Sulfhydryl Site-Specific Cross-Linking and Labeling of Monoclonal Antibodies by a Fluorescent Equilibrium Transfer Alkylation Cross-Link Reagent

Renato B. del Rosario, Richard L. Wahl, Stephen J. Brocchini, Richard G. Lawton, Richard H. Smith

Research output: Contribution to journalArticle

Abstract

The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of “equilibrium transfer alkylation cross-link (ETAC) reagents” is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with a,a-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(α,α-bis(p-tolylsulfonylmethyl) acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from lb indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (~ 23–34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 °C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3–4 A. Flow cytometry of the ETAC lb 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC la product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.

Original languageEnglish (US)
Pages (from-to)51-59
Number of pages9
JournalBioconjugate Chemistry
Volume1
Issue number1
DOIs
StatePublished - Jan 1 1990

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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