Article

A solid-phase enzyme-linked assay for vitamin B12

Department of Chemistry University of Kentucky 40506 Lexington KY USA
Microchimica Acta (Impact Factor: 3.74). 12/1988; 97(1):65-73. DOI: 10.1007/BF01197285
Source: OAI

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 solid-phase is inversely related to the concentration of B12 in the sample. Under optimized conditions, the method can detect B12 in the range of 310–10–110–8
M (using 100l sample) with high selectivity over other biological molecules.

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    • "The immobilized phase of the assay was prepared by coupling CN-Cbl to keyhole limpet hemocyanin (KLH) with the bromoacetyl bromide procedure , which improved the sensitivity compared with earlier work. A solid phase enzyme-linked competitive binding assay for CN- Cbl was described by Tsalta and coworkers in 1989 based on the competition between the analyte and glucose-6-phosphate dehydrogenase–vitamin B 12 conjugate for a limited number of R-protein binding sites immobilized on Sepharose particles [81]. Sample pretreatment with denaturing agents and neutralizing deactivating agents and a vitamin B 12 enzyme immunoassay was described by Watkins and coworkers in 1992 [82]. "
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    ABSTRACT: The use of two enzyme labels in a dual analyte simultaneous heterogeneous enzyme-linked competitive binding assay is examined. Reaction conditions for monitoring glucose-6-phosphate dehydrogenase (G6PDH) and [beta]-galactosidase ([beta]-gal) activities are found which enable the independent measurement of each enzyme activity in the presence of the other. The two enzymes are used as labels in the development of a model simultaneous heterogeneous competitive binding assay for detecting the biotin and vitamin B12 content of vitamin tablets. The simultaneous assay is shown to exhibit analytical dose-response characteristics comparable to those of the single analyte assays. The potential use of the G6PDH/[beta]-gal enzyme pair for devising other dual analyte competitive and non-competitive binding assays is discussed. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/31186/1/0000087.pdf
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