A solid-phase enzyme-linked assay for vitamin B12

Abstract

A new solid-phase enzyme-linked competitive binding assay for vitamin B12 (cyanocobalamin) is described. The assay is based on the competition between analyte B12 molecules and a glucose-6-phosphate dehydrogenase-vitamin B12 conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solidphase is inversely related to the concentration of B12 in the sample. Under optimized conditions, the method can detect B12 in the range of 3 x 10-1~ x 10-8 M (using 100 #1 sample) with high selectivity over other biological molecules.

Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins

“…This "standard" theory by Giddings et al has advantages because it requires only the densities of particles and the eluent and experimental operation conditions, such as the flow rate, the strength of external field, temperature, and the channel dimensions. The average particle sizes converted by this theory from the retention times measured by using an aqueous solution with 0.1% Aerosol OT (AOT) surfactant as the eluent were generally found to agree well with the values obtained from the suppliers, quasi-elastic light-scattering spectroscopy (QELS), and/or electron microscopy, although SdFFF tended to give sizes somewhat smaller than other techniques (7)(8)(9)(10)(11).…”

“…For this purpose, in accordance with the recommendations of Kabakoff and Greenwood for the antibody-based EMIT type of competitive assay (26), the amount of binder utilized to achieve optimum dose-responses should correspond to that which yields approximately 85 % of the maximum inhibition with a given amount of enzyme-ligand conjugate. Therefore, on the basis of the binder dilution curves shown in Figures 3 and 4, 5 Mg/tube of Jacalin and 13.6 Mg/tube of Con A were used in the subsequent competitive assay studies involving the MDH-galactose and MDHmannose conjugates, respectively.…”

“…It is suitable for the characterization and the fractionation of colloidal particles in the submicron range. The theoretical basis of SdFFF was originally presented by Giddings and his co-workers (4)(5)(6). This "standard" theory by Giddings et al has advantages because it requires only the densities of particles and the eluent and experimental operation conditions, such as the flow rate, the strength of external field, temperature, and the channel dimensions.…”

“…The use of enzyme labels is now commonplace in the development of competitive and noncompetitive binding immunoassays for the detection of a wide variety of biomolecules at trace levels (1)(2)(3)(4)(5). Enzyme-linked competitive binding assays may be classified as either heterogeneous or homogeneous.…”

“…Such modulation of enzyme activity is reversed in an amount proportional to the concentration of analyte ligand present in the sample. In previous work (7)(8)(9)(10)(11)(12), we have demonstrated the theoretical and practical advantages of using endogenous binding proteins rather than antibodies in the development of EMIT type assays for several biomolecules. We now extend this concept further by describing how natural carbohydrate-specific binding lectins can be used in conjunction with enzyme-saccharide conjugates to devise a very simple and rapid homogeneous assay method that can be used to both evaluate the specificity of lectin carbohydrate interactions and selectively detect certain carbohydrates and glycoproteins in solution.…”