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  Section: Plant Lab Protocols
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Methodology for Amino Acids and Proteins

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.

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.

Three ‘New Zealand’ White Rabbits (2.5kg body weight)
Purified Immunogen in an appropriate Buffer
Incomplete Freund’s Adjuvant
Infrared Lamp
Dilute the purified antigen (150mg) 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 Freund’s 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.
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.
Similarly, give three booster shots at 10-day intervals following the initial shot. Each time, 50mg protein (antigen) emulsified in a total volume of 0.5mL of incomplete Freund’s adjuvant is used.
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.
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.
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 4°C 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.
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.
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.
The antiserum is then tested  by immunodiffusion or immunoelectrophoresis for its ability to form immunoprecipitate.
The antiserum may be stored at -20°C in small aliquots in the presence of 0.1% sodium azide for longer duration.
The antiserum may be further processed to isolate IgG (for details see under ELISA).
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).
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.
Immunization can be done on any part of the body-skinfold of the neck and of back, foodpad etc.
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.
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.
Complete Freund’s adjuvant induces antibody, production greatly than the incomplete Freund’s adjuvant. CFA contains killed Mycobacterium cells over the IFA. These are commercially available from various suppliers.
1. Hurn, B A L and Chantler, S M (1980) Production of Reagent Antibodies In: Methods in Enzymology 70 (Eds. Van Vunakis, H and Langone, J J) Academic Press New York p 104.
2. Carlier, A R, Manickam, A and Peumans, W J (1980) Planta 149 227.
3. Manickam, A and Carlier, A R (1980) Planta 149 234.


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