Specificity of polyclonal antibodies raised against a novel 24,25-dihydroxyvitamin D-3-bovine serum albumin conjugant linked through the C-11 alpha or C-3 position

Novel hapten-carrier conjugants were prepared by coupling 11 alpha-hemiglutaryloxy-(24R)-24,25-dihydroxyvitamin D-3 or (24R)-23,25-dihydroxyvitamin D-3 [24,25(OH)(2)D-3] 3-hemiglutarate with bovine serum albumin (BSA), to obtain an antibody with high specificity and affinity for use in 24,25(OH)(2)D-3 immunoassay. The polyclonal antibodies showing high titre were each elicited in three or four rabbits against these two conjugants; the antibodies obtained from the former and the latter conjugants were expressed as Ab11 and Ab3, respectively. These had a much higher affinity for 24,25(OH)(2)D-3 than that of the vitamin D binding protein (DBP). Specificity of the antibodies was investigated by crossreactivities with 11 related compounds in a radioimmunoassay (RIA) system. The Ab11 well discriminated the 1 cc-hydroxylated metabolites such as 1,24,25(OH)(3)D-3 (less than or equal to 0.69%) and 1,25(OH)(2)D-3 (less than or equal to 0.25%), but significantly crossreacted with some side chain modified compounds such as (24S)-24,25-dihydroxyvitamin D-3 [24S,25(OH)(2)D-3] (greater than or equal to 67%), 25(OH)D-3 (greater than or equal to 14%) and 25,26(OH)(2)D-3 (greater than or equal to 23%). On the other hand, the Ab3 showed only negligible crossreactivities with the compounds having a different side chain structure such as 24S,25(OH)(2)D-3 (less than or equal to 3.0%), 25(OH)D-3 (<0.3%) and 25,26(OH)(2)D-3 (less than or equal to 0.53%). A significant crossreaction was found only with 1,24,25(OH)(3)D-3 (greater than or equal to 68%). These results demonstrated that the Ab3 are promising for developing an immunoassay system which is much more specific and sensitive than conventional competitive protein binding assays based on DBP. (C) 1997 Elsevier Science Ltd.