Production of Polyclonal Antisera

Helen M. Cooper1, Yvonne Patterson2

1 Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.4
DOI:  10.1002/0471142735.im0204s82
Online Posting Date:  August, 2008
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Abstract

Much of modern biology and biochemistry relies on the availability of highly specific antibodies for use in such ubiquitous techniques as immunohistochemistry, ELISAs, immunoprecipitation, and immunoblotting. Thus, the generation of large quantities of specific antibodies directed to proteins or peptides of interest is essential to the success of both basic and applied research programs. In addition, with the advent of antibody‐based proteomic strategies for profiling protein expression and post‐translational modification, a requirement for timely production of specific antibodies has emerged. Polyclonal antibodies derived from animals immunized with purified proteins or peptides are particularly valuable for use in the laboratory. This unit provides protocols for the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. Curr. Protoc. Immunol. 82:2.4.1‐2.4.10. © 2008 by John Wiley & Sons, Inc.

Keywords: polyclonal antiserum; adjuvant; antigen; immunization

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Immunization to Produce Polyclonal Antibodies Using Freund's Adjuvant
  • Alternate Protocol 1: Immunization to Produce Polyclonal Antiserum Using TiterMax
  • Support Protocol 1: Preparation of Serum from Blood
  • Commentary
  • Literature Cited
  • 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
  • 100‐ml beaker
  • Additional reagents and equipment for animal restraint (unit 1.3), parenteral injection (unit 1.6), and blood collection (unit 1.7)
CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced i.d. 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 TiterMax

  • TiterMax #R‐1 (CytRx Corporation, Sigma, or TiterMax; store <24 months at 4°C)
  • 1‐ml plastic syringes

Support Protocol 1: Preparation of Serum from Blood

  • Blood samples (see protocol 1)
  • Wooden applicator stick
  • Sigma 4K15 centrifuge and 11150 rotor or equivalent
  • Additional reagents and equipment for immunoblotting (unit 8.10), immunoprecipitation (unit 8.3), ELISA (unit 2.1), and double‐immunodiffusion assay in agar (unit 2.3)
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Figures

Videos

Literature Cited

Literature Cited
   Aguilar, J.C. and Rodríguez, E.G. 2007. Vaccine adjuvants revisited. Vaccine 25:3752‐3762.
   Benjamin, 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.
   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.
   Koh, Y.T., Higgins, S.A., Weber, J.S., and Kast, W.M. 2006. Immunological consequences of using three different clinical/laboratory techniques of emulsifying peptide‐based vaccines in incomplete Freund's adjuvant. J. Transl. Med. 4:42‐54.
   Larsson, K., Wester, K., Nilsson, P., Uhlén, M., Hober, S., and Wernérus, H. 2006. Multiplexed PrEST immunization for high‐throughput affinity proteomics. J. Immunol. Meth. 315:110‐120.
   Liang, M.T., Davies, N.M., Blanchfield, J.T., and Toth, I. 2006. Particulate systems as adjuvants and carriers for peptide and protein antigens. Curr. Drug Deliv. 3:379‐388.
   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.
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