EXERCISE 6
TO DEVELOP ANTISERA
Rabbit antiserum developed against Rhizobium is the basic reagent
for strain identification utilizing serological techniques.
In
this exercise, Rhizobium antigens are prepared and then used for
the development of antiserum in rabbits.
Key steps/objectives
l)
Culture rhizobia on YMA bottle flats
2)
Check for culture purity by Gram stain
3)
Harvest and prepare antigens for immunization
4)
Begin immunization; inject antigen intramuscularly
5)
Inject antigen intravenously
6)
Give intraperitoneal injections
7)
Trial bleed
8)
Determine the antiserum titers
9)
Harvest blood through cardiac puncture
10)
Give subcutaneous booster injections
(a)
Culturing rhizobia for antigen
(Key steps 1 and 2)
Inoculate selected strains of rhizobia on two 500 ml YMA flats
and incubate at 26oC.
Broth culture in 50 ml Erlenmeyer flasks
may also be used but the medium must be fully defined to avoid
complications with antigenic components from the yeast in YM
broth (see Appendix 3).
Check for purity (by Gram stain) at the end of the specified time
for growth, e.g., 3-5 days for fast-growers and 7-10 days for
slow-growers.
Strains that produce a lot of "gum" should be
harvested earlier.
(b)
Preparing antigens for immunodiffusion
(Key step 3)
When the cultures are ready for harvest, aseptically add about 10
ml of sterile, filtered saline and 20 sterile glass beads to the
YMA-slants.
Close the culture vessel and hold it level so that
the saline irrigates the entire surface.
Tilt back and forth so
that the glass beads dislodge the rhizobial cells into
suspension.
Transfer the suspension (but not the glass beads) to
sterile centrifuge tubes and spin down the cells at approximately
5,000 X g for 15-20 min.
Discard the supernatant and resuspend
the precipitate again in sterile saline.
The gummy substance in
the supernatant consists of polysaccharides and is found
especially in older cultures.
point.
It should be discarded at this
Do not repeat the centrifugation as excessive washing
would remove the soluble antigens essential to the
immunodiffusion reaction.
Resuspend the precipitate by dropwise
addition of sterile saline and with frequent agitation to obtain
a thick suspension of 1 x 1010 cells ml-1.
Store about one-half of the thick suspension in the refrigerator,
for reference.
Dilute the remainder to 1 x 109 cells ml-1 using
the McFarland standards (Appendix 6).
Dispense the diluted
suspension into small (5 ml) sterile serum vials in 2 ml portions
to be used for injections.
Add a preservative (1% merthiolate)
to each 2 ml sample and also to the thick suspension.
Merthiolate is used extensively in serology as a preservative.
When used in liquids at a final concentration of 1:10000, it does
not interfere with serological reactions.
The vials may be
stored at 4C for several weeks or kept frozen for several
months.
(c)
Preparing somatic antigens for the agglutination and
fluorescent antibody techniques
(Key step 3)
The insoluble somatic antigens found on the surface of the cells
are required.
Soluble and most of the flagellar antigens are
eliminated by frequent washing.
Harvest a fully grown culture from YMA-flats as before.
Cells
should be centrifuged, the supernatant discarded, and the pellet
resuspended in filter sterilized saline, using a vortex mixer.
This sequence of centrifugation and resuspension is repeated
three times and the cell concentration is adjusted to
approximately 1 x 109 cells ml-1.
Transfer the suspension to a
sterile serum bottle and close with a rubber septum.
Insert a
small gauge (about 23 gauge) needle through the septum to act as
an air and steam vent.
Heat the antigen for 1 h at 100C to
inactivate any remaining flagellar antigens.
This is
accomplished by partly immersing the serum bottle in boiling
water or by subjecting it to heat in a steam bath.
Add
merthiolate solution to the antigen suspension after heating.
(d)
Immunizing the rabbit
(Key steps 4, 5, and 6)
A variety of injection schedules have been used to produce
antisera of sufficiently high titers.
in Appendix 12.
Three examples are given
The schedule used in this chapter employs three
different routes of injection.
Pipette 2 ml of antigen and 2 ml of Freund's complete adjuvant
into a 50 ml beaker and emulsify by repeatedly drawing the
mixture into a glass or plastic syringe (no needle attached) and
expelling it through the orifice.
The right consistency is
reached when a drop of this emulsion does not disperse
immediately in water.
Freund's complete adjuvant is made from
mineral oil and killed cells of Mycobacterium tuberculosis or M.
butyricum.
It is used to enhance the effect of the antigen.
Inject 1 ml of the antigen-adjuvant emulsion into the thigh
muscle on each hind leg of the rabbit.
After 2 weeks, give an intravenous injection of 1 ml of antigen
without adjuvant.
After 4 weeks give an intraperitoneal injection of 1 ml antigen
without adjuvant.
