PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES AGAINST A NOVEL 1-ALPHA,25 DIHYDROXYVITAMIN D-3-BOVINE SERUM-ALBUMIN CONJUGATE LINKED THROUGH THE 11-ALPHA-POSITION

To obtain an antibody which is useful for developing a selective immun oaffinity chromatography (IAC) of 1 alpha,25-dihydroxyvitamin D-3 [1,2 5(OH)(2)D-3], which is an effective pretreatment tool for determining this metabolite in biological fluids,a variety of monoclonal antibodie s have been produced against a novel haptenic derivative, 11 alpha-hem iglutaryloxy-1 alpha,25-dihydroxyvitamin D-3, Linked to bovine serum a lbumin. BALB/c, A/J and C57BL/6 mice, as well as Wistar and SD rats, w ere immunized with the hapten-carrier conjugate and the titer of the a nti-1,25(OH)(2)D-3 antibody in serum was examined. Spleen cells from A /J mice or SD rats, which showed a high titer, were fused with P3/NS1/ 1-Ag4-1 myeloma cells. After serial screening by enzyme-linked immunos orbent assay and radioimmunoassay, sixteen kinds of hybridoma clones s ecreting different anti-1,25(OH)(2)D-3 antibodies were established. Sc atchard analysis revealed that 12 antibodies out of 16 have enough aff inity to 1,25(OH)(2)D-3 (K-a>10(9) M-1). By a cross-reaction study usi ng various related compounds, two antibodies, M114 and M129, both deri ved from A/J mice, were found to have suitable specificity. Although t hey showed relatively high cross-reactivities with the 1,25(OH)(2)D-3 derivatives having 24-hydroxylated (both 20%) or lactonized (30%, 8.9% ) side chain, all the tested 1 alpha-deoxy-type metabolites, some of w hich interfere with the 1,25(OH)(2)D-3 assays, were successfully discr iminated [25(OH)D-3: 0.67%, 0.40%; 24,25(OH)(2)D-3: 0.27%, 0.24%; 25(O H)D-3 3-sulfate: 0.11%, 0.14%]. These monoclonal antibodies will be ap plicable for developing not only IAC but also various immunoassay proc edures. (C) 1997 Elsevier Science Ltd. All rights reserved.