促红细胞生成素酶联免疫吸附测定法的研究

Research on Enzyme Linked Immunosorbent Assay for Erythropoietin

目的:建立血清促红细胞生成素(erythropoietin,EPO)的酶联免疫检测(ELISA)方法,观察其临床应用价值。方法:制备EPO多克隆抗体,异丙醇洗涤处理酶标板以增强其吸附能力,利用交叉反应法选择出抗原、抗体及酶标抗体的最佳的配对工作浓度。反应后,观察本方法的灵敏度、回收率、特异度、稳定性等指标,观察效果。以正常血清为对照组,测定缺铁性贫血组、乳腺癌组,并与放射免疫检测对比。结果:酶标板结合蛋白能力增强。抗体、酶标抗体、抗原最佳工作浓度分别为1∶1000,1∶6000,1∶800。灵敏度为0.46U/L,与生长激素、铁蛋白交叉反应率低;高浓度、低浓度样品平均回收率分别为96.3%、97.3%,批内和批间变异分别为8.31%、7.82%,稳定性好。乳腺癌组、缺铁性贫血组EPO水平均明显高于正常对照组。放射免疫检测及酶联免疫检测检出结果差别无统计学意义。结论:本血清EPO双抗体夹心ELISA法的建立在诊断相关疾病方面有一定的临床应用价值。

Objective： To establish an enzyme linked immunosorbent assay （ELISA）for erythropoietin（EPO）in serum, and observe its clinical application value thereof. Methods： Prepare the EPO polyclonal antibody, wash the plate with isopropyl alcohol, and then choose the suitable concentration of the antibody, enzyme labeled antibody, and antigen. After the reaction, check the sensitivity, recovery, specificity and stability of the method. The serum samples of anaemia and breast carcinoma individuals were detected. The results of radioimmunodetection were compared with that of normal control group. Results： The immo-assay plate showed strong adherence to proteins. The optimal concentrations of the antibody, enzyme labelled antibody and antigen were 1∶1 000, 1∶6 000 and 1∶800 separately. The sensitivity was 0.46 U/L. The cross-reaction with growth hormone and ferritin was low. The mean recoveries of samples with high and low concentrations were 96.3%, 97. 3% respectively. The coefficients of variation of intra-assay and inter-assay were just 8.31% and 7.82%, and the stability was good. The EPO levels were higher in anaemia and breast carcinoma groups than that of normal group. There was no significant difference between the results of the radioimmunodetection and ELISA. Conclusion： The double-antibody sandwich ELISA method was established for EPO in serum, which had certain clinical application value.