )
Mean
1 1 (2 )
-
————
0 (0 )
—™—™
————
0 (0 )
1 1 7 (1 7 )
6 7 (0 )
————
,
IgGg
Day p o s t - •par turn
I
5
0 (0 )
“ “ “—
1 2 5 (3 6 )
—-----
————
------
7 1 (8 )
88(28)
0 (0 )
0 (0 )
6 0 (0 )
— ——
1 2 7 (3 7 )
—---—
0 (0 )
0 (0 )
0 (0 )
7 3 (0 )
—---- -
0 (0 )
----
0 (0 )
---- —
3 1 (3 )
0 (0 )
2 3 (0 )
2 2 (0 )
48(9)
5 9 (1 1 )
—- - -
0 (0 )
r '-
0 (0 )
————
0 (0 )
” -----
2 5 (0 )
————
33
o (o )
——
0 (0 )
92
0 (0 )
——
0 (0 )
9 0 (5 )
— The im m unoglobulin d a ta f o r ea ch tim e p e r io d was o b ta in e d from n e o n a ta l p o o le d s e r a
T
0 I from on e h a l f o f th e l i t t e r .
A verage l i t t e r s i z e was 8 .
— F ig u r e s in p a r e n t h e s is i n d i c a t e th e p e r c e n t o f th e im m unoglobulin t h a t r e a c te d
s p e c i f i c a l l y w ith DNP.
46
IgG „.
IgG^ was p r e s e n t in a l l m ic e , in c lu d in g th e c o n t r o l .
a n im a ls , a t l e v e l s o s l i g h t l y b e lo w t h o s e o b ta in e d in norm al c o n v e n tio n ­
a l l y r e a r e d p o o le d s e r a .
S p e c i f i c a n tib o d y appeared in 2 a p im als a t
l e v e l s e q u a l to o r e x c e e d in g 87° o f th e serum c o n c e n t r a t io n s .
One
c o n t r o l an im al d id n o t d i s p la y a m ea su ra b le q u a n tity o f IgG^ w h ile
th e r e m a in in g 2 had m od erate l e v e l s o f t h i s im m unoglobulin s p e c i e s .
Im m unoglobulin l e v e l s in t h e n e o n a te s b orn to GF dams g iv e n a s i n g l e
i n j e c t i o n o f DNP^ y -BGG w ith CFA.
IgA .
One o f 8 n e o n a te s (T a b le V I) had IgA as w e l l a s s p e c i f i c
a n tib o d y in d e t e c t a b l e q u a n t i t i e s i n t h e serum .
The n e o n a t a l s e r a
w ith IgA was o b ta in e d from young b orn 58 days f o llo w in g a n t ig e n a t io n
o f th e dam.
S e r a o f 2 n e o n a t a l c o n t r o l l i t t e r s w ere w ith o u t d e t e c t a b l e
q u a n t i t i e s o f IgA im m u n oglob u lin .
IgM.
Only 2 o f 8 n e o n a t a l groups d is p la y e d IgM o r s p e c i f i c
a n tib o d y a t I day p o st-p a r tu r n .
a v era g ed 2% o f th e t o t a l IgM.
IgG^.
c e n tr a tio n s .
S p e c i f i c a n tib o d y p r e s e n t a t I day
None o f th e c o n t r o l s e r a d is p la y e d IgM.
Three o f 8 n e o n a ta l s e r a had IgG^ in n e a r ly norm al con­
The I n e o n a ta l group w h ich was a ssa y e d a t day I and a t
day 5 p o st-p a rtu rn f o r IgG^ had n e a r a d u lt l e v e l s a t day I , b u t showed
an 117° d e c r e a s e d u r in g th e f o llo w in g 4 d a y s .
There was no s p e c i f i c
a n tib o d y d e t e c t e d i n t h e IgG^ c l a s s in t h e s e n e o n a t a l s e r a .
47;
IgG^.
Four o f ^the 8 e x p e r im e n ta l n e o n a t a l grou p s d em on strated
IgGg e q u a l to m od erate l e v e l s in a d u lt l e v e l s (a v e r a g e 48%) compared
t o an a v e r a g e l e v e l o f 58% in th e nonimmune GF c o n t r o l g r o u p s.
s e r a c o n ta in e d s p e c i f i c a n tib o d y g r e a t e r th an 8%.
Four
N e a r ly a 4 - f o l d
in c r e a s e was o b s e r v e d i n th e s p e c i f i c a n tib o d y c o n c e n tr a t io n in one
group o f n e o n a t a l s e r a .
T h is in c r e a s e o c c u r r e d i n th e group borp 77
days a f t e r th e dam had b e e n a n t ig e n iz e d (T a b le V I) .
One o f 3 c o n t r o l
s e r a had a d u lt (90%) l e v e l s o f IgGg w h ile s p e c i f i c a n t ib o d ie s were
n o t d e t e c t a b l e in th e s e r a o f nopimmune GF n e o n a t e s .
IgGg was th e p red o m in a te c l a s s o f im m u n oglob u lin s in germ free
n e o n a t a l serum ..
A l s o , s p e c i f i c a n tib o d y was found m ost f r e q u e n tly
in IgGg ( F ig u r e 10) c l a s s and a t h ig h e r l e v e l s th a n in IgA , IgM, or
IgG^ c l a s s e s o f im m u n o g lo b u lin ..
A s y n o p s is o f th e s p e c i f i c a n tib o d y r e s p o n s e ( F ig u r e s 8 , 9 and 1 0 ).
T h ese f i g u r e s i l l u s t r a t e tfye number o f a n im a ls t h a t responded to
th e h a p te n as w e l l a s t h e number o f n e o n a t a l s e r a t h a t c o n ta in e d
s p e c i f i c a n tib o d y to DNP,
R e a c tin g t h e s e r a w ith h e a t - t r e a t e d DNPgy-
BSA was presum ed to d e c r e a s e th e c o n c e n tr a t io n o f th e im m unoglobulin
p r o p o r t io n I- to th e c o n c e n t r a t io n o f s p e c i f i c a n tib o d y .
