Antibody coating approach involving gamma globulins from non-immunized animal and second antibody antiserum

J Immunoassay. 1998 Nov;19(4):271-93. doi: 10.1080/01971529808005486.

Abstract

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins from non-immunized animal of the same species as that from which the antigen-specific antibody has been developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antigen specific antibody is immunoadsorbed on the immobilized second antibody. We found that the coating protocol developed allows the use of antigen-specific and second antibody antisera dilutions, thus avoiding the need for affinity purification of antibodies. Additionally, it provides solid-phase second antibody with increased binding capacity compared to the directly adsorbed onto the solid second antibody. The advantages of the proposed coating protocol were demonstrated through the development of a solid-phase radioimmunoassay for the determination of total triiodothyronine in human serum samples.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Antibodies / blood
  • Antibodies / immunology*
  • Antibody Specificity
  • Binding Sites, Antibody / immunology
  • Biomarkers / blood
  • Humans
  • Hydrogen-Ion Concentration
  • Immunization*
  • Immunoglobulin G / blood*
  • Immunoglobulin G / immunology
  • Immunosorbent Techniques
  • Osmolar Concentration
  • Polystyrenes
  • Rabbits
  • Radioimmunoassay / methods
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Temperature
  • Time Factors
  • Triiodothyronine / blood
  • Triiodothyronine / immunology

Substances

  • Antibodies
  • Biomarkers
  • Immunoglobulin G
  • Polystyrenes
  • Triiodothyronine