Validation of the anthrax lethal toxin neutralization assay
Validation of the anthrax lethal toxin neutralization assay
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DOI：
10.1016/j.biologicals.2003.09.003
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发表时间：
2004-03-01
期刊：
影响因子：
1.7
通讯作者：
Pace-Templeton, J
中科院分区：
文献类型：
--
作者：
Hering, D;Thompson, W;Pace-Templeton, J
提供了毒素中和试验性能特征的验证。该体外试验测量了含有炭疽致死毒素抗体的抗血清特异性保护J774A.1细胞免受炭疽芽孢杆菌致死毒素细胞毒性的功能能力。该比色测定基于活细胞对MTT的还原。人和兔抗炭疽疫苗吸收（AVA）产生的抗血清用于验证测定。结果显示出高水平的重复性和再现性，特别是对于生物测定。吸光度值的试验间变异性是最突出的阴性结果，然而，通过试验血清50%有效剂量（ED 50）与参比标准品ED 50的比值[中和比（NR）]证明了可接受的水平。即使在具有最小中和能力的样品中也保持准确性，并且当样品稀释导致Y最大值和Y最小值的准确预测时，注意到线性。特异性试验表明，正常血清对参比标准品中和毒素的能力没有可观察到的影响。对于参比标准品的时间、温度和冻融效应，该试验保持稳定，但对毒素不稳定。该试验在培养基和溶液储存影响下也保持稳定。细胞传代次数和细胞平板密度是耐用性研究期间确定的两个关键参数，可能是吸光度值试验间变异性的原因。根据FDA的生物分析方法验证行业指南和FDA的非临床实验室研究药物非临床研究质量管理规范（21 CFR第58部分）进行了该工作。(C)2003年由Elsevier Ltd代表国际生物制品协会出版。
A validation of the performance characteristics of a toxin neutralization assay is presented. This in vitro assay measures the functional ability of antisera, containing antibodies to anthrax lethal toxin, to specifically protect J774A.1 cells against Bacillus anthracis lethal toxin cytotoxicity. This colormetric assay is based upon the reduction of MTT by living cells. Human and rabbit antisera produced against anthrax vaccine absorbed (AVA) were used to validate the assay. Results showed a high level of repeatability and reproducibility, particularly for a bio-assay. Inter-assay variability in absorbance values was the most prominent negative finding however, an acceptable level was demonstrated with a ratio [neutralization ratio (NR)] of the test serum 50% effective dose (ED50) to the reference standard ED50. Accuracy was maintained even in samples with minimal neutralizing capacity, and linearity, was noted when sample dilutions resulted in accurate prediction of the Y-max and Y-min. Specificity tests demonstrated that normal sera did not have an observable effect on the ability of the reference standard to neutralize toxin. The assay remained stable against time, temperature, and freeze/thaw effects on the reference standards, but not on the toxin. The assay, also remained stable against media and solution storage effects. Cell passage number and cell plating density were two critical parameters identified during the robustness studies that may be responsible for inter-assay variability in absorbance values. The work was performed in accordance with the FDA's Bioanalytical Method Validation Guidance for Industry and the FDA's Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58). (C) 2003 Published by Elsevier Ltd on behalf of the International Association for Biologicals.