Development of a radioligand immunoassay for 1,25-dihydroxycholecalciferol receptors utilizing monoclonal antibody.
Development of a radioligand immunoassay for 1,25-dihydroxycholecalciferol receptors utilizing monoclonal antibody.
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利用单克隆抗体开发 1,25-二羟基胆钙化醇受体的放射性配体免疫测定法。
DOI：
10.1042/bj2210129
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Pike,JW
中科院分区：
文献类型：
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作者：
Dokoh,S;Haussler,MR;Pike,JW
利用从大鼠脾X小鼠骨髓瘤杂交瘤SP2/0- 9A 7悬浮培养物中获得的特异性、高亲和力（Kd = 1.8×10（-11 M））单克隆抗体（9A 7 γ），建立了1，25-二羟基胆钙化醇[1，25（OH）2D 3]受体的放射配体免疫分析法。基于1，25（OH）2[3 H]D3-受体（18 fmol/管）与放射惰性1，25（OH）2D 3-受体（0-240 fmol/管）之间对9A 7 γ上结合位点的竞争，建立标准曲线。在相同缓冲液中制备的样品含有0-100 fmol受体/管。1，25（OH）2[3 H]D3-受体与标准品或样品平衡孵育（4 ℃下16 h）后，抗体结合受体与预先连接至金黄色葡萄球菌的兔抗（大鼠免疫球蛋白）免疫沉淀并定量。该测定对2 fmol受体/管具有统计学灵敏度，测定内和测定间变异分别为7%和12%。观察到被占据、未被占据和变性受体在测定中同等竞争。这种定量技术已成功地应用于沉淀分析和DNA-纤维素色谱分离后的受体的表征。最后，通过该测定结合1，25（OH）2D 3结合测定对总受体的测量显示佝偻病、正常和注射1，25（OH）2D 3的小鸡分别有13%、20%和56%的受体处于占据形式。从这些结果中，我们认为，这种放射性配体免疫分析将是一个有用的工具，在进一步的研究，重点是定量组织和细胞提取物中的1，25（OH）2D 3受体。
A radioligand immunoassay for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] receptors was developed utilizing a specific, high-affinity (Kd = 1.8×10(-11 M) monoclonal antibody (9A7 gamma) obtained from suspension cultures of rat spleen X mouse myeloma hybrid SP2/0-9A7. A standard curve was established, based on the competition between 1,25(OH)2[3H]D3-receptor (18 fmol/tube) and increasing concentrations of radioinert 1,25(OH)2D3-receptor (0-240 fmol/tube) for the binding site on 9A7 gamma. Samples, prepared in identical buffer, contained 0-100 fmol of receptor/tube. After an equilibrium incubation of 1,25(OH)2[3H]D3-receptor with either standard or sample (16 h at 4 degrees C), antibody-bound receptor was immunoprecipitated with rabbit anti-(rat immunoglobulin) prelinked to Staphylococcus aureus and quantified. The assay is statistically sensitive to 2 fmol of receptor/tube, with intra- and inter-assay variations of 7 and 12% respectively. Occupied, unoccupied and denatured receptor were observed to compete equally in the assay. This quantitative technique has been successfully applied to the characterization of receptors after fractionation by sedimentation analysis and DNA-cellulose chromatography. Finally, the measurement of total receptor by this assay, in conjunction with 1,25(OH)2D3 binding assays, has revealed that rachitic, normal and 1,25(OH)2D3-injected chicks have respectively 13, 20, and 56% of receptor in the occupied form. From these results we consider that this radioligand immunoassay will be a useful tool in further research focusing on quantifying 1,25(OH)2D3 receptors in tissue and cell extracts.