BMT 205

IMMUNOLOGY

ANTIGEN-ANTIBODY REACTIONS & ANTISERUM PRODUCTION
PRACTICAL 1 - 6

NAME : EDWARD LEE KUAN MUN
I.C NO. : 900127-07-5645
MATRIX NO. : 109151
DATE : 25
TH
MAY 2012


Animal Testing and Antigen-Antibody Reactions
Objectives
1. Perform tests on animal serum
2. Prepare antigen, bacterial vaccine and lectin.
3. Perform blood group determination
4. Perform bacterial agglutination
5. Carry out antigen-antibody reactions (precipitation)

Method
Methods and procedures were carried out according to manual given.


Introduction
Immunization and antiserum production is the process where the animals (in our case,
rabbit and chicken) are immunized and its serum is extracted and several tests are conducted on the
extracted serum. Antiserum is blood serum that contains polyclonal antibodies. It is used to pass on
passive immunity to many diseases.
Suitable antisera are essential in all immunochemical procedures. Three important
properties of an antiserum are avidity, speficity and titer. Avidity measures the strength of
interactions of its antibodies with an antigen while specificity is the measure of the ability of its
antibodies to distinguish immunogen from related antigens. Titer of an antiserum is the optimal
dilution that is used in the procedure. It depends on the concentration of antibodies present and
their affinities for the antigen. (Bailey, 1985)
Immunogens are substances that induce an immune response when injected into a suitable
animal. Immunogenicity depends on various factors, among others; size, shape, chemical
composition and structural difference from any molecular species indigenous to the injected animal.
Particulate substances are usually very immunogenic but the resultant antisera do not possess a high
degree of specificity and do not store well. (Bailey, 1985)
For injection, highly purified samples are used although smaller molecules such as peptides
can be made immunogenic by chemical coupling to a protein that causes immune response by itself.
It is also usual to a mixture of the potential immunogen with an adjuvant to stimulate antibody
production. Adjuvants are substances which, when mixed with antigen to be injected, enhance
antigenicity and increase amount of antibody produced than injection with antigen alone. (Hyde,
1968)
The immunogen used to elicit antiserum production in chicken is a bacterial vaccine (Vibrio
cholerae), while protein antigen (Bovine Serum Albumin, BSA) is used for rabbits.
Vibrio cholerae are gram-negative, straight or slightly curved rods with a single polar
flagellum. They are facultative anaerobic organisms and can be grown readily with ordinary media.
The colonies are usually translucent, amorphous but may be wrinkled or rugose on nutrient agar. (T
& Norris, 1985)
Bovine serum albumin (BSA) used for rabbit immunization is a serum albumin derived from
cows. It has numerous applications aside from immunization such as ELISA and
immunohistochemistry. One important characteristic of BSA is that it does not affect other enzymes
which it does not need for stabilization. This makes it extremely suitable as in lacks effect in many
biochemical reactions and can be produced in large quantities at low cost as it can be readily purified
from bovine blood (byproduct of cattle industry).

