Conjugation of Enzymes to Antibodies

Scott E. Winston1, Steven A. Fuller1, Michael J. Evelegh1, John G.R. Hurrell2

1 ADI Diagnostics, Rexdale, Ontario, 2 Boehringer Mannheim, Indianapolis, Indiana
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.1
DOI:  10.1002/0471142727.mb1101s50
Online Posting Date:  May, 2001
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Abstract

Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen‐specific monoclonal or polyclonal antibody in which neither the antigen‐combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross‐linking HRPO, urease or alkaline phosphatase to immunoaffinity‐purified monoclonal or polyclonal antibodies (IgG).

     
 
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Table of Contents

  • Immunoassays
  • Basic Protocol 1: Conjugation of Horseradish Peroxidase to Antibodies
  • Alternate Protocol 1: Conjugation of Urease to Antibodies
  • Alternate Protocol 2: Conjugation of Alkaline Phosphatase to Antibodies
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Conjugation of Horseradish Peroxidase to Antibodies

  Materials
  • 1 mg/ml antibody solution (affinity‐purified polyclonal or monoclonal antibodies; unit 11.11)
  • recipe0.1 M phosphate buffer, pH 6.8
  • Horseradish peroxidase (HRPO; Sigma Type VI #P8375)
  • recipe0.1 M carbonate buffer, pH 9.2
  • recipeSodium periodate (NaIO 4) solution, freshly prepared
  • recipeSodium borohydride (NaBH 4) solution, freshly prepared
  • recipeSaturated ammonium sulfate [(NH 4) 2SO 4] solution
  • recipeTris/EDTA/NaCl (TEN) buffer, pH 7.2
  • Bovine serum albumin (BSA)
  • Glycerol
  • Dialysis membrane (see and appendix 3)
  • Pasteur pipet fitted with glass wool
  • Sephadex G‐25, medium (size of gel matrix)

Alternate Protocol 1: Conjugation of Urease to Antibodies

  Additional Materials
  • 20 mg/ml urease ( Sigma Type VII #U0376; source is important) in recipe0.1 M phosphate buffer
  • recipem‐Maleimidobenzoyl N‐hydroxysuccinimide ester in dimethylformamide (MBS/DMF solution)
  • 0.143 M 2‐mercaptoethanol (prepare from 14.3 M stock)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 12 × 75–mm glass tubes
  • 1.5 × 5–cm PD‐10 column ( Pharmacia)
  • Nitrogen tank

Alternate Protocol 2: Conjugation of Alkaline Phosphatase to Antibodies

  Additional Materials
  • 5 mg/ml antibody solution (affinity‐purified polyclonal or monoclonal antibodies, unit 11.11)
  • 10 mg/ml alkaline phosphatase (enzyme immunoassay grade; Boehringer Mannheim; source is important)
  • 25% glutaraldehyde in H 2O
  • recipeTris/ovalbumin solution
  • Sodium azide
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Figures

  •   FigureFigure 11.1.1 Conjugation of horseradish peroxidase (HRPO) to antibody (IgG) using the periodate oxidation method. The method involves three chemical steps: (1) sodium periodate (NaIO4) oxidation of the carbohydrate side chains of HRPO, (2) Schiff base formation between activated peroxidase and amino groups of the antibody, and (3) sodium borohydride (NaBH4) reduction of the Schiff base to form a stable conjugate.
  •   FigureFigure 11.1.2 Conjugation of urease to antibody (IgG) with m‐maleimidobenzoyl N‐hydroxysuccinimide ester (MBS). The first step involves benzoylation of the amino groups of the antibody (IgG) oxidate oxidation method. The second step involves the thiolation of the maleimide moiety by the sulfhydryl groups of the urease enzyme.

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Literature Cited

Literature Cited
   Healey, K., Chandler, H.M., Cox, J.C., and Hurrell, J.G.R. 1983. A rapid semi‐quantitative capillary enzyme immunoassay for digoxin. Clin. Chim. Acta. 134:51‐58.
   Jeanson, A., Cloes, J.‐M., Bouchet, M., and Rentier, B. 1988. Comparison of conjugation procedures for the preparation of monoclonal antibody‐enzyme conjugates. J. Immunol. Methods. 111:261‐270.
   Nakane, P.K. and Kawaoi, A. 1974. Peroxidase‐labeled antibody. A new method of conjugation. J. Histochem. Cytochem. 22:1084‐1091.
   Tijssen, P. and Kurstak, E. 1984. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase‐antibody conjugates for enzyme immunoassay. Anal. Biochem. 136:451‐457.
   Voller, A., Bidwell, D.E., and Barlett, A.1976. Enzyme immunoassays in diagnostic medicine. Bull. W.H.O. 53:55‐65.
Key Reference
   Van Vunakis, H. and Langone, J.J., eds. 1980. Immunochemical techniques. Methods Enzymol. 70:1‐525.
  An excellent collection of articles on immunoassay techniques, including several on enzyme‐antibody conjugation techniques.
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