Production of Polyclonal Antisera

Helen M. Cooper1, Yvonne Paterson2

1 Ludwig Institute of Cancer Research, Melbourne, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 16.2
DOI:  10.1002/0471143030.cb1602s03
Online Posting Date:  May, 2001
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Abstract

Polyclonal antibody preparations contain multiple antibody molecules recognizing different epitopes on an antigen molecule. Good quality polyclonal antisera are useful for a number of applications–e.g., immunoprecipitation, immunoblotting, and ELISAs–where the binding of more than one antibody molecule per antigen molecule improves the sensitivity of the assay. It is possible to produce polyclonal antibodies in a variety of species, and this make it possible to detect multiple immunoreactions. The protocols in this unit describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters using complete Freund's adjuvant (CFA) or other adjuvants. In addition, the unit presents a method for preparing serum from blood. Polyclonal antipeptide antisera can be produced by substituting carrier‐conjugated peptides for the purified protein antigens.

     
 
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Table of Contents

  • Basic Protocol 1: Immunization to Produce Polyclonal Antibodies Using Freund 's Adjuvant
  • Alternate Protocol 1: Immunization to Produce Polyclonal Antiserum Using Other Adjuvants
  • Support Protocol 1: Preparation of Serum from Blood
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Immunization to Produce Polyclonal Antibodies Using Freund 's Adjuvant

  Materials
  • Rabbit, rat, mouse, or hamster of appropriate strain
  • Complete Freund's adjuvant (CFA; Sigma)
  • 1 to 2 mg/ml purified protein antigen in PBS ( appendix 2A)
  • Incomplete Freund's adjuvant (IFA; Sigma)
  • 50‐ml disposable polypropylene centrifuge tubes
  • 3‐ml glass syringes with 19‐, 21‐, and 22‐G needles
  • Double‐ended locking hub connector (Luer‐Lok, Becton Dickinson) or plastic 3‐way stopcock
CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced intradermally or into the eyes and may cause profound sloughing of skin or loss of sight. Self‐injection can cause a positive TB test and lead to a granulomatous reaction. Use gloves and protective eyewear when handling CFA.

Alternate Protocol 1: Immunization to Produce Polyclonal Antiserum Using Other Adjuvants

  • TiterMax #R‐1 (CytRx; store <24 months at 4°C) or Ribi Adjuvant System (RAS; Ribi ImmunoChem; store at 2° to 8°C and do not freeze)
  • 1‐ml plastic syringes

Support Protocol 1: Preparation of Serum from Blood

  • Blood samples (see protocol 1)
  • Sorvall H‐1000B rotor or equivalent
  • Additional reagents and equipment for immunoblotting (unit 6.2), immunoprecipitation (unit 7.2), ELISA ( appendix 3A), or double‐immunodiffusion assay in agar ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Benjami, D.C., Berzofsky, J.A., East, I.J., Gurd, F.R.N., Hannum, C., Leach, S.J., Margoliash, E., Michael, J.G., Miller, A., Prager, E.M., Reichlin, M., Sercarz, E.E., Smith‐Gill, S.J., Todd, P.E., and Wilson, A.C. 1984. The antigenic structure of proteins: A reappraisal. Annu. Rev. Immunol. 2:67‐101.
   Bennett, B., Check, I.J., Olsen, M.R., and Hunter, R.L. 1992. A comparison of commercially available adjuvants for use in research. J. Immunol. Methods 153:31‐40.
   Deeb, , B.J., DiGiacomo, R.F., Kunz, L.L., and Stewart, J.L. 1992. Comparison of Freund's and Ribi adjuvants for inducing antibodies to the synthetic antigen (TG)‐AL in rabbits. J. Immunol. Methods 152:105‐113.
   Donovan, J. and Brown, P. 1995. Animal health assurance. In Current Protocols in Immunology (J.E. Coligan A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp.1.1.1‐1.1.3. John Wiley & Sons, New York.
   Freund, J., Casals, J., and Hismer, E.P. 1937. Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Proc. Soc. Exp. Biol. Med. 37:509.
   Johnston, B.A., Eisen, H., and Fry, D., 1991. An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits. Lab Anim. Sci. 41:15‐21.
   Klinman, N.R. and Press, J. 1975. The B cell specificity repertoire: Its relationship to definable subpopulations. Transplant Rev. 24:41‐83.
   Mallon, F.M., Graichen, M.E., Conway, B.R., Landi, M.S., Leonard, T.B., and Hughes, H.C. 1991. A comparison of antibody response using a synthetic adjuvant system and Freund's complete adjuvant in rabbits. Am. J. Vet. Res. 52:1503‐1506.
   Maloy, W.L., Coligan, J.E., and Paterson, Y. 1991. Production of antipeptide antibodies. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 9.4.1‐9.4.11. John Wiley & Sons, New York.
   McWilliam, A. and Niemi, S.M. 1988. Freund's adjuvant. Canadian Council on Animal Care Resource. 12:1.
   Smith, D.E., O'Brien, M.E., Palmer, V.J., and Sadowski, J.A. 1992. The selection of an adjuvant emulsion for polyclonal antibody production using a low‐molecular‐weight antigen in rabbits. Lab. Anim. Sci. 42:599‐601.
Key Reference
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  A comprehensive methods book with many modern techniques.
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