Presented by:
MUFIDATUR ROSYIDAH
(126090100111012)
IgA
Chicken
Immunoglobulin
IgM
IgY
Abundance
in Egg Yolk
Same physiological function in birds =
IgG in mammal
Benefit to diagnostics of pathogen infection
Production, characterization of mAbs, and
demonstrated in potential use
Fig.1 The structural organization of immunoglobulin.
A. Human IgG B. Chicken IgY
VH (variable domain of heavy chain); VL (variable domain of light chain); CL (constant
domain of light chain); CH 1, CH2, CH3 (constant domain of heavy chain); Cv1, Cv2, Cv3
and Cv 4 (constant domain of chicken heavy chain).
Egg Yolk
Cloroform extraction /
kit
Serum
Diluted with PBS (1:20)
Chicken
Cross reaction Test
•Turkey
•Peafowl
•Pheasant
•Parrot
•Sparrow
•Pigeon
Diluted with PBS
(1:50)
•Duck
•Goose
•Quail
Diluted with PBS
(1:50)
•Human
•Rabbit
•Pig
•Horse
•Cow
Non-Avian IgM
Production of mouse monoclonal antibodies
(mAbs)
Balb/ c Mouse (8
weeks old)
Protein test
Immunobinding
Assay
Imunized by chIgY
4x
(intraperitonial &
intravenal)
Washing by Buffer
Spleen cell vs NS-0
Meyloma cell
fusion
(+PEG 50 %)
Selected
hybridoma
Were cloned twice
Cont...
chIgY samples applied
to NTC membrane
strips
Blocking
(Tween 0,5%)
Incubation peroxidase
conjugated rabbit antichIgY antibodies, 30’
Washing in PBS +
substrate true Blue
Rinsing strips in distilled water
Positive control : blue spot
appeared
Dot immunobinding assay (DIBA) Cont...
Membrane
+ chIgY
Membrane
+ chIgY
Membran
e + chIgY
 Isotyping

Incubated in 1F5/3g2 mAb
diluted in PBS
In different pH value (3-12)
Incubated in 1F5/3g2 mAb 5-45
minutes
(increasing time interval was 5 min)
Incubated in 10 mM
periodic acid in 50mM
Na-acetate, pH 4,5, 1
(increasing time interval
was 5 min)
Incubation
in mAbs
of mAbs immunoenzyme assay :
Using isotyping reagent ISO-2
Optimal
condition
Minimal
incubation
time
Test Of
carbohydrates
SDS-PAGE and immunoblotting
chIgY samples were treated 2 % βmercaptoethanol
Apllied to gel
Protein in membran strips
Incubated in appropriate mAb solution
Immunoenzyme reaction
Conjugation of horseradish peroxidase
to 1F5/3G2 mAb
2 gr mAbs 1F5/3G2 diluted in 0,1 M
Na2CO3
Diluted in 160 µL glutaraldehyde,
overnight
Dialyzed again in NaHCO3 0,1 M, pH 9,2
Incubated with 4 gr enzyme, 24 h
Dialyzed in PBS
Blocking + Lysin 0,2 M
Indirect Immunoenzyme Assay
Tested
samples
incubated
withaMycoplasma
synoviae & M. Gallisepticum in agar block, 45’
Diluted in 160 washed in PBS
HRP-conjugatedn1F5/3G2 mAbs + IgY
(incubated)
Washing in PBS, drained and treated
with substrate containing DAB
Western blotting & Immunoenzyme
on reaction
Immunoadsoption of IgY
Undiluted and diluted serum (1:10)
mix with CNBr Sepharose 4B, coupled
with
mAbs
1F5/3G2,
room
temperature, 1 h
centrifugation, supernatan
were collected
Assayed for total IgY
4E4 clones
3C10 clones
IF5 clones
2F10 clones
Commercial
polyclonal HRPconjugated rabbit
anti-chIgY
Fig. 2 Reaction of mAb with chIgY, isolataed from chicken egg yolk (IgY) and with avian
sera (s, 1-7) or egg yolk (y, 8-10) and eith sea of some mammals (s, 11-16).
1: chicken, 2: turkey, 3: peafowl, 4 : pheasant, 5 : parrot, 6 : sparrow
7 : chicken, 8 : duck, 9 : goose, 10 : quail, 11: rabbit, 12 : pig, 13: cattle
14 : horse, 15 : mouse, 16 : human.
Fig. 3 Reaction of mAb M1 to HC of chicken IgM in DIBA with avian sera (s)
or egg yolk (y).
1 : chicken, 2 : turkey, 3 : peafowl, 4 : pheasant, 5 : japanese quail,
6 : sparrow, 7 : pigeon, 8 : parrot, 9 : duck, 10 : goose
 mAbs
chicken IgY use to detection of patogen
infection (Micoplacma gallisepticum)
 Remove
the IgY from yolk egg, to get IgA and IgM
Fig. 4. Detection of IgY antibodies
specific for in vivo expressed
Mycoplasma gallisepticum antigens using
HRP-conjugated 1F5/3G2 mAbs. In IIPA
agar
blocks
with
Mycoplasma
gallisepticum colonies were incubated in
tracheal washing of an infected chicken.
As secondary antibody HRP-conjugated
1F5/3G2 mAbs were used.
Arrows indicate various (2 and 3) and
sectorial (1 and 2) staining depending on
variably expressed antigens recognized by
local antibodies
Fig. 5. 1F5/3G2 mAb for detection of specific
IgY antibodies against protein antigens of
three major poultry pathogens using
immunoblotting.
Panel A, Mycoplasma gallisepticum; panel B, Mycoplasma
synoviae; panel C, Newcastle disease virus. After incubation
in sera of infected chicken membrane strips were incubated in
secondary antibodies: lanes 1, peroxidase conjugated rabbit
anti- -chIgY antibodies; lanes 2, HRP-conjugated 1F5/3G2
mAb. Molecular mass is indicated on the left side (in kDa);
arrows
indicate
major
immunogenic
proteins
i.e.
haemagglutinins pMGA (panel A) and haemagglutinis of M.
synoviae, named MSPB (panel B).
Note: HRP-conjugated 1F5/3G2 mAb gave much less background
staining, particularly in panel C