Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization
Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization
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DOI：
10.1016/j.jim.2008.01.007
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发表时间：
2008-04-20
影响因子：
2.2
通讯作者：
Quinn, Conrad P.
中科院分区：
文献类型：
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作者：
Li, Han;Soroka, Stephen D.;Quinn, Conrad P.
炭疽致死毒素(LTX)中和活性(TNA)的定量在评估炭疽疫苗的保护性抗体反应和评价炭疽的免疫治疗中是至关重要的。我们调整和重新设计了TNA检测方法，以建立一个统一、标准化、定量和验证的技术平台，用于J774A.1小鼠细胞系的LTX中和。该平台的关键设计特点是1)应用自由形式或受约束的4参数Logistic(4-PL)函数来模拟100%细胞存活率和95%细胞裂解的边界范围内和之间的中和响应，以及2)开发用于解释性终点的创新的分析曲线识别算法。以人血清ED50(中和50%的血清稀释度)作为主要报告值(RV)进行验证。术中精密度(以变异系数(%CV)表示)较高，分别为10.5%~15.5%和13.5%~14.5%。用Log(10)变换后的数据进行线性回归分析，人血清总核糖核酸测定的稀释线性，斜率=0.99，截距=-0.03，r(2)=0.985。从精密度和线性数据推算的精密度和线性数据，并使用加标回收率方法，测定准确度很高，百分比误差(%E)范围仅为3.4-20.5%E。检测下限(LLOD)为ED50=12，定量下限(LLOQ)为ED50=36。这种基于细胞的检测方法非常可靠，可以承受35-39℃的孵化温度，3%-7%的二氧化碳浓度和2.5-7.5 mg/ml的报告底物浓度。最多25代细胞培养的检测质量控制参数严格。使用人类参考标准AVR414和AVR801测定的长期(50个月)测定稳定性表明，高精密度、一致的准确度和没有检测到的测定漂移。一个定制的软件程序提供了两个额外的检测指标，定量滴度(Qt)和阈值滴度(TT)，这两个指标都显示出可接受的准确度、精密度和稀释线性。用TT建立检测反应阈值(RT)。分别对人、恒河猴和兔血清进行了测定，并对ED50值进行了聚集稀释线性分析(斜率=0.98，截距=0.003，r(2)=0.989)。我们提出了这种具有合格标准参考血清和定制解释软件的TNA检测格式，作为确定对炭疽疫苗的功能血清学反应和评估炭疽免疫疗法的统一平台技术。爱思唯尔出版公司(Elsevier B.V.)
Quantification of anthrax lethal toxin (LTx) neutralization activity (TNA) is pivotal in assessing protective antibody responses to anthrax vaccines and for evaluation of immunotherapies for anthrax. We have adapted and redesigned the TNA assay to establish a unifying, standardized, quantitative and validated technology platform for LTx neutralization in the J774A.1 murine cell line. Critical design features of this platform are 1) the application of a free-form or constrained 4 parameter logistic (4-PL) function to model neutralization responses within and between boundary limits of 100% cell survival and 95% cell lysis and 2) to exploit innovative assay curve recognition algorithms for interpretive endpoints. The assay was validated using human serum ED50 (dilution of serum effecting 50% neutralization) as the primary reportable value (RV). Intra-operator and intermediate precision, expressed as the coefficient of variation (%CV), were high at 10.5-15.5%CV and 13.5-14.5%CV respectively. TNA assay dilutional linearity was demonstrated for human sera using linear regression analysis of log(10) transformed data with slope=0.99, intercept=-0.03 and r(2)=0.985. Assay accuracy, inferred from the precision and linearity data and using a spike-recovery approach, was high with a percent error (%E) range of only 3.4-20.5%E. The lower limit of detection (LLOD) was ED50=12 and the lower limit of quantification (LLOQ) was ED50 = 36. The cell-based assay was robust, tolerating incubation temperatures from 35 to 39 degrees C, CO2 concentrations from 3% to 7% and reporter substrate (MTT) concentrations of 2.5-7.5 mg/ml. Strict assay quality control parameters were met for up to 25 cell culture passages. The long term (50 month) assay stability, determined using human reference standards AVR414 and AVR801, indicated high precision, consistent accuracy and no detectable assay drift. A customized software program provided two additional assay metrics, Quantification Titer (QT) and Threshold Titer (TT), both of which demonstrate acceptable accuracy, precision and dilutional linearity. The TT was also used to establish the assay reactivity threshold (RT). The application of the assay to sera from humans, Rhesuis macaques and rabbits was demonstrated separately and by aggregate dilutional linearity analysis of the ED50 (slope=0.98, intercept= 0.003, r(2)=0.989). We propose this TNA assay format with a qualified standard reference serum and customized interpretive software as a unifying platform technology for determination of functional serologic responses to anthrax vaccines and for evaluation of anthrax immunotherapeutics. Published by Elsevier B.V.