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Production of Polyclonal Antisera Using Freund's Complete and Incomplete Adjuvant

Catalog Number: 642851, 642861, 55828, 55829, 195187
Production of Polyclonal Antisera Using Freund's Complete and Incomplete Adjuvant


Introduction: All immunochemical procedures require a suitable antiserum or monoclonal antibody raised against the antigen of interest. Polyclonal antibodies are raised by injection an immunogen into a animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest. In producing antibodies, several parameters must be considered with respect to the final use to which the antibody will be put. These include (1) the specigens, (2) the avidity of the antibody, i.e., the strength of binding, and (3) the titer of the antibody, which determines the optimal dilution of the antibody in the assay system. A highly specific antibody with high avidity may be suitable for immunohistochemistry, where it is essential that the antibody remains attached during the extensive washing procedures, but may be less useful for immunoaffinity chromatography, as it may prove impossible to elute the antigen from the column without extensive denaturation.

To produce an antiserum, the antigen for the first immunization is often prepared in an adjuvant (usually a water in oil emulsion containing heat-killed bacteria), which allows it to be released slowly and to stimulate the animal's immune system. Subsequent injections of antigen are done with incomplete adjuvant that does not contain the bacteria. The species used to raise the antibodies depends on animal facilities, amount of antigen available, and the amount of antiserum required. Another consideration is the phylogenetic relationships between antigen and immunized species. A highly conserved mannalian protein may require an avian species in order to raise an antibody. Production of antibodies is still not an exact science and what may work for one antigen may not work for another.

A simple, generally applicable protocol for raising polyclonal antiserum to a purified protein of greater than 10,000 mol wt. is described. This method has been used to raise antibodies against a cytosolic protein, glutathione-S-transpeptidase. The latter was first soubilized by cleavage from the membrane with papain (1). Variations to this basic procedure are discussed in Chapters 2,3, and 5 of this vol. For proteins or peptides of low molecular weight (<5-10kDa) conjugation to a carrier protein is required for them to elicit antigenicity (seethis vol., Chapter 4). Variations on this basic technique can be found in selected references (2-5).

Materials:

  1. Phosphate buffered saline (PBS), pH 7.4: 8 g of NaCl, 0.2 g of KH2PO4, 2.8 g of Na2HPO412H2O and 0.2 g of KCl dissolved and made up to 1 L in distilled water.
  2. Antigen: Purified protein diluted to about 100 mg/mL in PBS.
  3. Complete and incomplete Freund's adjuvant.
  4. Two glass luer lock syringes: 2 mL is the best size.
  5. Three-way luer fitting plastic stopcock.
  6. 19-g Needles, 0.7 mmx22 mm Argyle medicut cannula.
  7. Xylene.
  8. Sterile glass universal tubes.
  9. Up to four rabbits about 4-6 months old. Various strains can be used, including half sandy lops or New Zealand whites (see Note 1).

Method:
  1. Take up 1 mL of complete adjuvant in one of the syringes and 1 mL of antigen solution containing approx 100 mg of the antigen in another. Attach both to the plastic connector (Fig. 1), making sure that the tap on the connector is open in such a way that only the two ports connecting the two syringes are open. Repeatedly push the mixture from syringe to syringe until it becomes thick and creamy (at least 5-10 minutes). Push all the mixture into one syringe, disconnect this and attach it to 19-g needle (see Notes 2 and 3).
  2. Ensure that the rabbit to be injected is held firmly, but comfortably. For the primary immunization, inject 500 mL deeply into each thigh muscle and also inject 500 mL into each of two sites through the skin on the shoulders.
  3. Repeat these injections biweekly for a further four weeks, but make the emulsion with incomplete adjuvant.
  4. Ten days after the last injection, test-bleed the rabbits from the marginal ear vein. Hold the animal firmly and gently swab the rear marginal vein with xylene to dilate the vein. Then cannulate the vein with an Argyle medicut cannula and withdraw the needle, leaving the plastic cannula in place. Draw blood out of the cnnula with a syringe until the required amount has been collected. Transfer the collected blood into a sterile glass universal container.
  5. Remove the cannula and stem the blood flow by sustained pressure on the puncture site with a tissue.
  6. Allow the collected blood to clot by letting it stand at room temperature for 2 h and then at 4oC overnight. Separate the serum from the blood by detaching the clot carefully with a spatula from the walls of the container and pouring the liquid into a centrifuge tube. Then centrifuge the clot at 2500g for 30 min at 4oC and remove any expressed liquid. Add this liquid to the clot-free liquid collected previously and centrifuge the whole pooled liquid as described above. Finally, remove the serum from the cell plellet with a Pasteur pipet (see Note 4).
  7. At this stage, test the antiserum using an appropriate assay (see Note 5). If the antibody has the requirements for the use to which it will be put, up to three further bleeds on successive days may be performed. If the antiserum is unsatisfactory, i.e., the reaction in very weak, inject the rabbit again one month after the test bleed, and again test-bleed 10 days after this injection.
  8. Store antibodies in small, preferably sterile, aliquots at a minimum of -20oC. Repeated freezing and thawing should be avoided. For long-term storage, aliquots may be freeze-dried and reconstituted when needed (see Note 6).

Notes:
  1. The production of antibodies in animals must be carried out in strict accordance with the legislation of the country concerned.
  2. Emulsions containing antigens are just as immunogenic to humans as to the experimental animal. Great care should be exercised during all the procedures.
  3. A stable emulsion has been produced when a drop of the preparation does not disperse when placed on water.
  4. Serum should be straw colored: a pink coloration shows that hemolysis has taken place. This should not affect the performance of the antibodies during most assay procedures.
  5. This can be done by the Ouchterlony diffusion technique (see Fig. 2 and ref. 6), by ELISA (see this vol., Chapters 29, 30 and 35), or by Western blot, either using the purified protein or a more complex mixture of proteins containing the antigen of interest separated on an SDS/PAGE gel (see this vol., Chapters 24-28).
  6. Some freeze-dried antisera are difficult to reconstitute, or occasionally may lose activity. Test a small sample before drying the whole batch. Any cloudiness after reconstitution is denatured lipoprotein and can be clarified by centrifugation and does not affect antibody binding.

References:
  1. Cook, N. D. and Peters, T. J., Purification of g glutamyl transferase by phenyl boronate affinity chromatography. Biochim. Biophys. Acta 828, 205-212 (1985)
  2. Catty, D. and Raykundalia, C., Production and quality control of polyclonal antibodies, in Antibodies vol. 1-A Practical Approach (Catty, D., ed.) IRL, Oxford (1988)
  3. Mayer, R. J. and Walker, J. H. (eds.), Immnochemical Methods in Cell and Molecular Biology.Academic, London (1987)
  4. Harlow, E. and Lane, D., Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1988)
  5. Langone, J.J. and Van Vunakis, H., Methods in Enzymology, vol. 93, Academic, New York (1983)
  6. Ouchterlony, O. and Nilsson, L.A., Immunodiffusion and immunoelectrophoresis, in Handbook of Experimental Immunology,3rd Ed. (Weir, D. H., ed.), Blackwell, Oxford, UK, pp.19.1-19.44 (1978)

End Note:
Authors: Jonathan A. Green and Margaret M. Manson, Methods in Molecular Biology, Vol. 10: Immunochemical Protocols Ed.: M. Manson 1992 The Humana Press, Inc. Totowa, NJ