Comparison of two methods of preparing enzyme-antibody conjugates: Application of these conjugates for enzyme immunoassay

✍ Scribed by Michael J. O'Sullivan; Ernesto Gnemmi; David Morris; Giorgio Chieregatti; Alan D. Simmonds; Madeleine Simmons; James W. Bridges; Vincent Marks

• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 783 KB
• Volume: 100
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90117-9

✦ Synopsis

Two methods of preparing enzyme-antibody conjugates were evaluated. High yields of conjugate were obtained with both methods. The first procedure utilizes the homobifunctional crosslinking reagent N,N'-o-phenylenedimaleimide.

Sulfydryl residues were introduced into second antibodies by reaction with methyl-mercaptobutyrimidate.

The modified antibodies were reacted with N&'-o-phenylenedimaleimide, excess reagent was removed by gel filtration, and the activated antibodies were cross-linked to /3-galactosidase. Up to 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of enzyme activity. The second method utilizes the heterobifunctionalmeta-maleimidobenzoyl-~-hydroxysuccinimide ester (MBS). The antibodies were reacted with MBS, excess reagent was removed by gel filtration and the activated antibodies were crosslinked to &galactosidase. Typically, approximately 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of both enzyme and antibody activity. Conjugates prepared using these two procedures were used as labels in an immunoassay system and were able to detect approximately 5 to 10 ng of first antibodies. The MBS procedure was simpler to perform, could more easily be adapted to large-scale work, and gave more reproducible results, and the conjugates produced were able to detect slightly lower concentrations of first antibody.

Enzymes have been used as labels in immunohistochemistry and in enzyme immunoassay (EIA)2 for a number of years. These labels are generally considered to provide less assay sensitivity than radiolabels. However, several workers have recently shown that when sufficient attention is given to the choice of enzyme, crosslinking method, and the detection system, very sensitive EIAs can be devised (l-4).