Sulfhydryl site-specific cross-linking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation cross-link reagent.
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 alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees 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 1b 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 1a 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.
del Rosario
R B
RB
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109-0028.
Wahl
R L
RL
Brocchini
S J
SJ
Lawton
R G
RG
Smith
R H
RH
D000098:Acetophenones / Q000138:chemical synthesis; D000477:Alkylating Agents; D000818:Animals; D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D014018:Tissue Distribution
D000098:Acetophenones; D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007457:Iodine Radioisotopes; D013438:Sulfhydryl Compounds