A Method for the Determination of Human Serum Phenobarbital Based on a Competitive Binding Enzyme-Immunoassay

概要

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A competitive binding enzyme-immunoassay (EIA) for phenobarbital (PB) in human sera has been devised. An enzyme labeled antigen, β -D-Galactosidase-m-diazo-phenobarbital (13-GAL-PB) was prepared by reacting m-diazonium chloride of PB with 13-galactosidase of E. coll. The insolubilized antibody was prepared by combining yeast cell walls with immunoglobulins from sera of rabbits immunized with bovine serum albumin-m-diazo-PB. After 60 min competitive immunoreaction between the β P-GAL-PB and PB (antigen in samples) for the insolubilized antibody, the enzyme activity of free or bound β -GAL-PB in the supernatant or the precipitate separated by centrifugation, was measured by using o-nitrophenyl-β -D-galac-topyranoside as a chromogenic substrate. One to 400 ng of PB in 5 μl of serum samples (=0.25-80μ /g/ml of serum) could be successfully determined within 2 hours. Concentrations of PB in 106 sera from epileptic patients measured by the EIA and the conventional gas liquid chromatography method were found to be correlated well (r=0.95, Y=0.96 x +1.08). The method is very simple and can be practically used without any special equipments in the ordinal clinical laboratories.
Japan Society of Clinical Chemistryの論文