Hering Donna, Thompson William, Hewetson John, Little Stephen, Norris Sarah, Pace-Templeton Judith
Product Development and Regulatory Affairs, US Army Medical Research Institute of Infectious Diseases 1425 Porter Street, Fort Detrick, Frederick, MD 21702-5011, USA.
Biologicals. 2004 Mar;32(1):17-27. doi: 10.1016/j.biologicals.2003.09.003.
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 (ED(50)) to the reference standard ED(50). 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).
本文介绍了一种毒素中和试验性能特征的验证。这种体外试验可测量含有抗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影响的功能能力。这种比色法基于活细胞对MTT的还原作用。使用针对吸附了炭疽疫苗（AVA）产生的人源和兔源抗血清来验证该试验。结果显示出高度的重复性和再现性，特别是对于生物测定而言。然而，各试验间吸光度值的变异性是最突出的负面发现，不过，通过测试血清50%有效剂量（ED(50)）与参考标准ED(50)的比值[中和率（NR）]证明其处于可接受水平。即使在中和能力最小的样本中也能保持准确性，并且当样本稀释能准确预测Y(max)和Y(min)时可观察到线性关系。特异性试验表明，正常血清对参考标准物中和毒素的能力没有可观察到的影响。该试验在参考标准物受到时间、温度和冻融影响时保持稳定，但毒素则不然。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接种密度是稳健性研究中确定的两个关键参数，可能是各试验间吸光度值变异性的原因。这项工作是按照美国食品药品监督管理局（FDA）的《工业生物分析方法验证指南》和FDA的《非临床实验室研究良好实验室规范》（21 CFR Part 58）进行的。