S in c e a n t i ­
body a v i d i t y w i l l i n f l u e n c e tb e r e s u l t s o f p r e c i p i t i n a s s a y s , th e
c o n c e n t r a t io n o f s p e c i f i c a n tib o d y d e term in ed i s a r e l a t i v e m easure­
ment and sh o u ld n o t b e m is in t e r p r e t e d a s an a b s o lu t e v a lu e .
48
I
I Dam
3
IgA
Figure 8
N eonate
Ig M
IgGj
Ig G g
Frequency o f s p e c i f i c an tibody in the sera o f CR female
and n eonates f o llo w in g a s i n g l e i n j e c t i o n o f DNP^-BGG.
49
PU Neonate
ill
1
5
IgA
F igu re 9
1
5
Ig M
Dam
1
5
IgGj
1
5
IgG2
Frequency o f s p e c i f i c an tib od y in the sera o f CR females
and n eon ates f o llo w in g two i n j e c t i o n s of DNP^. ^-BGG.
50
Dam
Neonate
Ig A
F igu re 10
Ig M
Ig Gj
Ig Gg
Frequency o f s p e c i f i c an tib od y in the s e r a o f GF fem ales
and n eon ates f o llo w in g a s i n g l e i n j e c t i o n o f DNP^-BGG.
51
As s e e n in F ig u r e S more a n im a ls resp on d ed to th e h a p te n ic
a n t ig e n w ith a n tib o d y p r o d u c tio n in c l a s s e s IgM, IgG^ and IgG
s p e c ie s .
2
Many n e o n a te s a c q u ir e d m a te r n a l a n tib o d y to DNP v i a p l a c e n t ­
a l t r a n s m is s io n w h ich was m a n if e s te d by h ig h l e v e l s o f s p e c i f i c a n t i ­
body a t day I .
The s p e c i f i c a l l y immune IgM and IgG^ w ere found in
few n e o n a te s a t day I b u t a t day 5 n e a r ly h a l f o f th e a n im a ls p o s s e s s
s p e c i f i c a n tib o d y .
T h is can b e i n t e r p r e t e d a s p a s s iv e t r a n s f e r o f
im m unoglobulin v i a th e c o lo s tr u m o f t h e dam.
I n t e r e s t i n g l y , th e number o f dam s e r a w ith s p e c i f i c a l l y immune
IgG^ d e c r e a s e d a t day 5 w h ich ig in a c c o rd w ith th e o b s e r v a t io n s o f
o t h e r w o r k e r s.
F o llo w in g 2 i n j e c t i o n s o f a n t ig e n t h e r e was a h e ig h te n ­
ed r e s p o n s e on th e IgA and IgM f r a c t i o n s b u t a d e p r e s s e d r e sp o n s e in
IgG
and IgG0 a s t h e secon d d o se seem ed to su p p r e ss t h e s e immuno­
g lo b u lin s .
A g a in , IgA was n o t t r a n s m itt e d to th e o f f s p r i n g .
IgM was
t r a n s m itt e d w eak ly v i a p l a c e n t a l and l a c t e a l r o u t e s ; IgG^ was t r a n s ­
m it te d s t r o n g ly v i a th e l a c t e a l r o u te and IgGg s t r o n g ly v i a p l a c e n t a l
r o u te.
The pred om in an t im m unoglobulin in th e GF dam s e r a was IgM,
f o llo w e d by IgG^ and th e n IgG g.
L a c t e a l s e c r e t i o n s w ere r e s p o n s ib le
i n p a r t f o r d o n a tin g IgG^ t o n e o n a t a l c i r c u l a t i o n .
IgA and IgGg were
in f r e q u e n t ly t r a n s f e r r e d p l a c e n t a l l y w h ich was in d ic a t e d by a low
in c id e n c e o f t h e s e s p e c i e s a t day. I p o s t-p a r tu m .
P a s s iv e h e m a g g lu tin a tio n t i t r a t i o n s ^ (T a b le .V II)
A t o t a l o f 32 serum sp ecim en s from immunized dams and n e o n a te s
w ere t e s t e d f o r hemaggI u t i n a t i n g or. .h e m o ly tic a n tib o d y a c t i v i t y .
B oth a d u lt ( fe m a le ) and n e o n a ta l s e r a were t e s t e d . ' Of th e 32
sp ecim en s a n a ly z e d , 12 gave p o s i t i v e r e a c t i o n s , a l l o f w h ich were
s e r a from ’.'the dams.
T h ere w ere no t i t r e s found in th e n e o n a ta l
serum o f in th e few c o l o s t r a l sam p les t e s t e d .
T h ree o f 7 fe m a le s frpm th e e x p e r im e n ta l group t h a t had r e c e iv e d
2 a n t i g e n a t io n s d e m o n str a ted h e m a g g lu tin a tio n t i t r e s r a n g in g from 1:8
to 1 :3 2 .
A q u e s t io n a b le h e m o ly sin t i t r e was o b se r v e d i n o n ly I o f
th e s e r a .
One o u t o f 5 s e r a from fe m a le s r e c e i v i n g a s i n g l e a n t ig e n a t io n
d em o n stra ted a p o s i t i v e r e a c t i o n .
H e m a g g lu tin a tin g and h e m o ly tic
t i t r e s w ere 1 :1 2 8 and 1 :1 6 r e s p e c t i v e l y .
Of p a r t i c u l a r i n t e r e s t was th e r e s p o n s e t o th e h a p te n e l i c i t e d
by th e g erm free f e m a le s .
E ig h t o f th e 11 s e r a t e s t e d gave s tr o n g
r e a c t io n s w ith th e PHA t e c h n iq u e .
The t i t r e s were h ig h e r (r a n g e 1 :1 2 8
to 1 :5 1 2 ) i n th e s e r a o b ta in e d from fe m a le s s a c r i f i c e d l e s s than 55
days p o s t - i n j e c t i o n .
The t i t r e s o f s e r a f o llo w in g t h i s p e r io d were
lo w er ( 1 : 4 to 1 : 6 4 ) w h ich in c lu d e d sp ecim en s from 55 t o 106 days
p o s t-in je c tio n .
H em olysin t i t r e s w ere o f low m agn itu d e and were
found i n serum c o l l e c t e d a f t e r 77 d a y s .
53
T a b le '.VII.
