Photoreduction of monoclonal antibodies for conjugation and fragmentation

Biotechniques. 2000 Feb;28(2):318-22, 324-6. doi: 10.2144/00282rr02.

Abstract

The conjugation of enzymes, fluorescent or radioactive labels, cross-linkers and other moieties to antibodies is a commonly performed procedure in biochemical research. Using reduced disulphides, conjugation can be an inconvenient, multistep, time- and material-consuming process. We have developed a reduction technique based on UV irradiation, which lacks these drawbacks. Antibodies are irradiated in a sealed vial for a few minutes by a common laboratory UV source in the presence of stannous ions, following the depletion of atmospheric oxygen. The preparation may subsequently be conjugated with thiol-reactive probes such as maleimide derivatives, with no need for any prior purification or concentration. This simple, rapid and effective reduction and conjugation process results in a fully functional immunoglobulin conjugate that can be used for a variety of biochemical applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / isolation & purification
  • Antibodies, Monoclonal / radiation effects*
  • Biotechnology
  • Chromatography, High Pressure Liquid
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / isolation & purification
  • Immunoglobulin Fab Fragments / radiation effects
  • Mice
  • Oxidation-Reduction
  • Photochemistry
  • Radioligand Assay
  • Sulfhydryl Compounds / analysis
  • Ultraviolet Rays

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Fab Fragments
  • Sulfhydryl Compounds