基于"双抗体夹心法"全自动酶免工作站检测方法的建立

Establishment of method with Automatic ELISA Workstation based on "Two-site sandwich enzyme immunoassay"

目的 建立"双抗体夹心法"测定抗原含量全自动酶免工作站检测方法,并验证可替代手工操作.方法 根据"双抗体夹心法"检测试剂盒操作规程对全自动酶免工作站进行程序设计,除样品解吸附操作外,样品稀释、多次孵育、多次洗板、加入显示抗体与酶标抗体、加入显色液及酶标仪测定OD值均实现了自动化操作.同时进行了单板和多板自动化检测与人工检测的比对,并对显色底物的稳定性、回收试剂的稳定性及全自动酶免工作站操作精密度和方法精密度进行了考察.结果 单板和多板自动化检测与人工检测的结果比对偏差符合规定;显色液稳定考察结果表明可以提前加入显色液,无需中断程序;回收试剂稳定性满足多次使用,可有效降低检验成本;精密度考察结果表明采用全自动酶免工作站进行60批样品的测定,操作精密度和方法精密度均符合要求.结论 "双抗体夹心法"为半定量方法,步骤繁琐,手工操作影响因素多,并且该方法是目前多种疫苗进行效力检测的主要方法.通过开发全自动酶免工作站检测方法替代手工操作,可有效降低手工操作带来的偏差,提升检验结果的准确度和重现性,并显著提升检验效率.当前新冠疫情仍在全球肆虐,疫苗作为有效遏制疫情蔓延的最有效的武器,需求量的急剧增加对检验效率和检验准确性提出了更高要求.因此,开发全自动酶免工作站替代手工操作在提升检验效率、保障疫苗供应方面具有重要的意义.

0bjective To establish an aulomalic method for the lest of antigen content by"Two sile sandwich enzyme immunoasay"and venify that the resul is parallel and consistent with manual opention.Methods The Autonatie ELISA W orkstation was programmed according 10 the operating proeedures of"Two-site sandwich enxyme immunoassay"testing kit.The entire process has been automated.incduding the sample diuion,inenubation.plale washingaddition of various reagents and determination of abeorbance,in addition to the sample desorption operation.At the same time,the comparison between single-plate and multi-plate was carried out by the method of automatie detection and manual deetion,and the stabiliy of ehromogenic subtraethe stability of reeovered reagent,the operating prerision of workstation and the method precision were inestigated.Results The deviation of the results of single-plale and muli-plate both performned auomaically and manualy met the requirementsthe investigation of the stability of chromogenic substrale showed that it could be added in advance withou interrupting the procedure;the stability of the reeovered reagent met the requirements for multiple uses,whieh could reduce the lest cosl effeetively;the results of precision investigation with 60 batches of samples showed that both of the operation pree ision and method precision met the require ments.Conclus ion"Two-site sandwich enzyme immunoasay"is a semi-quantitative method with cum bersome steps and many influencing factors of manual operation.Moreover,his method is the main method for potency lesI of many vaccines at present.By developing an automatic detec tion method,instead of manual operation,the deviation can be effectively reduced,the accuracy and re producibility of test results can be improved,and the lest efficiency can be signifieanly improved.The COV ID-19 pandemie is still rampant globally by now.As the most effective weapon to ferirely curb the expansion of the pandemicthe danatie incerease in demand places higher demands on lest eficieney and accuracy.Therefore it is of great signifcance to develop an automatie process with the workstation instead of manual operation in improv ing the worklow efficiency to ensure the supply of vaccines.