Day from
la s t
in je c tio n
a/
—
—/
a/
H em m agglu tin atin g and h e m o ly sin t i t r e s — in serum
o b ta in e d from fe m a le s i n j e c t e d one o r two tim e s i n t r a d e r m a lly w ith DNPf, ^-BGG.
Code
b,
number —
H e m a g g lu tin a tin
titr e
H em olysin
titr e
60
IA -5
1 :8
69
ID-5
1 :32
(1 :8 ? )
82
IE -I
1 :3 2
-------
60
2H-5
1 :1 2 8
33
GFB-5
1 :512
47
GFC-rl
1 :1 2 8
————
54
GFD-I
1:128
-T--—
77
GFH-I
1 :64
1 :8
78
GFJ-I
1 :4
1 :4
82
GFK-I
1 :64
90
GFL-I
1 :4
1 :8
106
GFQ-L
1 :6 4
1 :8
1 :16
————
N in e n e o n a t a l s e r a were a s s a y e d and none o f th e s e r a d em on strated
d e t e c t a b l e a c t i v i t y t h e r e f o r e th e y a r e n o t in c lu d e d i n . t h e t a b l e .
D e f i n i t i o n o f cod e: The number p r e c e d in g th e l e t t e r i n d i c a t e s
th e number o f i n j e c t i o n s g iv e n to th e dam, 1 = 2 i n j e c t i o n s .
The l e t t e r i n d i c a t e s th e seq u en ce i n w hich th e an im al was
p r o c e s s e d . The l a s t . number i n d i c a t e s th e day p o s t-p a r tu m .
'54
D e t e c t io n o f im m unoglobulin s p e c ie s in th e c o lo s tr u m .
S p e c i f i c a n tib o d y l e v e l s in c o lo s tr u m ranged from 0-2% o f th e
t o t a l im m unoglobulin' c o n c e n t r a t io n s .
T h is f s b e lo w th e l i m i t s o f
e r r o r , a t t h e s e l e v e l s . h e n c e th e s p e c i f i c a n tib o d y c o n c e n tr a t io n s
a r e n o t in c lu d e d in th e t a b l e s .
H igh l e v e l s o f IgA (a v e r a g e 427=) w ere found in 3 o f th e 7
c o l o s t r a l sam p les from Stom achs o f n e o n a te s born to dams g iv e n a
s i n g l e d o se o f DNP^^-BGG.
Of s i g n i f i c a n c e , IgM was n o t d e t e c t e d in
th e c o l o s t r a l sam p les o f t h i s g ro u p .
IgG^ was p r e s e n t a t m o d e ra te ly
h ig h l e v e l s (a v e r a g e 257=) in 6 o f th e co l o s t r a l sa m p le s.
IgGg was
a ls o found in m od erate l e v e l s b u t in o n ly 4 o f th e 7 c o l o s t r a l sam p les
assayed .
A f t e r 2 i n j e c t i o n s th e c o l o s t r a l sp ecim en s fro m u n eo n a ta l stom achs
d e m o n str a ted h ig h l e v e l s o f IgA , a v e r a g in g 457=, and w ith a h ig h (837=)
i n c id e n c e .
A g a in , IgM was n o t d e t e c t e d in any c o l o s t r a l sa m p le s.
IgG^ was p r e s e n t in I o f 6 sam p les a t an a v era g e l e v e l o f 237=.
IgGg
was n o t a s h ig h a s IgA b u t was d e t e c t e d w ith n e a r ly th e same
fr e q u e n c y (667=).
The IgA and IgG^ l e v e l s c o r r e l a t e w e l l w ith th e
serum l e v e l s in th e dams (T a b le I I I ) .
The fre q u en cy o f IgGg in
c o lo s tr u m r e l a t e s w e l l to th e i n c r e a s i n g fre q u en cy o f IgGg in n e o n a t­
a l serum s from day I to day 5 p o st-p a rtu rn (F ig u r e 10).
55
C o lo s t r a l sp ecim en s from n e o n a te s born to germ free fe m a le s
f o llo w in g a s i n g l e i n j e c t i o n d is p la y e d com parable l e v e l s o f IgA
(a v e r a g e 45%) b u t a t a l e s s e r fr e q u e n c y (37%) th an t h o s e se e n in th e
c o n v e n t io n a l r e a re d c o u n t e r p a r t s .
A g a in , th e s in g u la r f e a t u r e o f
t h e s e s t u d i e s was a la c k o f d e t e c t a b l e l e v e l s o f IgM.
The c o n c e n t r a t io n o f IgG^ was somewhat lo w er (20%) th an l e v e l s
in th e CR c o u n te r p a r ts and was found in o n ly 37% o f th e sp e c im e n s.
T h is com pares to an a v e r a g e l e v e l o f 25% and a fr e q u e n c y o f 85% in
th e c o n v e n t io n a l g ro u p .
IgGg l e v e l s in c o lo str u m in th e germ free
group a v era g ed 24% o f norm al serum c o n c e n tr a t io n s and w ere found in
o n ly 2 o f t h e 6 n e o n a t a l groups exam in ed .
The o c c u r r e n c e o f IgGg
in c o lo s tr u m and th e m o d e r a te ly h ig h l e v e l s r e f l e c t th e n o te d
in c id e n c e and d e c r e a s in g c o n c e n tr a t io n found in th e s e r a o f th e dam
(T a b le V I) .
The c o r r e l a t i o n b etw een th e numbers o f c e l l s in mammary g la n d s
o f dams a s s o c i a t e d w it h im m u noglobulin s w ith th e l e v e l s o f th e ■
im m u n oglob u lin s m easured in c o lo str u m from n e o n a ta l stom achs i s
p r e s e n te d in F ig u r e 11 ( a b ar graph d e r iv e d from d a ta shown in T a b le s
V III and I X ).
A d i r e c t r e l a t i o n s h i p i s ap p aren t f o r IgG^ and IgGg
and to a l e s s e r e x t e n t to th e IgA c o n c e n t r a t io n .
