Lindsey Changhong Y, Pace-Templeton Judith G, Millard Charles B, Wannemacher Robert W, Hewetson John F
US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, MD 21702-5011, USA.
Biologicals. 2006 Mar;34(1):33-41. doi: 10.1016/j.biologicals.2005.08.018. Epub 2005 Nov 2.
An enzyme-linked immunosorbent assay (ELISA) for the determination of anti-ricin immunoglobulin G (IgG) concentration in mouse sera was systematically validated. The results obtained throughout the validation process strongly demonstrated that the ELISA was reliable, reproducible, and suitable for its intended use. The assay had a high level of precision within and between runs, was specific for the anti-ricin IgG, and showed no interference with a number of different serum matrices. The assay exhibited excellent accuracy, linearity, and stability. The mean recovery of four test samples with different known concentrations was 100.9+/-11.3%, 102.7+/-10.8%, 99.0+/-7.2%, and 95.9+/-11.3%, respectively (n=10). The mean recovery of the observed anti-ricin IgG concentration of three quality control samples run on 73 plates to their nominal concentrations was 100.1+/-7.3%, 100.2+/-5.8%, and 103.7+/-8.1%; and the coefficient of variation (CV) was 7.3%, 5.8%, and 7.8%, respectively. The back-calculated anti-ricin IgG concentration, %CV, and relative error of seven standards from the calibration curves run in the entire validation study were analyzed (n=7 x 73). The results indicated that the four-parameter logistic (4PL) equation, y=(a-d)/(1+(x/c)b)+d, provided an accurate representation of a sigmoidal relationship between the measured response and the logarithm of observed concentration of anti-ricin IgG in mouse sera for this ELISA. The lower limit of quantification and upper limit of quantification of the calibration curve were 3.3 ng/ml and 82.8 ng/ml, respectively. The measurable range of the assay would cover all possible anti-ricin IgG concentrations in mouse sera stimulated with a ricin vaccine candidate, when the test sera are measured at a 1:800 starting dilution followed by four additional fourfold serial dilutions.
对用于测定小鼠血清中抗蓖麻毒素免疫球蛋白G（IgG）浓度的酶联免疫吸附测定（ELISA）进行了系统验证。在整个验证过程中获得的结果有力地证明了该ELISA可靠、可重复且适用于其预期用途。该测定在批次内和批次间具有高度的精密度，对抗蓖麻毒素IgG具有特异性，并且对多种不同的血清基质无干扰。该测定表现出优异的准确性、线性和稳定性。四个已知不同浓度测试样品的平均回收率分别为100.9±11.3%、102.7±10.8%、99.0±7.2%和95.9±11.3%（n = 10）。在73个平板上运行的三个质量控制样品的观察到的抗蓖麻毒素IgG浓度相对于其标称浓度的平均回收率为100.1±7.3%、100.2±5.8%和103.7±8.1%；变异系数（CV）分别为7.3%、5.8%和7.8%。分析了在整个验证研究中运行的校准曲线的七个标准品的反算抗蓖麻毒素IgG浓度、CV和相对误差（n = 7×73）。结果表明，四参数逻辑（4PL）方程y = (a - d)/(1 + (x/c)b) + d，准确地表示了该ELISA中测得的响应与小鼠血清中抗蓖麻毒素IgG观察浓度的对数之间的S形关系。校准曲线的定量下限和定量上限分别为3.3 ng/ml和82.8 ng/ml。当以1:800的起始稀释度测量测试血清，然后再进行四次四倍系列稀释时，该测定的可测量范围将涵盖用候选蓖麻毒素疫苗刺激的小鼠血清中所有可能的抗蓖麻毒素IgG浓度。
