SPECIFICITY OF THE POLYCLONAL ANTIBODIES RAISED AGAINST A NOVEL 25-HYDROXYVITAMIN D-3-BOVINE SERUM-ALBUMIN CONJUGATE LINKED THROUGH THE C-11 ALPHA-POSITION

To obtain a specific antibody for use in 25-hydroxyvitamin D-3 [25(OH)D-3] immunoassay, a novel hapten-carrier conjugate was prepared by coupling 11 alpha-hemiglutaryloxy-25(OH)D-3 with bovine serum albumin (BSA). Three polyclonal antibodies (Ab(11)) showing high titer and affinity for 25(OH)D-3 (K-a = 0.96-2.6 x 10(9) M(-1)) were elicited in rabbits by repeated immunization with the conjugate. Specificity of the Ab(11) was investigated by cross-reactivities with 11 related compounds in a radioimmunoassay using a tritium-labeled antigen and compared with that of conventional antibodies (Ab(3)) raised against 25(OH)D-3 3-hemiglutarate conjugated with BSA. The Ab(3) could not discriminate the A-ring modified metabolites [1,25(OH)(2)D-3 (87-290%) and 25(OH)D-3 3-sulfate (S) (130-180%)], although the cross-reactivities with the side chain modified metabolites were satisfactorily low [24,25(OH)(2)D-3 (2.3-7.4%), 25(OH)D-2 (less than or equal to 1.1%)]. On the contrary, the Ab(11) easily discriminated 1,25(OH)(2)D-3 (0.10-2.4%) and 25(OH)D-3 3S (<0.3%), whereas significant cross-reactivities were found with 24,25(OH)(2)D-3 (110-120%) and 25,26(OH)(2)D-3 (66-130%) having a dihydroxylated side chain. These results show that the Ab(11) are complementary to the A-ring portion of the 25(OH)D-3 molecule which is opposite from the side chain structure recognized by the Ab(3). Thus, the Ab(11) will compensate for insufficient specificity of the Ab(3) and are expected to be a useful tool for the pretreatment of biological samples in the development of various analyses of vitamin D metabolites including specific 25(OH)D-3 immunoassays using the Ab(3).