Specificity of the polyclonal antibodies raised against a novel 25-hydroxyvitamin D3-bovine serum albumin conjugate linked through the C-11 alpha position.
To obtain a specific antibody for use in 25-hydroxyvitamin D3 [25(OH)D3] immunoassay, a novel hapten-carrier conjugate was prepared by coupling 11 alpha-hemiglutaryloxy-25(OH)D3 with bovine serum albumin (BSA). Three polyclonal antibodies (Ab11) showing high titer and affinity for 25(OH)D3 (Ka = 0.96-2.6 x 10(9) M-1) were elicited in rabbits by repeated immunization with the conjugate. Specificity of the Ab11 was investigated by cross-reactivities with 11 related compounds in a radioimmunoassay using a tritium-labeled antigen and compared with that of conventional antibodies (Ab3) raised against 25(OH)D3 3-hemiglutarate conjugated with BSA. The Ab3 could not discriminate the A-ring modified metabolites [1,25(OH)2D3 (87-290%) and 25(OH)D3 3-sulfate (S) (130-180%)], although the cross-reactivities with the side chain modified metabolites were satisfactorily low [24,25(OH)2D3 (2.3-7.4%), 25(OH)D2 (< or = 1.1%)]. On the contrary, the Ab11 easily discriminated 1,25(OH)2D3 (0.10-2.4%) and 25(OH)D3 3S (< 0.3%), whereas significant cross-reactivities were found with 24,25(OH)2D3 (110-120%) and 25,26(OH)2D3 (66-130%) having a dihydroxylated side chain. These results show that the Ab11 are complementary to the A-ring portion of the 25(OH)D3 molecule which is opposite from the side chain structure recognized by the Ab3. Thus, the Ab11 will compensate for insufficient specificity of the Ab3 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)D3 immunoassays using the Ab3.
Kobayashi
N
N
Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Hisada
A
A
Shimada
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D002112:Calcifediol / Q000276:immunology*; D003429:Cross Reactions; D005260:Female; D006241:Haptens / Q000276:immunology; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000276:immunology*
D000906:Antibodies; D006241:Haptens; D012710:Serum Albumin, Bovine; D002112:Calcifediol