No. O
F FLUORESCING CELLS/50 FIELDS
56
16I
8
4-
IgM
M
EAN LEVELO
FIM
M
UNO
G
LO
BULIN (%of NSP)
Num
ber of cells inm
am
mary gland with fIuorescenated anti-heavy chain sera
Ig A
IgM
IgGj
IgGg
Figure Il Levels of immunoglobulins in colostraI samples
from neonatal stomachs
57
Days from
la s t
in je c tio n
(a )
The number o f c e l l s p e r 50 m ic r o s c o p ic f i e l d s ( o i l
im m ersion ) e x h i b i t i n g f lu o r e s c e n c e in mammary t i s s u e
s e c t i o n s t r e a t e d w ith f lu o r e s c e n t a n t i- h e a v y c h a in
s e r a (Ig A , IgM, IgGj., Ig G g ).
IgA .
IgM
H
OQ
Q
T a b le V IlX .
IgGg
CR fe m a le s t h a t r e c e iv e d a s i n g l e a n t ig e n a t i o n .
18 - /
39
5
17
12
A ''
40
3
3
5
18
41
9
5
14
17
44
4
8
19
9
60
12
8
10
3
83
13
8
4
20
■ 9
6
11
13
Mean v a lu e
(b )
CR fe m a le s t h a t r e c e iv e d two i n j e c t i o n s .
47
17
4
12
13
80
0
12
0
18
1-15
16
8
24
145
4
40
5
4
16
13
9
N o n - s p e c i f i c a l l y immune m ic e .
25 .
16
33
12
9
MO
56
14
12
17
44
14
Mean v a lu e
54
61
Mean
(c)
14 '
14
GF fe m a le s t h a t r e c e iv e d a s i n g l e i n j e c t i o n o f DNP.
34
16
13
14
19
47
2
9
4
4
54.
4
0
25
13
58
I
0
I
0
77
nt
nt
nt
nt
58
T a b le 'VrX JJ.
C o n tin u ed .
Days from
la s t .
in je c tio n
IgA.-
IgM
• IgG1
IgG2
78
4
2
3
0
87
60
20
2
11
99
I
.0
0
0
106
4
0
3
0
12
8
6
6
N e g a tiv e c o n t r o l W
7
3
5 '
2
E x p er im e n ta l ,
c o n tr o l —
6
0
3
0
Mean v a lu e
a/
The f ig u r e s r e p r e s e n t th e t o t a l number o f c e l l s / t w o mammary
g la n d s .
b/
N e g a tiv e c o n t r o l anim al was a n o n - in j e c te d anim al from th e GE
u n it.
SlI
E x p er im e n ta l c o n t r o l anim al had b e e n i n j e c t e d w ith s a l i n e and OFA.
n t = no t i s s u e specim en
N ote:
T a b le V II (b ) - B oth c o n t r o l a n im a ls c a n n a b iliz e d a p o r t io n
o f th e ir l i t t e r s .
59
T ab le IX.--
Day from
la s t
in je c tio n
(a )
Im m unoglobulin l e v e l s i n c o lo str u m h a r v e s te d from
stom achs o f n e o n a t e s .
Code
,
number —
C o n c e n tr a tio n e x p r e s s e d as 7o o f
norm al serum p o o l
IgA
IgM
IgG1
IgG2
Im m unoglobulin l e v e l s in c o lo str u m h a r v e s te d from
stom achs o f n e o n a te s born t o CR dams g iv e n a s i n g l e d o se
o f DNPz--^-BGG.
39
2B-1
0
0
22
0
39
2B-5
39
0
17
0
40
2C-5 ■
43
0
25
31
44
2E-5
0
0
0
■ 31
44
- 2F -1
43
0
17
25
44
2F -5
0
0
28
28
60
2H-5
0
0
19
0
(b )
59
Im m unoglobulin l e v e l s in c o lo s tr u m h a r v e s te d from stom achs
o f n e o n a te s born to CR dams g iv e n two i n j e c t i o n s o f DNP^y■BGG.
IE -I
0
19
23
41
60
IA -I
43
0
0
22 -
60
IA -5
43
0
0
24
80
ID -I
41
0
0
28
115
61
(c )
33
0
0
0
57
IE -I
N o n - s p e c if i c a l l y immune m ic e .
0
0
0
1C-5
0
l
e
v
e
l
s
in
c
o
lo
str
u
m
h
a
r
v
e
s
te
d
from.
stom
achs
Im m unoglobulin
o f n e o n a te s born to germ free dams g iv e n a s i n g l e a n t i g e n a t io n 'w itlf DNP —BGG•
67
0
0
42
0
GF2B-1
33
GF2B-5
0
0
22
25
44
GF2D-1
43
0
' 0
0
58
GF2E-1
0
0
0
0
77
GF2H-1
0
0
0
■-
0
60
T a b le IZ.'! ,
Day from
la s t
in je c tio n
Code
number
C o n c e n tr a tio n e x p r e s s e d a s % o f
norm al serum p o o l
IgM
IgA
IgG2
IgG1
78
GF2I-1
92
GF2L-1
0
0
N o n - s p e c i f i c a l l y immune m ice
GF2A-1
0
0
33
a/
—
C on tin u ed .
51
0
19
0
19
23
0
0
•
D e f i n i t i o n o f c o d e : The number p r e c e d in g th e l e t t e r i n d i c a t e s
th e number o f i n j e c t i o n s g iv e n to th e dam, 1 = 2 i n j e c t i o n s .
The l e t t e r i n d i c a t e s th e se q u e n c e ih o w h ich th e anim al was
p r o c e s s e d . The l a s t number i n d i c a t e s th e day p o s t-p a r tu m .
DISCUSSION
The r e s u l t s o f t h e s e s t u d ie s i n d i c a t e t h a t th e im m unoglobulin
l e v e l s in s e r a o f th e dams were in f lu e n c e d by th e a n t ig e n i c regim en s
u sed in th e v a r io u s e x p e r im e n ta l g r o u p s.
In CR m ice i n j e c t e d w ith a
s i n g l e d o se o f DNP^y-BGG, IgA and IgG^ were found in h ig h e r con cen ­
t r a t i o n th an IgM o r IgG^.
On th e o t h e r hand, h ig h e r s p e c i f i c a n t i ­
body l e v e l s w ere p r e d o m in a n tly found in th e IgM and IgG^ c l a s s e s o..
th an in IgA and IgGg im m u n o g lo b u lin s.
