Boc-O-methyl-L-serine N-hydroxysuccinamide ester

This article describes the synthesis of labels and hapten-protein conjugates for use in bio- and chemiluminescent immunoassay systems, together with the problems encountered. The effects of maleimide upon acetate-, adenylate- and pyruvate kinase activity have been studied, as well as upon the luciferin-luciferase monitoring system. Maleimide inhibited both acetate and adenylate kinase but showed no inhibition of pyruvate kinase and the monitoring reagent. Four heterobifunctional reagents were tested for their capability in forming pyruvate kinase-donkey-anti-rabbit IgG conjugates which retained enzyme and antibody activity. The best results were obtained with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and succinimidyl-6-(p-maleimidophenyl)-hexanoate. The relationship between the amounts of succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and IgG was studied with respect to enzymic activity of the conjugate. The Michaelis-Menten constants for both conjugated and non-conjugated pyruvate kinase were calculated and compared. It was found that the maximal velocity (Vmax) of the conjugated enzyme was lower than that of the non-conjugated enzyme although the "apparent" Km value was the same for both conjugated and non-conjugated pyruvate kinase. The pyruvate kinase-anti rabbit IgG conjugate was tested for its ability to bind to rabbit-IgG coated polystyrene balls. In addition to bioluminescent labels, the synthesis of chemiluminescent markers was undertaken and optimised. The three substances used for labelling were diazoluminol, diazoisoluminol and N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide the latter being used as an N-hydroxysuccinamide "active" ester. The ratio of label to IgG was studied for diazoluminol and N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide active ester after it had been discovered that diazoisoluminol was not suitable for coupling to antibodies. The optimal molar ratios label: IgG were for diazoluminol 40:1 and for N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide active ester 60:1. Increasing the substitution rate led to a lessening of the dynamic range, shown by an increase in the ratio between unspecific binding (noise) to maximal binding (signal) in an assay. The synthesis of hapten-protein conjugates for covalent coupling to polystyrene balls was undertaken as this formed part of the preparation for the assays described in Part III. The optimal production of gentamicin-bovine serum albumin and thyroxine-transferrin conjugates has been described in detail.

Objective: Fluorophore-labeled contrast imaging agents are moving toward clinical use for a number of applications. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. To prepare for clinical use as a near-infrared fluorophore, a toxicity study was conducted on IRDye 800CW carboxylate. Methods: Male and female Sprague-Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; Indocyanine Green was used as a comparative control. Animals were injected with varying doses of the test and control articles and observed for up to 14 days. Clinical chemistry, hematological, and pharmacokinetic analyses were performed on subgroups of animals. Organs were analyzed for content of the test article. Tissues were analyzed microscopically for pathological changes. Results: Based on hematologic, clinical chemistry, and histopathologic evaluation, single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5, and 20 mg/kg or 20 mg/kg intradermally produced no pathological evidence of toxicity. Conclusion: A dose of 20 mg/kg was identified as the no observed adverse effect level following IV or ID routes of administration of IRDye 800CW.

RIAs have a number of advantages which make them ideally suited for use in diagnostic microbiology. These advantages include sensitivity, objectivity, and versatility. However, the widespread application of RIAs has been limited by the instability of the reagents required for the performance of available solid-phase RIAs. The relatively short half-life of gamma-emitting isotopes is particularly a problem in cases where multiple antigens must be assayed, since distinct radioactively labeled reagents are required for each antigen to be measured. The problems associated with the use of standard RIAs could be avoided if the specific immunoglobulin directed at the antigen were labeled with a stable, nonradioactive isotope and if a generally reactive radioactive ligand were bound in a subsequent reaction. We have thus developed RIA systems that use immunoglobulin linked with biotin by reaction with biotin N-hydroxysuccinamide ester [1]. The biotin bound to the solid phase is subsequently measured by reaction with unlabeled avidin and 3H-labeled biotin (New England Nuclear Corp, Boston). The reaction is quantitated by the measurement of tritiated biotin in a standard scintillation counter. This reaction format takes advantage of the stability of biotin-immunoglobulin conjugates, the high affinity of biotin to avidin, and the fact that a single molecule of avidin can react with four molecules of biotin [2]. We devised an avidin-biotin RIA that uses goat and guinea pig antisera directed at human rotavirus and used it to detect rotavirus in 44 stool specimens obtained from children with acute gastroenteritis during the winter months [3].(ABSTRACT TRUNCATED AT 250 WORDS)