In order to harvest a considerably large amount of antiserum, several injections were
necessary to induce the production of antiserum in the rabbit and chicken. The production of
antibody from the first injection is usually low. Here we elicit an Amanestic response, where a final
injection several weeks after the first will induce a higher amount of antibody titre. Immune
response can be described as occurring in two phases. The primary response, which produces only
small amounts on antibody molecules results from the initial administration of immunogen. This is
because the antibody producing system is still being primed. Subsequent injection of the
immunogen results in the secondary phase of the response whereby large amounts of antibody
molecules are produces by the primed system. However, these phases are not clear in practice.
(Bailey, 1985)
The collection of antiserum is followed by testing it to test the presence of antibody for the
antigen. The method used is agglutination. Here we observe the clumping of particles, which
represent the binding of antibodies to the antigen. This agglutination is visible without help of
microscopes. Another method of observation is via precipitation. However, unlike agglutination, the
precipitation reactions need dissolved antigens instead of particulate antigens. In this experiment,
we use the Ring test to identify solutions that contain antigen. A white ring will be seen in
solutions that contain antigen. By manipulation of values such as highest dilution that produces the
white ring, we can determine the antibody titre.
Raising polyclonal antisera is also determined by the animal used. Individual animals can
respond quite differently to the same immunization process. (Bailey, 1985) When choosing the
animal species to be used in antiserum production, a few factors must be considered, such as;
amount of antiserum needed, ease of bleeding, cost of purchase and maintenance of animal. It
seems that only vertebrate animals are capable of synthesizing antibodies in response to antigen
exposure. Also, antigens must be foreign to the circulation of the animal, for instance, the greater
the phylogenetic disparity of the antigen and the experimental animal, the greater will be the
immune response elicited by the antigen. In our practical we used, bovine antigen on rabbit and
bacterial vaccine on chicken. (Umbreit, 1968)
Correct grouping of human blood is imperative for numerous reasons; among them
transfusion compatibility is paramount. The term blood group is a classification of an individuals
blood based upon the inherent presence or absence of antigens on the surface of their red blood
cells (RBCs). In the blood plasma, antibodies exist which are formed against foreign antigens to
protect the body from perceived threats. This reciprocal relationship, Landsteiners Law, means that
individuals who lack a RBC antigen (A, B or D for example) will possess the corresponding serum
antibody, whilst those who possess the antigen must not. There are 30 discrete blood type systems
recognised by the ISBT Committee on Terminology for Red Cell Surface Antigens. Among these the
ABO and Rh systems are of primary importance when transfusing blood, as incompatibility may lead
to an acute haemolytic reaction with catastrophic results such as shock and renal failure leading to
death. (Ballerini, Wei Shen, & Xu Li, 2011)
Precipitation test is another type of antigen-antibody reaction, where the antigen is in a
soluble form. In this test, antibody interacts with the soluble antigen in the presence of an
electrolyte. A lattice will be formed from the reaction between the antibodies and the antigens.
Precipitins are antibodies that aggregate soluble antigen. Its important to note that the antibody to
be tested with this method must be bivalent and the antigen must be either bivalent or polyvalent.
The antigen must have at least two copies of the same epitope or have different epitopes that react
with different antibodies in polyclonal antisera. (Parija, 2009)
Results
PRACTICAL 5 : Antigen and Antibody Reactions; Agglutination
I. Lectins preparation and standardization

Titre =

= 1 / 2
-4

= 16

II. Blood Group Determination of Group Members
Blood group No. of students
A 12
B 13
O 35
AB 6

III. Bacterial agglutination
Titre =

= 1 / 2
-7

= 128
PRACTICAL 6 : Antigen-Antibody reactions; Precipitation
I. Antigen dilution
Titre =

