EV71抗原BA-ELISA检测方法的建立及应用

目的建立EV71抗原生物素-亲和素酶联免疫(BA-ELISA)检测方法,并进行验证及初步应用。方法以抗EV71抗体为包被抗体,利用生物素-亲和素系统建立双抗体夹心ELISA法,并对其灵敏度、特异性、准确性及精密性进行验证。采用该方法检测组织培养及临床样品中EV71抗原的含量。结果所建立的BA-ELISA法在EV71蛋白含量625～156.25ng/ml之间线性关系最佳,R2达0.9976,灵敏度为78.125～156.25ng;与包括CoxA16在内的42种肠道病毒及相关病毒均无交叉反应;检测10份EV71阳性样品和10份阴性样品,结果符合率均为100%;检测EV71原液和高、中浓度样品的变异系数分别为3.09%、6.69%和6.43%;可检出约103～104CCID50的活病毒及手足口病患者的大便悬液和部分咽拭子悬液中的病毒。结论用生物素标记EV71抗体与亲和素系统建立的双抗体夹心ELISA法具有较高的特异性、灵敏度、准确性和精密性,为临床检验及抗原分析提供了参考。

Development and Application of BA-ELISA Method for EV71 Antigen

Objective To develop, verify and preliminarily apply a BA-ELISA method for EV71 antigen. Methods A double antibody sandwich ELISA method was developed based on biotin-avidin system using antibody against EV71 as coating antibody, and verified for sensitivity, specificity, accuracy and precision. The EV71 antigen contents in tissue culture and clinical samples were determined by the developed method. Results The developed BA-ELISA method showed good linearity at a EV71 protein content of 625 ～ 156. 25 ng / ml, and its R2 value and sensitivity were 0. 997 6 and 78. 125 ～ 156. 25 ng respectively. The method showed no cross reactions with 42 kinds of enteroviruses and the relevant viruses, including Cox A16. Both the coincidence rates of determination results of 10 known EV71 positive and 10 known EV71 negative samples were 100%. The coefficients of determination results of bulk EV71 and EV71 antigen positive samples at high and moderate concentrations were 3. 09%, 6. 69% and 6. 43% respectively. The detection limit of live EV71 in tissue culture by the developed method was about 103 ～ 104 CCID50. The EV71 in fecal suspension and a part of pharynx swabs of patients with hand-foot-mouth disease were detected by the developed method. Conclusion The developed BA-ELISA method showed high specificity, sensitivity, accuracy and precision, which provided a basis for clinical laboratory and antigen analysis of EV71.