Mawer E B, Berry J L, Bessone J, Shany S, Smith H, White A
Steroids. 1985 Aug-Sep;46(2-3):741-54. doi: 10.1016/0039-128x(85)90054-6.
The preparation of high-affinity and high-specificity monoclonal antibodies to 1 alpha,25-dihydroxyvitamin D is described. Monoclonal antibodies were derived from Balb-c mice immunised with either 1 alpha-hydroxy-25,26,27-trinor-24-cholecalcioic acid or with 1 alpha-hydroxy-26,27-dinor-cholecalciferol-25-oxime, and spleen cells were hybridised with mouse myeloma cells. From six fusions nine monoclonal antibodies (MAb's) were selected from 676 antibody-secreting hybrids. Antibodies varied widely in their ability to bind 1 alpha,25-dihydroxyvitamin D3 (50% displacement of radioligand ranged from 25 - 900 pg); two had particularly useful characteristics for 1 alpha,25-dihydroxyvitamin D assay. MAb 5F2 has high affinity (Ka = 1.39 X 10(10) M-1) and does not discriminate between 1 alpha,25-dihydroxyvitamin D2 and D3, thus enabling the two forms to be measured together. MAb 1G7 is highly specific, having no cross-reactivity with 25-hydroxy-, 24,25-dihydroxy- or 25,26-dihydroxyvitamin D at concentrations found in normal human serum; this MAb has the potential to eliminate the lengthy extraction procedures involved in currently available assays for 1 alpha,25-dihydroxyvitamin D.
本文描述了针对1α,25 - 二羟基维生素D制备高亲和力和高特异性单克隆抗体的方法。单克隆抗体来源于用1α - 羟基 - 25,26,27 - 三降 - 24 - 胆钙化酸或1α - 羟基 - 26,27 - 二降 - 胆钙化醇 - 25 - 肟免疫的Balb - c小鼠，其脾细胞与小鼠骨髓瘤细胞进行杂交。在六次融合实验中，从676个分泌抗体的杂交瘤中筛选出了九种单克隆抗体（MAb's）。这些抗体结合1α,25 - 二羟基维生素D3的能力差异很大（放射性配体50%被取代时所需的浓度范围为25 - 900 pg）；其中两种抗体对于1α,25 - 二羟基维生素D的检测具有特别有用的特性。单克隆抗体5F2具有高亲和力（Ka = 1.39×10(10) M-1），并且不区分1α,25 - 二羟基维生素D2和D3，因此能够同时检测这两种形式。单克隆抗体1G7具有高度特异性，在正常人血清中的浓度下，与25 - 羟基 - 、24,25 - 二羟基 - 或25,26 - 二羟基维生素D均无交叉反应；这种单克隆抗体有可能省去目前1α,25 - 二羟基维生素D检测方法中繁琐的提取步骤。
