Production of Anti‐Peptide Antisera

John Mark Carter1

1 null, Kensington, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.3
DOI:  10.1002/0471142735.im0903s55
Online Posting Date:  August, 2003
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Abstract

The protocols described in this unit can be used for generating and testing anti‐peptide antisera, assuming that a scientist has already selected and conjugated peptides for experimental vaccines. Described are immunization and bleeding of experimental animals, a simple ELISA for antiserum screening, and affinity purification of the serum, including preparation of columns.

Keywords: affinity chromatography; antibody; anti‐peptide; antiserum; ELISA; immunization; peptide; purification; vaccine

     
 
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Table of Contents

  • Basic Protocol 1: Immunization Schedule for Producing Anti‐Peptide Sera in Rabbits
  • Basic Protocol 2: Indirect Elisa to Determine Anti‐Peptide Antibody Titer
  • Alternate Protocol 1: Elisa with Biotinylated Peptide
  • Basic Protocol 3: Using an Affinity Column with Acidic, Basic, or Chaotropic Elution
  • Support Protocol 1: Preparation of Peptide Immunoaffinity Column Using Preswollen NHS‐ or CNBr‐Activated Resins
  • Support Protocol 2: Preparation of Peptide Affinity Column Using a Thiol Resin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Immunization Schedule for Producing Anti‐Peptide Sera in Rabbits

  Materials
  • Rabbits
  • Peptide‐carrier conjugate (unit 9.2)
  • PBS, pH 7.5 (see recipe)
  • Freund's complete adjuvant (FCA; Pierce Biotechnology)
  • Freund's incomplete adjuvant
  • Additional reagents and equipment for blood collection (unit 1.7), immunization and bleeding of rabbits (unit 2.4), obtaining antibody titer (see protocol 2 or protocol 3), and immunoaffinity chromatography (see protocol 4; optional)
NOTE: Refer to Chapter 1 for information concerning care and maintenance of rabbits.

Basic Protocol 2: Indirect Elisa to Determine Anti‐Peptide Antibody Titer

  Materials
  • 0.2 to 2.5 µM synthetic peptide in 0.l M sodium carbonate (see recipe)
  • Blocker solution (see recipe)
  • PBST (see recipe)
  • Anti‐peptide antiserum (see protocol 1)
  • Positive (e.g., serum) and negative (e.g., blocker solution) controls
  • Probe conjugate: goat anti‐rabbit globulin conjugated to alkaline phosphatase (Kirkegaard & Perry Laboratories)
  • Alkaline phosphatase substrate solution (see recipe)
  • 96‐well, flat‐bottom, optically‐clear, polystyrene microtiter plates
  • Microplate absorbance reader with filter/monochromator for 405 to 415 nm

Alternate Protocol 1: Elisa with Biotinylated Peptide

  • 2.5 µg/ml streptavidin in PBS or 0.1 M sodium carbonate (see reciperecipes)
  • 1 µg/ml biotinylated peptide in PBS or carbonate buffer

Basic Protocol 3: Using an Affinity Column with Acidic, Basic, or Chaotropic Elution

  Materials
  • 50 mM potassium phosphate (pH 7.5)/0.4 M NaCl with and without azide (see recipe)
  • Antiserum known to contain anti‐peptide antibodies (see protocol 1Basic Protocols 1 and protocol 22)
  • Neutralization buffer: 1 M Tris·Cl, pH 7.5 ( appendix 2A)
  • Elution buffer (choose one of the following):
    • Acidic elution buffer: 0.1 M glycine (pH 2.8)/0.15 M NaCl, 0.1 M acetic acid, or 0.1 M sodium citrate, pH 3.0 (see recipe)
    • Basic elution buffer: 0.1 M diethanolamine, pH 9.8 (see recipe)
    • Chaotropic elution buffer: 4 M sodium isothiocyanate, pH 7.5, 6 M urea, pH 7.5, 5 M guanidine·HCl, pH 7.5 (see recipe)
  • PBST (see recipe)
  • Affinity column (see protocol 5Support Protocols 1 and protocol 62)
  • 0.45‐µm syringe filter with 3‐ or 5‐ml syringe with Luer lock
  • Additional reagents and equipment for dialysis ( appendix 3H)
NOTE: 50 mM potassium phosphate buffer (pH 7.5)/0.4 M NaCl with azide should only be used for column storage.

