化学发光免疫分析法检测人血清孕酮

Determination of Progesterone in Human Serum by Chemiluminescent Immunoassay

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。

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.