The l e v e l s o f a l l fo u r immuno­
g l o b u l in . c l a s s e s w ere in e x c e s s o f 100% o f th e norm al serum p o o l and
w ere m a in ta in e d th r o u g h o u t th e 83 day s tu d y .
F o llo w in g two i n j e c t i o n s in CR f e m a le s , IgA and IgG^ immuno­
g l o b u l i n s , o b ta in e d th e h ig h e s t serum c o n c e n t r a t io n s .
T here were no
a p p r e c ia b le d i f f e r e n c e s o b se r v e d in s p e c i f i c a n tib o d y l e v e l s in th e
fo u r im m u n o g lo b u lin s.
Of p a r t i c u l a r i n t e r e s t w ere th e n o t i c e a b l e lo w er l e v e l s o f
t h r e e (Ig A , IgG^ and IgGg) o f fo u r im m u n oglob u lin s in germ free fem ale
s e r a f o llo w in g a s i n g l e i n j e c t i o n o f DNP^y-BGG.
F in d in g norm al l e v e l s
o f IgM was n o t s u r p r is in g s in c e s im i la r l e v e l s f o r t h i s im m unoglobulin
in t h e serum o f GF a n im a ls have b e e n r e p o r te d in th e l i t e r a t u r e by
A s o f s k y , H y lto n and I k a r a (1 9 6 8 ) and more r e c e n t ly by Nash e t a l
(1 9 6 9 ).
S p e c i f i c a n tib o d y l e v e l s w ere found to v a r y w it h in th e
im m unoglobulin c l a s s e s gp e v id e n c e d by t h e predom inance o f s p e c i f i c
a n tib o d y in IgA f o llo w e d by lo w er l e v e l s o f s p e c i f i c
a n tib o d y in
62
IgG ^, IgG g, and IgM im m u n o g lo b u lin s.
The l e v e l s o f s p e c i f i c a n t i ­
body o b se r v e d in GF fe m a le s were s im i la r in m agnitude to th e serum
l e v e l s o f s p e c i f i c a n tib o d y se e n in th e CR f e m a le s .
In f a c t , th e
l e v e l o f s p e c i f i c IgA a n tib o d y in GF fe m a le s was on th e avera g e
th e h i g h e s t o b se r v e d in any a d u lt fem a le serum .
An i n t e r e s t i n g
f in d in g was th e la c k o f a c o r r e l a t i o n b etw een th e t o t a l immuno­
g l o b u l in c o n c e n t r a t io n and th e s p e c i f i c a n tib o d y l e v e l s in m ost o f
th e a d u lt fem a le s e r a .
N e o n a te s born to dams t h a t had r e c e iv e d a s i n g l e i n j e c t i o n
d em o n stra ted a d u lt l e v e l s o f IgG^ a t b i r t h .
T h is i n d i c a t e s ad eq u ate
p l a c e n t a l t r a n s m is s io n to c o n s t i t u t e t h e n e o n a ta l serum w ith t h i s
c la s s ' o f im m unoglobulin a t b i r t h .
There was good e v id e n c e t h a t th e
n e o n a te s w ere a b le to su p p lem en t t h e i r l e v e l s o f c i r c u l a t i n g IgG g,
from l a c t e a l s e c r e t i o n s and, h e n c e , presu m ab ly o b ta in e d by t r a n s ­
p l a c e n t a l p a s s a g e p o s s e s s e d a d u lt l e v e l s by day f i v e p o st-p a r tu m .
A lth o u g h g e n e r o u s q u a n t i t i e s o f IgM appeared to be a c q u ir e d l a c t e a l l y
( o r p erh a p s from de novo s y n t h e s i s in th e n e o n a te ) th e amounts were
n o t s u f f i c i e n t to e s t a b l i s h a d u lt serum l e v e l s .
In g e n e r a l, n e o n a ta l
a c q u i s i t i o n o f IgA la g g e d b eh in d th e o t h e r th r e e im m unoglobulin
c l a s s e s i n n e o n a t a l serum .
The l e v e l s o f s p e c i f i c a n tib o d y and th e
i n c r e a s e s w hich o c c u r r e d from .d ay I t o day 5 p o st-p a r tu m m imic th e
r i s e s e e n in th e t o t a l im m unoglobulin c o n c e n t r a t io n s .
The predom inant
63
l e v e l s o f s p e c i f i c a n t ib o d ie s a t b o th day I a n d ■day 5 w ere o b se r v ed
in th e IgGg c l a s s .
A h ie r a r c h y in p l a c e n t a l and l a c t e a l t r a n s m is s io n
o f c e r t a in im m u n oglob u lin s s p e c ie s was s e e n .
The h ie r a r c h y appears
to be r e l a t e d to the:'.im m unoglobulin c l a s s r a th e r th an to th e con cen ­
t r a t i o n o f im m u n oglob u lin s in s e r a o f th e dams.
For ex a m p le , th e
low IgA c o n c e n t r a t io n and low s p e c i f i c a n tib o d y l e v e l s in n e o n a ta l
serum a t day I and day 5 d id n o t r e f l e c t , t h e e le v a t e d l e v e l s o b se r v e d
in th e dam s e r a .
In th e e x p e r im e n ta l group r e c e i v i n g two i n j e c t i o n s th e h ie r a r c h y
i s f u r t h e r su p p o rted by th e f a c t t h a t two im m unoglobulin c l a s s e s
p a s s e d w e l l p l a c e n t a l l y , in f a c t in c o p io u s am ounts, s i n c e o n ly IgG^
and IgGg a r e p r e s e n t a t day I and day 5 p o s t-p a r tu m .
A ls o , v e r y
little
IgG^ was p a s s a g e d l a c t e a l l y , e s p e c i a l l y s p e c i f i c IgG^ a n t i ­
body.
T h is i s in c o n t r a s t to IgGg w h ich was a c q u ir e d from l a c t e a l
s e c r e t i o n s d u r in g p o st-p q r tu m f e e d i n g s .
A f t e r f i v e days o f f e e d in g ,
IgA d id n o t appear in t h e s e r a o f th e n e o n a te s even th ough th e dams
had h ig h IgA l e v e l s in t h e i r s e r a .