(e)
Trial bleeding for titer determination
(Key steps 7 and 8)
Seven days after the last injection, conduct a test-bleed through
the marginal ear vein.
(Appendix 12).
Transfer the blood into a sterile screw-cap test tube.
Allow the
blood to clot at room temperature for approximately 2 h.
Detach
the blood clot from the test tube wall by moving a wooden
applicator stick around the clot.
Refrigerate overnight to
separate the serum from the clot.
Decant clear serum into a test tube, minimizing carry over of red
blood cells.
Since only a very small amount of blood is obtained
in the trial bleeding, centrifugation may not be practical.
Determine the agglutination titer (Exercise 7).
(f)
Collecting blood and giving booster injections
(Key steps 9 and 10)
If the titer is satisfactory (not less than 1:1600), bleed the
rabbit from the heart by cardiac puncture using a bleeding rack
as shown in Figure A.14 of Appendix 12.
blood.
Obtain 30-50 ml of
Transfer the blood into a sterile, screw-cap test tube of
50 ml capacity.
After the blood has been clotted and refrigerated, decant the
serum and centrifuge at 5,000 x g for 15 min (under
refrigeration, if possible) to clear the serum of red blood
cells.
Transfer the clear serum supernatant into an appropriate
container for storage by freezing.
ml portions in suitable sized vials.
Serum should be stored in 1-2
This may not be necessary
if the blood is to be processed for conjugation with fluorescein
isothiocyanate (FITC).
Sera from different rabbits receiving the
same antigen may be pooled.
If the titer was too low in the trial bleeding (less than
1:1600), give a booster injection of 1 ml antigen subcutaneously
immediately after the titer determination.
Bleed 1 week later by
cardiac puncture.
If more antiserum is desired, the level of immunoglobulins in the
rabbit can be maintained by booster injections 7-14 days after
each bleeding.
However, in such a case, it would be advisable to
make intraperitoneal injections of sterile saline each time after
the blood has been taken to replenish the liquid level in the
animal.
The volume of saline injected should be equal to the
volume of blood taken from the rabbit.
Note: Storage vials should also be adequately labelled to
indicate rhizobial strain, serum batch-number and date.
Records
of injection schedules and agglutination titers determinations
should be noted.
Weight, age, sex and other relevant information
on the animals are also usually recorded.
Requirements
(a)
Culturing rhizobia for antigens
Microscope, incubator
Microscope slides, immersion oil
Inoculation loop, flame
Gram stain solutions (Appendix 3)
Wash bottle with distilled water
YMA flats in 500 ml medicine bottles or
Erlenmeyer flasks (250 ml) containing 100 ml of a defined
medium (Appendix 3)
Cultures of rhizobia
(b)
Preparing antigens for immunodiffusion
Centrifuge, balance, vortex mixer (optional)
Sterile glass beads (4 mm diameter, sterilized and stored in
test tubes at 20 beads per tube)
Pipettes (10 ml)
Centrifuge tubes (30-50 ml capacity) with caps, rack
Saline, sterile, (membrane filtered 0.85% NaCl w/v)
(c)
Preparing antigens for the agglutination and the FA
technique
Centrifuge, balance, vortex mixer (optional)
Stove or bunsen burner with tripod and gauze screen, or
steam bath
Centrifuge tubes 30-50 ml with caps, rack
Pipette (1 ml)
Glass beads as in (b)
Saline, sterile
Serum bottle with rubber septum, syringe needle (small
gauge)
1% membrane filtered merthiolate (also called Thimerosal)
solution (B.D.H., Gallard-Schlesinger Chem. Mfg. Corp.,
Carle Place, N.Y., or Sigma ChemicalCorp.)
Inoculated YMA flats from (b)
(d)
Immunizing the rabbit
Large towel (approximately 100 cm x 75 cm)
Small sterile beaker (50 ml capacity)
Glass syringes (10 ml capacity)
Plastic syringes (1-5 ml capacity)
Syringe needles 20 gauge, 22 gauge and 26 gauge (sterile)
Freund's complete adjuvant (DIFCO Laboratories, Detroit,
Michigan, USA)
Rhizobial antigens from (c)
(e)
Trial-bleeding for titer determination
Refrigerator
Large towel, scalpel, vaseline, razor blade
Cotton wool or tissue paper, alcohol (70%)
Test tubes with caps, rack
Wooden applicator sticks or thin glass rods
Requirements for titer determination (Exercise 7)
(f)
Bleeding for collection and injecting boosters
Centrifuge, balance, freezer
Bleeding rack (Appendix 12)
Syringes (with 18 gauge needles)
Screw-cap test tubes (50 ml), rack
Centrifuge tubes (50 ml with caps), rack
Vials 5 ml (for storage of serum)
Merthiolate solution (Appendix 4)
Alcohol 70%, cotton wipes or soft tissue paper
Glass vials (20 ml) for bulk storage