■在这项研究中,开发并验证了内部酶联免疫吸附测定(ELISA).基于使用国际参考抗小鼠血清NIBSC(美国国家生物标准和对照研究所)97/642绘制的标准曲线,使用参考线(RFL)方法计算ELISA的滴度。
■在开发步骤中,描绘信噪比以选择显示最合适比率的缓冲液。在验证步骤中,标准范围,精度,稀释线性度,和特异性得到证实,比较了RFL和平行线(PLL)方法的精度和稀释线性。
■对于百日咳毒素(PT),平板的涂层浓度达到0.1µg/mL,丝状血凝素抗原(FHA)为0.15µg/mL,Pertactin(PRN)为0.25µg/mL。PT的信噪比为22.02,14.93FHA,和8.02的PRN与0.25%山羊血清在磷酸盐缓冲盐水(PBS)作为稀释缓冲液,和在PBS中的2%脱脂乳作为封闭缓冲液。根据精度结果,我们通过1、0.2和1.5EU/mL浓度评估PT的定量下限,FHA,和PRN符合ICH(国际协调理事会)M10标准的25%的准确性和40%的总误差。具体而言,将同源血清掺入异源血清中,准确性符合标准。RFL和PLL计算结果没有差异(PT的p值=0.3207,FHA为0.7394,PRN为0.2109)。
■本研究中使用RFL计算方法验证的ELISA是用于小鼠体液免疫原性测试的相对准确的测定。
UNASSIGNED: In this study, an in-house enzyme-linked immunosorbent assay (ELISA) was developed and validated. The titer of ELISA was calculated using the reference line (RFL) method based on the standard curve drawn using the international reference anti-mouse serum NIBSC (National Institute for Biological Standards and Control) 97/642.
UNASSIGNED: In the development step, signal to noise was depicted to select the buffers that showed the most appropriate ratio. In the validation step, standard range, precision, dilution linearity, and specificity were confirmed, and RFL and parallel line (PLL) methods were compared in precision and dilution linearity.
UNASSIGNED: Coating concentration for plate was achieved at 0.1 µg/mL for pertussis toxin (PT), 0.15 µg/mL for filamentous hemagglutinin antigen (FHA), and 0.25 µg/mL for pertactin (PRN). The signal to noise ratio was 22.02 for PT, 14.93 for FHA, and 8.02 for PRN with 0.25% goat serum in phosphate-buffered saline (PBS) as a dilution buffer, and 2% skim milk in PBS as a blocking buffer. Based on the precision results, we assessed the lower limit of quantification by 1, 0.2, and 1.5 EU/mL concentration for PT, FHA, and PRN which met the ICH (International Council for Harmonization) M10 criteria of a 25% accuracy and total error of 40%. In specificity, homologous serum was spiked into heterologous serum and the accuracy met the criteria. There was no difference in the results between RFL and PLL calculations (p-value=0.3207 for PT, 0.7394 for FHA, 0.2109 for PRN).
UNASSIGNED: ELISA validated with RFL calculation method in this study is a relatively accurate assay for mouse humoral immunogenicity test.