C u r io u s ly , IgA serum l e v e l s in
th e dams w ere n o t i c e a b l y h ig h e r th an th e o t h e r im m u n oglob u lin s in
t h i s and o t h e r e x p e r im e n ta l g r o u p s.
A lth o u g h th e h ie r a r c h y f o r
l a c t e a l t r a n s m i s s i o n .o f im m u n oglob u lin s to n e o n a ta l
sera is
i n d ic a t e d i t was n o t as c l e a r l y d e f in e d in t h i s e x p e r im e n ta l group.
64
Of p a r t i c u l a r i n t e r e s t was th e low l e v e l s o f t h r e e o f th e fo u r
im m unoglobulin c l a s s e s in GE n e o n a t e s .
N ot u n lik e th e CR c o u n te r ­
p a r t s , IgG^ was th e pred om in an t im m unoglobulin a t day I .
L e v e ls o f
IgGg were in c r e a s e d f o ll o w i n g f e e d in g s d u r in g th e 5 day p o st-p a r tu m
p e r io d .
Of s i n g l e im p ortan ce was a co m p le te la c k o f s p e c i f i c IgG^
a n tib o d y in n e o n a t a l serum though th e dams had resp on d ed w e ll to
a n t ig e n a t io n and had norm al l e v e l s o f IgG^ were o b se r v e d in serums
o f th e dam.
There was a c o n sp ic u o u s la c k o f IgA and IgM in th e s e r a
o f th e GE n e o n a te s a t day I p o st-p a r tu m n e i t h e r o f w h ich in c r e a s e d
w ith l a c t e a l i n g e s t i o n .
L e v e ls o f im m u n oglob u lin s were m easured in c o lo str u m o b ta in e d
from th e stom achs o f th e n e o n a t e s .
little
I n t e r e s t i n g l y , t h e r e was v e r y
d i f f e r e n c e d>ri th e c o n c e n t r a t io n o f th e v a r io u s im m unoglobulin
c l a s s e s from one n e o n a t a l group t o t h e o t h e r .
The a v e r a g e v a lu e s f o r
th e v a r i o u s ' im m u n oglob u lin s in c o lo s tr u m were as f o l l o w s : IgA 46%,
IgM n o t d e t e c t e d , IgG^ 23%, IgGg 27%.
I t i s ap p aren t from t h i s d a ta
t h a t IgA was th e p red o m in a te im m unoglobulin found:".in c o lo str u m
o b ta in e d from t h e stom ach s o f th e n e o n a t e s .
E xcep t f o r th e la c k o f
IgM in c o lo s tr u m th e l e v e l s o f th e o t h e r im m u noglobulin s w ere in
g e n e r a l r e l a t e d to t h e l e v e l s found in th e s e r a o f th e dams.
I t is
p r o b a b le t h a t th e la c k o f IgM ccould b e a t t r i b u t e d to th e p r o c e d u r e .
IgM may have b e e n bound w ith l i p i d m a t e r ia l d u r in g th e p r o c e s s in g
65
s t e p s and would have b e e n removed from th e c o lo str u m w ith th e l i p i d
fr a c tio n .
A s im i la r f i n d i n g , a la c k o f IgM in c o lo s tr u m , h as been
r e p o r te d by S t e c h s c h u lt e and A u s tin (1 9 7 0 ) in th e c o lo s tr u m o f r a t s .
The p r o c e d u r e s th e y em ployed in p r o c e s s in g c o lo str u m were v e r y
s im i la r to t h o s e u se d in t h i s s tu d y .
In GF a n im a ls t h e r e l a t i o n s h i p o f c o l o s t r a ! l e v e l s to th e l e v e l s
o f im m u n oglob u lin s in th e s e r a o f th e dams was n o t a s d i r e c t as t h a t
o b se r v e d in th e CR f e m a le s .
C o lo s t r a l l e v e l s o f IgG^ and IgGg,
a lth o u g h h o t to o d i f f e r e n t in m a g n itu d e , were in r e v e r s e o r d e r o f
th e l e v e l s se e n in t h e s e r a o f th e dams.
T hese d a ta b e t t e r c o r r e l a t e
w ith th e d a ta o b ta in e d from th e e x p e r im e n ta l group r e c e i v i n g two
i n j e c t i o n s th an to th e CR group t h a t had r e c e i v e a s i n g l e a n t i g e n a t io n .
The im m unoglobulin in v o lv e d in g u t im m unity was i d e n t i f i e d in
th e se s tu d ie s .
When c o l o s t r a l l e v e l s o f im m unoglobulin compare
f a v o r a b ly w it h t h o s e found in th e serum o f th e dam b u t n o t th e n e o n a te
th e i n t e s t i n a l m ucosa and i t s a b s o r p t iv e c a p a b i l i t i e s u n d o u b ted ly
c o n t r ib u t e to s e l e c t i v e s c r e e n in g and r e t e n t io n o f c e r t a in immuno­
g lo b u l in s to prom ote th e so c a l l e d g u t im m unity.
M orris (1 9 6 5 )
d em o n stra ted a s e l e c t i v e m echanism e x i s t s in th e i n t e s t i n a l t r a c t o f
th e mouse w h ich was. a l s o found in r a t s ( H a l li d a y , 1 9 5 4 , 1957)
(B ra m b ell e t a l 1 9 5 8 ) .
A r e c e n t s tu d y by P o r te r (1 9 6 9 ) in d ic a t e d
66
t h a t p r e c o l o s t r a l p i g l e t serum was a lm o st e n t i r e l y d i f i c i e n t o f
im m u n o g lo b u lin s, h ow ever, p o s t - c o l o s t r a l serum c o n c e n t r a t io n s o f IgG
and IgM w ere s im i la r to a d u lt l e v e l s w h ile IgA g e n e r a ll y ex ceed ed
a d u lt l e v e l s 3 -4 t im e s .
T h e r e fo r e , th e im m u noglobulin s p a s s e d v i a
c o lo s t r u m .t o n e o n a t a l s e r a , and some more r e a d il y th a n o t h e r s .
Thus,
a d is c r im in a t in g m echanism h as b e e n e lu c i d a t e d in c e r t a i n r o d e n ts
and in la r g e a n im a ls .
