ABSTRACT
p-Maleimidophenyl isocyanate (PMPI, 1) is a heterobifunctional cross-linking agent useful for thiol to hydroxyl coupling. Several maleimide-activated compounds were prepared and characterized and then shown to be reactive with thiol-containing proteins. Examples include activation of vitamin B12, digoxigenin, digitoxigenin, estradiol, progesterone, and some serine-containing peptides.
Subject(s)
Cross-Linking Reagents/chemistry , Cyanates/chemistry , Maleimides/chemistry , Sulfhydryl Compounds/chemistry , Alkaline Phosphatase/chemistry , Amino Acid Sequence , Cross-Linking Reagents/chemical synthesis , Cyanates/chemical synthesis , Digoxigenin/chemistry , Drug Stability , Estradiol/chemistry , Haptens/chemistry , Maleimides/chemical synthesis , Molecular Sequence Data , Peptides/chemistry , Vitamin B 12/chemistryABSTRACT
A fluorometric enzyme immunoassay using an alkaline phosphatase-conjugated monoclonal antibody was developed to quantitate feline leukemia virus (FeLV) infection. Monoclonal antibodies, directed against the FeLV structural protein p27, were conjugated with alkaline phosphatase using a modified maleimide method. The enzyme immunoassay requires only 4 days to reproducibly measure FeLV production instead of the 12 days required for the commonly used transformation assay using C81 cells. A linear correlation was found between the virion associated reverse transcriptase activity and the amount of intracellular p27 as determined by the fluorometric enzyme immunoassay. An immunocytochemical assay using the same conjugated monoclonal with a different substrate gave visible plaques in infected cell monolayers and was therefore used to titrate FeLV in plaque-forming units. The results obtained by all the procedures followed single hit kinetics for FeLV infection. The fluorometric enzyme immunoassay was adapted to measure FeLV neutralizing antibodies, allowing a sensitive and accurate determination of neutralizing titers.
Subject(s)
Fluorometry , Immunoenzyme Techniques , Leukemia Virus, Feline/analysis , Neutralization Tests , Animals , Antibodies, Monoclonal , Cats , Cell Line , Fibroblasts/analysis , Fluorometry/methods , Gene Products, gag , Immunohistochemistry , Leukemia Virus, Feline/immunology , Neutralization Tests/methods , Retroviridae Proteins/analysis , Viral Plaque Assay/methodsABSTRACT
Medium-performance anion-exchange chromatography was applied to the purification of murine IgG class monoclonal antibodies from ascites fluid. The separations were performed under mild conditions at pH 8 using relatively low sodium-chloride concentrations. Recoveries for monoclonal antibodies of subclasses IgG1, IgG2a, and IgG2b were about 90%. The IgG preparations were free of other ascites fluid proteins.
