Production of Polyclonal Antisera

Helen M. Cooper1, Yvonne Paterson2

1 Queensland Brain Institute and University of Queensland, Brisbane, Australia, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Appendix 3I
DOI:  10.1002/0471142956.cya03is43
Online Posting Date:  January, 2008
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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 against 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 been exemplified. Polyclonal antibodies derived from animals immunized with purified proteins or peptides are particularly valuable for use in the laboratory. This appendix provides protocols for the production of polyclonal antisera specific for protein antigens using rabbits, rats, mice, and hamsters. Curr. Protocol. Cytom. 43:A.3I.1‐A.3I.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
  • Figures
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Basic Protocol 1: Immunization to Produce Polyclonal Antibodies Using Freund's Adjuvant

  • 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 Titermax

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

Support Protocol 1: Preparation of Serum from Blood

  • Blood samples (see protocol 1)
  • Sigma 4K15 centrifuge and 11150 rotor (or equivalent)
  • Additional reagents and equipment for immunoblotting (Gallagher et al., ), immunoprecipitation (Bonifacino et al., ), and ELISA (Hornbeck, )
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Literature Cited

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Key Reference
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   Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M. and Strober, W. (eds.) 2007. Current Protocols in Immunology. John Wiley & Sons, Hoboken, N.J.
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