Those im m unoglobulin c l a s s e s t h a t a r e n o t
found in th e serum o f th e n e o n a te s may be r e t a in e d by th e i n t e s t i n a l
t r a c t f o r " in s it u " p r o t e c t i o n .
T hus, i t i s m is le a d in g to m easure
th e a c q u ir e d imm unity o f th e n e o n a te and th e immunol o g i c a l s t a t u s o f
newborns by im m unoglobulin a s s a y s perform ed o n ly on n e o n a t a l serum.
IgA i s found v e r y i n f r e q u e n t ly in serum o f newborns a s i l l u s t r a t e d
in t h e s e s t u d i e s even though IgA may b e th e predom inan t immuno­
g l o b u l in in c o lo s tr u m .
The fr e q u e n c y o f t h e o c c u r r e n c e o f s p e c i f i c a n tib o d y in n e o n a ta l
serum d o e s n o t n e c e s s a r i l y r e l a t e t o th e fr e q u e n c y o f th e s p e c i f i c
a n tib o d y l e v e l s in th e serum o f th e dam, n or i s l a c t e a l tr a n s m is s io n
in f lu e n c e d by th e fr e q u e n c y o f s p e c i f i c a n tib o d y found in dam s e r a .
The im m unoglobulin c l a s s e s m ost f r e q u e n t ly p a s s e d p l a c e n t a l Iy as
in d ic a t e d by h ig h in c id e n c e o f s p e c i f i c a n tib o d y a t day one in
n e o n a t a l s e r a a r e a s f o ll o w s : IgG^, IgG ^, th e n IgM, i n n e o n a te s born
to dams t h a t had r e c e iv e d a s i n g l e i n j e c t i o n o f a n t ig e n ; IgG g, IgG^
67
f o llo w e d by IgM appear a t th e same fr e q u e n c y in n e o n a te s born to dams
t h a t had r e c e iv e d two i n j e c t i o n s w h ich i s in c o n t r a s t t o th e f in d in g s
in th e GF n e o n a te s w here th e o r d e r o f o c c u r r e n c e was IgG g, IgM and
th e n IgA fo llo w e d by a co m p le te la c k o f IgG^ s p e c i f i c a n t ib o d ie s .
There i s a s i m i l a r i t y in th e o c c u r r e n c e o f t h e IgGg c l a s s in b o th
g r o u p s.
The GF n e o n a te s d i f f e r from th e CR c o u n te r p a r ts o n ly in t h a t
th e y d id n o t r e c e i v e IgG^ s p e c i f i c a n t ib o d ie s w ith th e same freq u en cy
a t day I .
The in c r e a s e d fr e q u e n c y o f s p e c i f i c a n tib o d y from day I to day
5 p o st-p a rtu rn a r e compared;
In n e o n a te s born t o dams t h a t r e c e iv e d
a s i n g l e i n j e c t i o n th e m ost d e m o n str a b le in c r e a s e i s s e e n in th e IgM
c l a s s f o llo w e d by in c r e a s in g fr e q u e n c y o f IgGg th e n IgA immuno­
g lo b u lin s .
In th e n e o n a te s born to dams i n j e c t e d t w ic e w ith a n tig e n
th e g r e a t e s t in c r e a s e in fr e q u e n c y was se e n in IgGg f o llo w e d by IgG^
th e n IgM.
GF n e o n a te s d em o n stra ted l a c t e a l t r a n s f e r o n ly in one
c l a s s o f im m u n o g lo b u lin s, th e IgGg c l a s s .
IgGg ap p ears to b e t h e s p e c i f i c a n tib o d y c l a s s p a s s in g l a c t e a l l y
w ith t h e g r e a t e s t fr e q u e n c y w h ich i s n o t u n lik e th e fr e q u e n c y
p a t t e r n s se e n f o r p l a c e n t a l t r a n s m is s io n .
T h e r e fo r e , th e m ost e f f i c i e n t im m unoglobulin f o r s y s te m ic p a s s iv e
im m unity o f n e o n a t a l m ice would be IgGg f o llo w e d by IgG ^, w h e r e a s, th e
im m u n oglob u lin s p ro m o tin g l o c a l g u t im m unity would b e IgA and IgM.
SUMMARY
F o llo w in g in tr a d e r m a l i n j e c t i o n s o f DNP-BGG in c o n v e n t io n a lly
r e a r e d and germ free S w is s Manor m ic e , a d u lt serum and n e o n a t a l serum
a s w e l l a s c o lo s tr u m from n e o n a t a l stom achs were a s s a y e d f o r
DNP-BGG
a n tib o d y and l e v e l s o f IgA , IgM, IgG^ and IgG g.
A f t e r a s i n g l e a n t ig e n a t i o n , CR fe m a le s d em o n stra ted serum
im m unoglobulin l e v e l s in th e f o ll o w i n g d e sc e n d in g o r d e r o f m a g n itu d e :
IgG g, IgA , IgM f o llo w e d by IgG^.
S p e c i f i c a n t ib o d ie s w ere predom in­
a n t in IgM f o llo w e d by IgG ^, IgA , and IgG g.
In s e r a o f fe m a le s th at-
had r e c e iv e d two a n t i g e n a t i o n s , im m unoglobulin c o n c e n t r a t io n s were
h i g h e s t in th e IgA f r a c t i o n f o llo w e d by IgG ^, IgM and IgGg w h ile
s p e c i f i c a n tib o d y l e v e l s w ere h ig h e s t in IgM and IgG^ fo llo w e d by
IgA and IgG g.
G erm free fem a le s e r a d is p la y e d a h ig h e r c o n c e n tr a t io n o f IgM
im m unoglobulin th an IgA , IgG^ o r IgG g.
S p e c i f i c a n tib o d y was found
a t h i g h e s t l e v e l s in IgA w ith d e c r e a s in g l e v e l s in th e f o llo w in g
o r d e r , IgG ^, IgM and IgG g.
P l a c e n t a l t r a n s f e r o f IgGg and IgG^ was in d ic a t e d when n ea r
norm al l e v e l s o f t h e s e im m u n oglob u lin s w ere found in n e o n a t a l serum
a t day I p o st-p a r tu r n .