= 1 / 10
-2

= 100

Discussion
Agglutination occurs when two different blood types are mixed or when bacteria are
exposed to specific antisera. The binding of the epitope to the antibody triggers the binding of a
component of the complement on the antibody to bind to the Fc region of the IgG heavy chain and
this is followed by a change in conformation of the antibody. (Redei, 2008)
For lectin and bacterial agglutination, the highest dilutions that produced agglutination are
2
-4
and 2
-7
, respectively. These values correspond to titer values of 16 and 128 respectively. These
values indicate of the number of times the solution can be diluted and still contain detectable
amounts of the antibody. This means a higher titer value is produced by antisera with higher
amounts of antibody. However, it must be noted that according to Coico R. and Sunshine G., in their
book, Immunology : A Short Course, agglutination may not occur at high concentrations of
antibody, although at higher dilutions, agglutination does take place. Tubes in which agglutination
does not occur because of high concentrations of antibody are called prozone. Here antibodies are
present in excess and every epitope on antigen may bind to only a single antibody molecule, hence
preventing cross-linking between different particles.
Blood test was conducted for every student using the slide test, where blood is mixed with
Anti-A and Anti-B serum to determine whether the blood samples contained A and/or B antigen.
Blood samples that reacted (agglutinate) with Anti-A serum are from blood group A. This is similar
for blood group B samples. If agglutination occur when blood samples are reacted with Anti-A and
Anti-B, this means that two types of antigen are present on the blood sample, suggesting that the
blood group is AB. If agglutination does not occur with both Anti-A and Anti-B, this means none of
the A or B antigen are present, suggesting that the blood is form group O.
Blood group O has highest frequency in the population of students, followed by relatively
similar frequency of blood group A and B. Blood group AB has lowest frequency. This trend is similar
globally, where blood group O has relatively higher frequency than other blood groups followed by A
and B and followed by AB.
As for the ring test, the titer value obtained is 100, where the highest dilution that produces
agglutination is 10
-2
. In this test, the antigen solution is layered on top of antiserum. Precipitation
between the two layers is marked by the appearance of a white ring around the tube at the
junction where the two layers meet. A control tube was prepared but it was used during the
practical because there was a handling error when putting the antigen layer into one of the test
tubes causing the occurrence of bubbles in that particular test tube.
Similar to agglutination, a high titer value in the precipitation test corresponds to a high
antibody concentration. A more visible white ring marks the presence of higher amounts of
antibody compared to a less visible white ring.
Precautions
Basic procedures such as scrubbing work table and bench before and after procedures must
be carried out to avoid infection to students. Aseptic techniques like flaming the inoculating loop
after use must be practiced in the laboratory. Work in pairs to reduce careless error and verify
results.
When preparing agar media, it was carefully prepared so that it is not exposed to air as it
may cause contamination of the media.
Carefully layer the antigen on antiserum when performing the ring test so as to avoid the
formation of bubbles. Also avoid accidentally mixing the two layers as this would make the white
ring to appear less clear-cut.
Add only one drop of red blood cell for lectin practical. Contents of the wells are mixed with
toothpick, starting from the highest dilution to avoid changes in concentration of its components.
Cover wells with parafilm to avoid exposure to air and contaminants.
Injection site on rabbit and chicken must be swabbed with alcohol before and after injection.
Massage/tap the injection site when drawing blood to make the veins more obvious. Attempt to
draw blood at one try as multiple blood drawings may cause clots to form in the animals.
Injection must be administered subcutaneously. Subcutaneous injections can allow the
immunogen to enter the circulation quicker. Immunogens may be digested if administered orally.
Avoid the formation of skin lumps by not injecting on different places at the same time.
Female rabbits are less aggressive, and although smaller and yielding smaller quantities of
antiserum are preferable to male rabbits for antiserum production. Many biomedical facilities use
communal floor pens for donor animals and female rabbits adapt better to this form of housing.
(Burns, 2009)
The use of excessive amounts of antigen in immunizations should be avoided, as this can
lead to a poor immunological response. Swamping of the system can lead to selective deletion of the
B cell clones of interest and a reduction in the specific antibody titer. High doses of antigen in the
secondary and subsequent immunizations can cause anaphylactic shock and death of the donor
animal. (Burns, 2009)
It has been reported that increased stress levels in animals can depress the immune
response, and appropriate measures should be taken to ensure that immunizations and bleeds are
performed with the minimum of stress to the animals. General husbandry in terms of housing, noise
levels, and other environmental factors should also be examined to ensure that animals for
polyclonal production are maintained under suitable conditions. (Burns, 2009)
In order to avoid the observational error caused by the presence of prozone it is
imperative to test antiserum at several dilutions.

Conclusion
Objectives of the practical were met. Tests on rabbit and chicken antiserum such as
precipitation and agglutination were run. Blood group determination was carried out. Antiserum was
successfully produced and blood groups of students were determined.



References

Bailey, G. S. (1985). Production of Antisera. In J. M. Walker (Ed.), Proteins (Vol. 1). Humana Press.
Ballerini, D. R., Wei Shen, & Xu Li. (2011). An inexpensive thread-based system for simple and rapid
blood grouping.
Burns, R. (2009). Immunisation Strategies for Antibody Production. In R. Burns, Plant Pathology.
Humana Press.
Coico, R., & Sunshine, G. (2009). Immunology: A Short Course. John Wiley & Sons.
Hyde, R. M. (1968). Antiserum Production in Experimental Animals. In Advances in Applied
Microbiology (Vol. 9). Academic Press.
Parija. (2009). Textbook of Microbiology & Immunology. India: Elsevier.
Redei, G. P. (2008). Encyclopedia of Genetics, Genomics, Proteomics, and Informatics (3rd ed., Vol.
1). Springer.
T, B., & Norris, J. R. (1985). Methods in Microbiology (Vol. 16). Academic Press.
Umbreit, W. W. (1968). Advances in Applied Microbiology (Vol. 9). Academic Press.