Production and characterization of monoclonal antibodies against a novel 1alpha,25-dihydroxyvitamin D3-bovine serum albumin conjugate linked through the 11alpha-position.
To obtain an antibody which is useful for developing a selective immunoaffinity chromatography (IAC) of 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is an effective pretreatment tool for determining this metabolite in biological fluids, a variety of monoclonal antibodies have been produced against a novel haptenic derivative, 11alpha-hemiglutaryloxy-1alpha,25-dihydroxyvitamin D3, linked to bovine serum albumin. BALB/c, A/J and C57BL/6 mice, as well as Wistar and SD rats, were immunized with the hapten-carrier conjugate and the titer of the anti-1,25(OH)2D3 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 immunosorbent assay and radioimmunoassay, sixteen kinds of hybridoma clones secreting different anti-1,25(OH)2D3 antibodies were established. Scatchard analysis revealed that 12 antibodies out of 16 have enough affinity to 1,25(OH)2D3 (Ka>10(9) M[-1]). By a cross-reaction study using various related compounds, two antibodies, M114 and M129, both derived from A/J mice, were found to have suitable specificity. Although they showed relatively high cross-reactivities with the 1,25(OH)2D3 derivatives having 24-hydroxylated (both 20%) or lactonized (30%, 8.9%) side chain, all the tested 1alpha-deoxy-type metabolites, some of which interfere with the 1,25(OH)2D3 assays, were successfully discriminated [25(OH)D3: 0.67%, 0.40%; 24,25(OH)2D3: 0.27%, 0.24%; 25(OH)D3 3-sulfate: 0.11%, 0.14%]. These monoclonal antibodies will be applicable for developing not only IAC but also various immunoassay procedures.
Kobayashi
N
N
Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Imazu
T
T
Ebisawa
A
A
Shimada
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification*; D002117:Calcitriol / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006241:Haptens / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology*; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D006241:Haptens; D012710:Serum Albumin, Bovine; D002117:Calcitriol