静注COVID-19人免疫球蛋白(pH4)原料血浆筛选用试剂盒(ELISA)的确定及其定量检测方法的建立、验证及应用

Determination of kit(ELISA)for screening material plasma of human COVID-19 immunoglobulin(pH4)for intravenous injection and establishment,validation and application of quantitative detection method

目的通过比较特异性、敏感度、线性范围对目前市售的5家新型冠状病毒IgG抗体检测试剂盒(厂家A、B、C、D、E)进行筛选,建立定量检测方法并验证,用于静注COVID-19人免疫球蛋白(pH4)所需原料血浆的检测。方法将已知有中和活性的新型冠状病毒康复者恢复期血浆作为内控标准品,进行2倍系列稀释共7个稀释度(S1~S7),按照5个厂家试剂盒说明书操作,比较最高浓度S1的吸光值,用四参数拟合曲线比较5家试剂盒的线性范围;用候选的3家试剂盒初步建立定量检测方法并检测部分有代表性的阴性和阳性样本,比较结果的相关性;综合考虑选定一家试剂盒,将内控标准品进行系列稀释检测,确定其稀释度赋值,拟合标准曲线建立定量检测方法,并验证其线性范围、准确度和精密度,用于建立血浆筛查的模式。结果内控标准品的线性范围显示,厂家A可用倍比6个稀释度,S1吸光值为2.8;厂家B可用5个倍比稀释度,S1吸光值为2.4;厂家C可用5个倍比稀释度,S1吸光值为1.4;厂家D可用倍比3个稀释度,S1吸光值为3.3;厂家E可用5个倍比稀释度,S1吸光值为3.3。线性范围相近的厂家A、B、E试剂盒利用内控标准品建立了定量检测方法,曲线的决定系数R^(2)和各稀释度的回收率均符合要求,厂家A检测有代表性的阴性样本本底较高,特异性不好;3家试剂盒与中和试验检测有代表性的阳性样本结果的相关性均>0.9。选定用厂家B试剂盒,用其3个批次试剂盒4次检测的内控标准品的稀释度赋值为32,四参数拟合曲线线性成立,稀释度的线性范围为1~16,R^(2)>0.99,准确度、精密度符合要求。建立了单孔检测的筛浆模式,确定了复测样品的稀释度。结论厂家B试剂盒优化为定量检测试剂盒,经验证后可用于静注COVID-19人免疫球蛋白原料血浆的筛选。

Objective To screen five currently commercially available 2019-nCoV IgG antibody detection kits(A,B,C,D and E)by comparing the specificity,sensitivity and linear range,and establish and validate a quantitative detection method for material plasma of human COVID-19 immunoglobulin(pH 4)for intravenous injection.Methods The COVID-19 convalescent plasma with neutralizing antibody activity was used as the internal control standard and 2-fold serially diluted to seven dilutions(S1~S7).According to the instructions of the kits from five manufacturers,the absorbances of S1 at the highest concentration were compared,while the linear ranges were compared by using a four-parameter fitting curve.A quantitative detection method was developed with three candidate kits and used for determination of partial representative negative and positive samples,and the correlation of results was evaluated.A kit was selected based on comprehensive consideration,with which the dilution of the internal control standard was assigned after serial dilution to fit the standard curve and establish a quantitative detection method.The established method was validated for linear range,accuracy and precision,and used for establishmetnt of a plasma screening mode.after verifying its linear range,accuracy and precision.Results The linear range of the internal control standard showed that six dilutions were detected by kit A,while the absorbance of S1 was 2.8;five dilutions were detected by kit B,while the absorbance of S1 was 2.4;five dilutions were detected by kit C,while the absorbance of S1 was 1.4;three dilutions were detected by kit D,while the absorbance of S1 was 3.3;however,five 5 dilutions were detected bu kit E,while the absorbance of S1 was 3.3.By using kits A,B,and E with similar linear ranges,a quantitative detection method was established with the internal control standard,of which the R^(2) of the curve and the recovery rates of various dilutions met the relevant requirements.The representative negative samples tested by kit A showed high background and poor specificity.The correlations between the determination results by three kits and the neutralization test of the representative positive samples were more than 0.9.Kit B was selected finally,by which the dilution of the internal control standard determiend with three batches for 4 times was assigned as 32,and the four-parameter fitting curve was linearly established,and the linear range of dilution was 1~16,with a R^(2) value of more than 0.99,indicationg that the accuracy and precision were qualified.The plasma screening mode was establilshed as single test each sample,and the dilution of retest sample was determiend.Conclusion The kit from manufacturer B is optimized as a quantitative detection kit,which may be used for the material plasma screening of human COVID-19 immunoglobulin for intravenous injection after validation.