肝素诱导血小板减少症抗体的酶联免疫吸附检测方法研究  

Application of ELISA in Detecting Heparin-Induced Thrombocytopenia Antibodies

作　　者：王红梅[1] 田晶晶[1] 段生宝[1] 丁少华[1] 蒙青林[1] 陈晔洲 陈强[2] 李勇[1] 

机构地区：[1]中国科学院苏州生物医学工程技术研究所,中科院生物医学检验技术重点实验室,江苏苏州215163 [2]苏州市立医院,江苏苏州215002

出　　处：《现代检验医学杂志》2014年第6期65-68,共4页Journal of Modern Laboratory Medicine

摘　　要：目的：研究建立肝素诱导的血小板减少症抗体（HIT）的酶联免疫吸附检测方法。方法人血小板4因子（PF4）与肝素形成的复合物作为包被抗原，加入待检血浆孵育洗涤后，加入酶标二抗，孵育洗涤后加入底物显色，终止反应后测定吸光度A450nm／A630nm ，根据阴阳性标准品判定检测结果，并与 IBL方法进行比较分析，对该方法进行优化和性能评估。结果用制备的人PF4建立检测方法，通过正交试验得到待检样品和酶标抗体的最佳稀释度分别为1∶100和1∶1500，对三份阳性标准血浆重复测定，该方法的批内和批间平均变异系数分别为7.67％和7.76％（P均＜10％）。对100人份无肝素使用史的健康人血浆测定，该方法的阴阳性参考值分别为0.304和0.456。与 IBL检测方法同时检测100份血液透析病人样品，检测结果经统计学分析，该方法的灵敏度、特异度和准确度分别为90％，97.78％和97％，阴阳性预测值分别为81.8％和98.88％，K＝0.84（K 值在0.81～1），Pexac＝0.012＜0.05，差异有统计学意义，一致性为优，95％可信区间为92.59％～99.17％。结论建立的 H IT抗体的酶联免疫吸附检测方法与 IBL检测方法相比性能相当、一致性程度良好，可以满足临床对 H IT患者的检测和诊断需求。Objective To develop an ELISA method for determination of heparin-induced thrombocytopenia （HIT）antibody. Methods The compound formed between human platelet factor 4 （PF4）and heparin was used as the coating antigen,incu-bating the patients plasma with the coating antigen in the well,after washing,the second antibody labeled HRP was added in the well to incubate and washing again,the chromogenic substrates was added in the well to incubate,when the stop reaction was finished,the absorbance A450/A630 was detected,and the test results were judged according to standard,this method was compared with IBL method and was optimized and evaluated the performance.Results An indirect ELISA method was de-velop with the purified human PF4,the optimal dilution of sample and second antibody were 1∶100 and 1∶1 500 which de-tected by the orthogonal test,the intra-and inter-assay average coefficients of variation were 7.66% and 7.76%（〈10%） respectively that detected by repeated measurement the three positive standard plasma.Through measureing the 100 healthy human plasm with no history of using heparin,the positive and negative predictive reference values were 0.304 and 0.456. IBL and this method detected 100 hemodialysis patients samples at the same time,and the result of statistical analysis was that,the sensitivity,speciality and accuracy of this method were 90%,97.78% and 90%,respectively.The negative and posi-tive predictive value were 81.8% and 98.88% respectively,and the difference was statistically significant [K=0.84（0.81～1）and Pexac=0.012〈0.05].The difference was statistically significant,consistency was optimal,95% confidence interval was 92.59%～92.59%.Conclusion Comparing with the IBL,the method reported by this article had the similar perform-ance and good consistency,and it could satisfy the clinical detection and diagnosis of HIT patients.

关 键 词：血小板4因子 肝素 肝素诱导血小板减少症 酶联免疫吸附法 

分 类 号：R446.61[医药卫生—诊断学]