An Enzyme-Linked Immunosorbent Assay (ELISA) for Pantothenate

An enzyme-linked immunosorbent assay (ELISA) for pantothenate has been developed. Antibodies induced in rabbits against bovine serum albumin-pantothenate conjugate were specifically purified by affinity chromatography. This process served to reduce the amount of endogenous pantothenate attached to the antibody, as well as to purify the antibody. The purified antibodies were covalently linked to alkaline phosphatase (Sigma type VII) with glutaraldehyde (0.05% aqueous solution). An immobilized pantothenate substrate was first obtained by attaching human serum albumin-pantothenate conjugate to the surface of polystyrene culture tubes by passive adsorption. The binding of the enzyme labelled antibody (E-AB} to this substrate is proportionately inhibited by free pantothenate as standards or as samples for analysis. The inhibition of E-AB immobilization was quantitated at 405 nm by the hydrolysis of p-nitrophenyl phosphate as indicated by the formation of p-nitrophenol. A standard curve was plotted on log logit paper, and was linear in the range of 2 through 1000 ng pantothenate. Initial experiments show that the ELISA will be useful in assessing pantothenate in deproteinized blood samples and in food extracts