Preparation of antiserum

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Production of antiserum
The production of antiserum is a very important step for the immunological experiments. An
immunogen (antigen) is a substance that when injected into a suitable animal gives rise to an
immune response, this property depends upon many factors of the immunogen molecule its
size, shape, chemical composition and structural difference from any related molecular
species indigenous to the injected animal. In general, proteins of molecular weight above 500
daltons and certain large polysaccharides are effective immunogens. Low molecular weight
substances (hapten) coupled to a protein with immunogenicity can also be an immunogen. It
is always preferable to use the immunogen in a highly purified form so that highly specific
antibodies against the antigen is produced.

Antiserum so produced in the animal contains antibodies in response and to counter the
immunogen. The immune response occurs in two places. First administration of the
immunogen induces the primary response during which only small amounts of the antibodies
molecules are produced. Subsequent administration of the immunogen results in the
secondary phase during which large amounts of antibody molecules are produced by the
lymphocytes. However, there appears no clear demarcation between these two phases.

Different types of animals are used for the production of antiserum. Rabbit is generally used.
Besides, rat, guinea pig, sleep, goat, horse, elephant etc. have also been used for the
purpose. The choice of the animal depends upon many factors such as the amount of antigen
available, the degree of immune response, the quantity of antiserum to be produced,
maintenance of the antiserum etc.

There are a number of different methods described for the production of antiserum. A method
that has been successfully used to produce antiserum using microgram amounts of protein is
described below.

Principle
Antibody synthesis is a defense reaction of higher vertebrates evolved to combat any foreign
material that has entered the body. Any substances which possesses an arrangement of
atoms at its surface that differs from the surface configuration of normal host component is
recognized as an antigen. Antibodies are glycoproteins commonly called immunoglobulins
(IgG) and are present in blood and other local secretions of the organs where antigen is
present. The cellular events triggered by antigen lead to the differentiation and proliferation
of specific antibody producing lymphocytes against a particular antigen. There are five major

immunoglobulin classes. An antigen may stimulate the production of one or more classes of
immunoglobulins. All the immunoglobulins have two identical light chains and two identical
heavy chains. The antibody combining site is located in the amino terminal portion of the
molecule which is also called the V (variable)-region. The primary structure of V-region varies
with the type of antibody produced against an antigen.

Materials
Three New Zealand White Rabbits (2.5kg body weight)
Purified Immunogen in an appropriate Buffer
Incomplete Freunds Adjuvant
Infrared Lamp

Procedure
1.

Dilute the purified antigen (150g) to 0.2mL in phosphate-buffered saline. (PBS:

100mg CaCl2; 120mg MgSO4; 200mg each of KCl and KH2PO4; 8.0g NaCl; 1.15g
Na2HPO4 per liter) are emulsified with 0.8mL of incomplete Freunds adjuvant by
vortexing thoroughly. Draw the mixture inside a syringe. The syringe is emptied
and refilled with the mixture 3-5 times to produce a uniform, stable emulsion which
can then be administered to a rabbit.

2.

Prepare the rabbit for immunization by shaving away the hair at one or two places
on the hind thighs. Inject the emulsion in these places subcutaneously using a
21-gauge needle.

3.

Similarly, give three booster shots at 10-day intervals following the initial shot.
Each time, 50g protein (antigen) emulsified in a total volume of 0.5mL of
incomplete Freunds adjuvant is used.

4.

Eight days after the last booster shot, arrest the animal in a suitable cage for
bleeding. Rub the surface of back of an ear with alcohol-mistuned tissue to expand
the veins.

5.

Nick the marginal vein with a scalpel blade and collect the blood in a glass vessel
(about 15mL can be collected in 10min). every minute or so clean the clot at the
puncture wound with alcohol-moistened tissue for continuous bleeding. Alternately,
bleeding is carried out under infrared light. At the end, thoroughly clean the stained
surface of the ear to avoid any infection. A test-bleeding may be carried out even
before the third booster shot to examine the antiserum for the immune response
and titre.

6.

The blood is allowed to clot standing for 3-4h at room temperature. The serum is

separated from the clot by low speed centrifugation and stored at 4C in the
presence of 0.1% sodium azide as antibacterial agent. The serum is usually straw
yellow-colored. If RBCs are partly lysed then the serum is colored red.
7.

If on testing, the serum shows the characteristics required for its particular usage,
then further bleeding up to three times can be made on successive days. Then the
rabbit is allowed to rest for 4-6 weeks before further bleeding.

8.

The animal can be used for about six months to collect blood and then abandoned.
If after boostering, the antiserum is not of the desired quality, it is better to
disregard the particular rabbit and look for the other rabbits for the antiserum. If
the whole lot is unsatisfactory possibly due to the weak immunogens, either repeat
the immunization or use other animals.

9.

The antiserum is then tested by immunodiffusion or immunoelectrophoresis for its

ability to form immunoprecipitate.

10.

The antiserum may be stored at -20C in small aliquots in the presence of 0.1%
sodium azide for longer duration.

11.

The antiserum may be further processed to isolate IgG (for details see under
ELISA).

Notes
1.

A good antiserum should possess three important qualities: avidity (measure of

the strength of the interactions of its antibodies with antigen), specificity (ability
of the antibody to recognize its antigen from related molecules) and titre (the
concentration of antibodies present, and on their affinities for the antigen).

2.

When large amounts of pure immunogen are available, a high initial

immunization dose can be used. However, lower the dose of antigen, the greater
is the avidity of the antiserum.

3.

Immunization can be done on any part of the body-skinfold of the neck and of
back, foodpad etc.

4.

A long gap of 3-10 weeks is also normally used between the initial and booster
immunizations in order to avoid development of a state of tolerance to the
antiserum by the animal.

5.

Since the antisera produced by conventional methods consist of mixtures of

different antibody molecules, the properties of antisera collected during the
prolonged period of immunization may change. Hence, each bleeding should be
tested for the antiserum qualities before any use.

6.

Complete Freunds adjuvant induces antibody, production greatly than the

incomplete Freunds adjuvant. CFA contains killed Mycobacterium cells over the
IFA. These are commercially available from various suppliers.