Support Protocol 1: Preparation of Peptide Immunoaffinity Column Using Preswollen NHS‐ or CNBr‐Activated Resins

  Materials
  • Peptide bearing a free amino group
  • N,N‐dimethylformamide (DMF)
  • CNBr‐activated Sepharose 4 FF (Amersham Pharmacia Biotech) or
  • NHS‐preactivated resin:
    • Affi‐Gel 10 (BioRad; for neutral and basic peptides) or
    • Affi‐Gel 15 (BioRad) or NHS‐activated Sepharose 4 FF (Amersham Pharmacia Biotech; for acidic peptides)
  • 1 M ethanolamine·HCl, pH 8 (see recipe)
  • 50 mM potassium phosphate (pH 7.5)/0.4 M NaCl with and without sodium azide (see recipe)
  • 15‐ml nonpolystyrene centrifuge tube with cap
  • 30‐ml Glass‐fritted funnel with medium frit or 30‐ml Buchner funnel
  • 1 × 10–cm glass or plastic chromatography column
NOTE: The manufacturers of chromatography resins provide detailed instructions for use of their products. The protocols included here are simplified versions of those methods. Users of the products described in these protocols are advised to consider the literature provided by the manufacturer.NOTE: 50 mM potassium phosphate buffer (pH 7.5)/0.4 M NaCl with azide should only be used for column storage.

Support Protocol 2: Preparation of Peptide Affinity Column Using a Thiol Resin

  Materials
  • Peptide with reduced disulfide bonds (unit 9.2)
  • PBE (see recipe)
  • SulfoLink (Pierce) or thiopropyl Sepharose 4B (Amersham Pharmacia) resin
  • Cysteine
  • 50 mM potassium phosphate buffer, pH 7.5/0.4 M NaCl (see recipe)
  • 30‐ml glass‐fritted funnel with medium frit or 30‐ml Buchner funnel
  • 15‐ml tube with cap
  • 1 × 10–cm glass or plastic chromatography column
NOTE: 50 mM potassium phosphate buffer (pH 7.5)/0.4 M NaCl with azide should only be used for column storage.
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Figures

Videos

Literature Cited

   Engvall, E. and Perlman, P. 1971. Enzyme‐linked immunosorbent assay: Quantitative assay of immunoglobulin G. Immunochemistry 8: 871‐879.
   Geerligs, H.J., Weijer, W.J., Bloemhoff, W., Welling, G.W., and Welling‐Wester. S. 1988. The influence of pH and ionic strength on the coating of peptides of herpes simplex virus type 1 in an enzyme‐linked immunosorbent assay. J. Immunol. Methods 106:239‐244.
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Sutcliffe, J.G., Schinnick, T.M., Green, N., Liu, F.‐T., Niman, H.L., and Lerner, R.A. 1980. Chemical synthesis of a polypeptide predicted from a nucleotide sequence allows detection of a new retroviral gene product. Nature 287:801‐805.
   Van Regenmortel, M.H. 1994. The recognition of proteins and peptides by antibodies. In Immunochemistry (C.J. van Oss and M.H. Van Regenmortel, eds.) pp. 227‐300. Marcel Dekker, N.Y.
   Walter, G., Scheidtmann, K.‐H., Carbone, A., Laudano, A.P., and Doolittle, R.F. 1980. Antibodies specific for the carboxy‐ and amino‐terminal regions of simian virus 40 Large tumor antigen. Proc. Natl. Acad. Sci. U.S.A. 77:5197‐5200.
Key References
   Van Regenmortel, 1994. See above.
  Excellent detailed procedures for conjugation and immunization.
   Harlow and Lane, 1988. See above.
  Comprehensive discussion of immunochemistry methods.
Internet Resources
   http://www.biorad.com/webmaster/pdfs/9115_Activated_Affinity.pdf
  Instruction Manual for Affi‐Gel 10 and Affi‐Gel 15.
   http://www.piercenet.com/files/ACF20A5.pdf
  Instructions for Pierce SulfoLink Gel.
   http://www.bio.com/protocolstools/
  BioProtocol website with many detailed protocols for biotechnology.
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