Kobayashi N, Higashi T, Saito K, Murayama T, Douya R, Shimada K
Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
J Steroid Biochem Mol Biol. 1997 May;62(1):79-87. doi: 10.1016/s0960-0760(97)00016-2.
Novel hapten-carrier conjugants were prepared by coupling 11 alpha-hemiglutaryloxy-(24R)-24,25-dihydroxyvitamin D3 or (24R)-24,25-dihydroxyvitamin D3 [24,25(OH)2D3] 3-hemiglutarate with bovine serum albumin (BSA), to obtain an antibody with high specificity and affinity for use in 24,25(OH)2D3 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)2D3 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 Abll well discriminated the 1 alpha-hydroxylated metabolites such as 1,24,25(OH)3D3 (< or = 0.69%) and 1,25(OH)2D3 (< or = 0.25%), but significantly crossreacted with some side chain modified compounds such as (24S)-24,25-dihydroxyvitamin D3 [24S,25(OH)2D3] (> or = 67%), 25(OH)D3 (> or = 14%) and 25,26(OH)2D3 (> or = 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)2D3 (< or = 3.0%), 25(OH)D3 (< 0.3%) and 25,26(OH)2D3 (< or = 0.53%). A significant crossreaction was found only with 1,24,25(OH)3D3 (> or = 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.
通过将11α-半戊二酰氧基-(24R)-24,25-二羟基维生素D3或(24R)-24,25-二羟基维生素D3[24,25(OH)2D3]3-半戊二酸与牛血清白蛋白(BSA)偶联，制备了新型半抗原-载体结合物，以获得用于24,25(OH)2D3免疫测定的具有高特异性和亲和力的抗体。分别针对这两种结合物在三或四只兔子中诱导出了具有高滴度的多克隆抗体；从前者和后者结合物获得的抗体分别表示为Ab11和Ab3。这些抗体对24,25(OH)2D3的亲和力远高于维生素D结合蛋白(DBP)。通过在放射免疫分析(RIA)系统中与11种相关化合物的交叉反应性研究了抗体的特异性。Ab11能很好地区分1α-羟基化代谢物，如1,24,25(OH)3D3(≤0.69%)和1,25(OH)2D3(≤0.25%)，但与一些侧链修饰化合物，如(24S)-24,25-二羟基维生素D324S,25(OH)2D3、25(OH)D3(≥14%)和25,26(OH)2D3(≥23%)有明显交叉反应。另一方面，Ab3与具有不同侧链结构的化合物，如24S,25(OH)2D3(≤3.0%)、25(OH)D3(<0.3%)和25,26(OH)2D3(≤0.53%)的交叉反应性仅可忽略不计。仅与1,24,25(OH)3D3(≥68%)有明显交叉反应。这些结果表明，Ab3有望用于开发一种免疫分析系统，该系统比基于DBP的传统竞争性蛋白质结合分析更具特异性和敏感性。
