Some observations on the measurement of ‘binding’ of cyanocobalamin by ‘intrinsic factor’ preparations

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Abstract
Cyanocobalamin bound to extracts of pig pyloric mucosa (intrinsic-factor concentrates) is active for Escherichia coli in plate-assay techniques; therefore the use of plate assays, and possibly other assays, with microorganisms for measuring binding activity is unsound. The ultrafiltration method, previously described by us, for measuring binding capacity was extended by the use of radioactive cyanocobalamin. Further evidence of the reliability of this method is presented and its advantages over the dialysis method are discussed. The technique is not subject to interference by such substances as lysozyme, heparin, chondroitin sulfate and nucleic acids, as are methods depending on uptake of free vitamin by bacteria. The cyanocobalamin-protein complex is stable to dialysis and only slowly exchanges its bound vitamin in vitro, even in the presence of a large excess of free cyanocobalamin.