ENZYME-IMMUNOASSAY FOR PLASMA 25-HYDROXYVITAMIN D-3 EMPLOYING IMMUNOAFFINITY CHROMATOGRAPHY AS A PRETREATMENT METHOD

An enzyme immunoassay (EIA) of 25-hydroxyvitamin D3 [25(OH)D3] has been developed as a new methodology for measuring its plasma levels. Three anti-25(OH)D3 antibodies elicited against 25(OH)D3 3-hemisuccinate or -hemiglutarate conjugated with bovine serum albumin were used for this purpose. Two enzyme-labeled antigens were prepared by linking these haptens to beta-galactosidase employing the N-succinimidyl ester method. An EIA system, selected from several combinations of the antibodies and labeled antigens, exhibited higher sensitivity and specificity than those of the conventional competitive protein binding assay. However, direct measurement of plasma specimens gave lower values than those obtained from the ones which were pretreated with a Sephadex(R) LH-20 column followed by a normal-phase high-performance liquid chromatography. This problem has been overcome by employing immunoaffinity chromatography (IAC). In IAC the homologous anti-25(OH)D3 antibody with that used in the EIA was immobilized as a pretreatment method. The IAC/EIA system developed in this study afforded reliable plasma 25(OH)D3 levels which were confirmed by serial dilution study and the recovery test. The 25(OH)D3 levels of healthy volunteers in the summer measured by this method were 25.2 +/- 6.2 ng/ml (n = 10).