I n c r e a s e d l e v e l s o f IgGg and IgG^ a t day 5
p o st-p a rtu rn in d ic a t e d a c q u i s i t i o n from l a c t e a l s e c r e t i o n s . ' In o n ly
one e x p e r im e n ta l g r o u p , t h o s e n e o n a te s b o m to dams t h a t had r e c e iv e d
a s i n g l e i n j e c t i o n , d id th e serum l e v e l s o f IgM in c r e a s e d u rin g th e
69
5 day p o s t -p a r t u m ■s u c k lin g p e r io d .
E x c e p t f o r IgM t h e r e was a d ir e c t r .c o r r e la t io n b etw een th e number
o f c e lls
in mammary g la n d t i s s u e r e a c t in g w ith f l u o r e s c e in a te d a n t i ­
heavy c h a in s e r a and th e c o n c e n tr a t io n o f th e im m u n oglob u lin s in
c o lo s tr u m .
A ssa y s o f c o l o s t r a l sam p les d em o n stra ted a la c k o f IgM and a
predom inan ce o f IgA .
I n t e r e s t i n g l y , IgA d id n o t appear in n e o n a ta l
serum e x c e p t in v e r y low c o n c e n t r a t io n s a t e i t h e r day I o r day 5
p o s t-p a r tu m .
T h e r e fo r e , IgA i s r e le g a t e d to " lo c a l" g u t immunity
s in c e i t d o e s n o t r e a d i l y p a s s th e n e o n a t a l i n t e s t i n e .
A h ie r a r c h y f o r th e t r a n s m is s io n o f im m u n o g lo b u lin s, r e la t e d
to th e s p e c i e s r a th e r th a n to serum o r c o l o s t r a l im m unoglobulin
c o n c e n t r a t io n s , was o b s e r v e d .
IgA and IgM were p o o r ly tr a n s m itte d
a c r o s s th e p la c e n t a w h ile IgA was p o o r ly tr a n s m itt e d a c r o s s th e
• i n t e s t i n a l m ucosa o f t h e n e o n a t e .
IgG^ and IgG^ r e a d i l y p a s s th rough
b o th t h e p l a c e n t a l and t h e h y p o t h e t ic a l mammary g la n d b a r r i e r s .
APPENDIX
71
Appendix A
The d ia m e te r o f p r e c i p i t i n r in g s in th e m o d ifie d M a s s e y e ffZ i s s w i l l e r te c h n iq u e o f im m u n o r a d ia ld iff u s io n u s in g known a n t i- h e a v y
c h a in and p o o le d norm al mouse serum are r e c o r d e d in th e t a b le b e lo w .
The serum d i l u t i o n s t h a t gave th e maximum r e a c t io n s a r e shown in
p a r e n t h e s i s . Mean v a lu e s and sta n d a rd d e v i a t i o n s f o r t h e v a r io u s
im m unoglobulin m easurem ents were c a lc u l a t e d and r e co r d e d a t th e bottom
o f th e t a b l e ;
F ig u r e 3 , in th e t e x t , d e m o n str a te s th e l i n e a r i t y o f
th e r e a c t io n u s in g s e r i a l l y d i l u t e d norm al p o o le d serum and an appro­
p r i a t e s i n g l e d i l u t i o n o f a n t i- h e a v y c h a in s e r a .
IgM
IgA
ABS-/
UNAB-/
3 .9
3 .9
4 .0
3 .5
4 .0
3 .5
4 .0
—
3.2
3 .2
4 .3
3 .9
5 .5
5 .2
<— — —
5 .0
4 .2
X
—
4 .0 .
= 0.9..
25 0 = 1 .8 ./.
UNAB-/
3 .4
3 .2
2 .8
4 .0
4 .6
3 .2
4 .0
3 .3
5 .4
4 .7
4 .9
4 .1
4 .0
4 .2
4 .2
0 .6
1.2
IgGi
ABS-/
3 .3
3 .2
2 .7
4 .0
3.2
3 .1 .
™
6 .1
6 .1
6 .0
™ ™ ™
3 .0
3 .4
3 .5
4 .8
5 .0
4 .2
4 .7
4 .0
4 .2
4 .6
3 .8
3 .6
0 .8
1 .6
0 .7
5 .0
0 .5
1 .0
4 .6
4 .2
1 .4
ABS-/
6 .6
6 .6
6«o
™
—
UNAB-
IgG2
™ ™ ™
UNAB4 .5
4 .0
4 .0
— — ™
— — *1
4 .0
5 .0
6 .0
™ — —
— — —
4 .0
5 .5
— — —
4 .4
0 .5
1 .0
N o te:
4 .5
4 .5
— ■ —
4 .4 .
5 .1
—
6 .0
3 .6
5 .2
— — —
* — —
4 .0
4 .2
4 .0
0 .3
4 .6
0 .6
1 .2
0 .9
UNAB = un ab sorb ed p o o le d norm al serum
ABS = ab sorb ed p o o le d norm al serum
a./
b/
ABS-/
F in a l d i l u t i o n o f serum i s 1 : 4 .
F in a l d i l u t i o n o f serum i s 1 : 3 2 .
The a n t i s e r a w ere o b ta in e d from Mel=-Par ( F a l l s Church,
V i r g in ia ) and d i l u t e d in s a l i n e in th e f o llo w in g m anner:
A n ti-I g A 1 / 4 0 , A n ti-IgM 1 / 4 0 , A n ti-Ig G 1 /5 0 , A n ti-I g G 0
1 /5 0 .
1
2
72
Appendix B
(a )
A s i n g l e f l u o r e s c i n g c e l l i s se e n in a t i s s u e s e c t i o n r e a c te d
w ith f lu o r e s c in a t e d g o a t a n ti-m o u se IgA .
(b )
A group o f f l u o r e s c i n g c e l l s i s o b se r v e d in a t i s s u e s e c t i o n
r e a c t e d w ith f lu o r e s c e i n a t e d g o a t a n ti-m o u se IgA .
73
Appendix C
(a )
Equipment l a y o u t .
(b )
P r e c i p i t i n r in g s in agar d i s c s
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LIBRARIES
co p .?
'' t l s o n , lic b a r d Adar
The nature o f
i r m u n o g o l c u l i n s p e c i e s ir]
p a s s iv e immunity o f
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