The potency of all modern tissue culture human rabies vaccines is measured based on the National Institute of Health (NIH) potency test that is laborious, time-consuming, involves large test variations and requires sacrifice of large number of animals. To circumvent these limitations, several researchers and WHO expert working groups have discussed development of alternative in vitro methods to replace the NIH potency test. Although several immunochemical methods have been proposed to quantify rabies glycoprotein (G-protein) using multiple murine monoclonal antibodies, we report an In vitro competitive inhibition ELISA (CIA) method based on the use of a neutralizing rabies glycoprotein site III directed novel therapeutic human rabies monoclonal antibody (RAB1) that shows equivalence to the mice NIH potency test in recognition of neutralization site of the glycoprotein. In vitro potency testing of WHO 7th International Standard for rabies vaccine (IS) by CIA using RAB1 and In-house reference standard (IHRS) as a standard to assess its suitability for the assessment of validation parameters showed accurate and precise values with <15% coefficient variance. The method was validated using 5PL standard curve with linearity r² > 0.98 and LLOQ of 0.125 IU/ml indicating sensitivity of the method. The method was found to be precise, robust and accurate to quantitate intact rabies glycoprotein in final vaccine and showed a strong correlation (Pearson's r = 0.81) with the NIH potency values of licensed Vero cell rabies vaccine. The CIA test using RAB1 was able to accurately quantitate degradation of rabies vaccine and assess loss in antigenicity of lyophilized and reconstituted liquid rabies vaccine under thermal stress conditions. The method was able to differentiate between potent and reduced potency vaccine samples. The new in vitro competitive inhibition ELISA method using RAB1 thus can be a valid alternative to the NIH test.

所有现代组织培养人狂犬病疫苗的效力都是根据美国国立卫生研究院 (NIH) 效力测试进行测量的，该测试费力、费时、涉及大量测试变化并需要牺牲大量动物。为了规避这些限制，一些研究人员和 WHO 专家工作组讨论了替代体外方法的开发，以取代 NIH 效力测试。虽然已经提出了几种免疫化学方法来使用多种鼠单克隆抗体量化狂犬病糖蛋白（G 蛋白），但我们报告了体外竞争性抑制 ELISA (CIA) 方法基于使用中和性狂犬病糖蛋白位点 III 定向的新型治疗性人狂犬病单克隆抗体 (RAB1)，该抗体在识别糖蛋白中和位点方面与小鼠 NIH 效力测试等效。中央情报局使用 RAB1 和内部参考标准 (IHRS) 作为评估其对验证参数评估的适用性的标准，对 WHO 狂犬病疫苗 (IS) 的第 7 号国际标准进行的体外效力测试显示准确和精确的值 <15%系数方差。使用线性 r ^{2 的}5PL 标准曲线验证该方法 > 0.98 和 LLOQ 为 0.125 IU/ml，表明该方法的灵敏度。发现该方法对最终疫苗中完整的狂犬病糖蛋白进行定量是精确、稳健和准确的，并且 与获得许可的 Vero 细胞狂犬病疫苗的 NIH 效力值具有很强的相关性（Pearson's r = 0.81）。使用 RAB1 的 CIA 测试能够准确量化狂犬病疫苗的降解，并评估冻干和重组液体狂犬病疫苗在热应激条件下的抗原性损失。该方法能够区分有效和降低效力的疫苗样品。因此，使用 RAB1的新体外竞争性抑制 ELISA 方法可以成为 NIH 测试的有效替代方法。