化学发光免疫分析法检测人血清孕酮

目的研究用化学发光免疫分析法(CLIA)检测人血清孕酮(PROG)。方法采用竞争抑制法,用高亲和力的多克隆抗体包被,碱性磷酸酶标记孕酮,金刚烷增敏化学发光体系作为酶底物来检测PROG。并考察试剂的灵敏度、精密性、准确性、特异性和测定范围。将试剂盒置4℃存放14个月,检测质控血清的PROG含量,考察试剂的稳定性。并与进口试剂进行比较。结果CLIA法检测PROG灵敏度达0.1ng/ml;特异度99.5%;测定范围在0.1～200ng/ml之间;用PROG浓度分别为高、中、低的质控血清测定精密性,批内和批间变异系数均小于10%,回收率在91.7%～108.4%之间;与雌二醇、雌三醇和雌酮的交叉反应率低于0.1%,与睾酮和可的松无交叉反应;试剂在4℃存放14个月,质控血清的测定值均在规定范围内;与进口全自动化学发光试剂BeckmanAccessTM相比有较好的相关性。二者测定结果的符合率为99.2%。结论化学发光法灵敏度高,特异性好,稳定性强,检测范围宽,有很好的准确性和精密性,是现有放射免疫法的理想替代方法。

Determination of Progesterone in Human Serum by Chemiluminescent Immunoassay

Objective To determine the progesterone(PROG)in human serum by chemiluminescent immunoassay(CLIA).MethodsDetermine the PROG in human serum samples by competitive inhibiting ELISA using the polyclonal antibody with high affinity,alkaline phosphatase-labeled progesterone(PROG-ALP)and adamantine-sensitized chemiluminescent substrate.Evaluate the sensitivity,precision,accuracy,specificity and detection limit of the developed kit.Determine the PROG content in internal quality control reference serum with the kit after storage at 4℃ for 14 months to evaluate its stability.Compare the determination results with those of imported kit.Results The sensitivity,specificity and detection limit of the developed kit were 0.1 ng/ml,99.5% and 0.1～200 ng/ml respectively.Both the inter-and intra-variation coefficents of determination results of PROG by the kit in internal quality control reference serum were less than 10%.The recovery rate was 91.7%～108.4%.The kit showed no cross reaction with testosterone or cortisone,and all the cross reaction rates with dihydrotheelin,theelod and estrone were less than 0.1%.The determination results of internal quality control reference serum with the kit after storage at 4℃ for 14 months were within the range required.The determination result by the developed kit showed good relationship to that by imported full-automatic CLIA kit.The consistence rate of determination results by the developed and imported kits was 99.2%.Conclusion The developed chemiluminescent immunoassay was sensitive,specific,stable,accurate,precise and of wide detection limit,which might be an ideal substitute for the current CLIA.