Research and Development Department, Serum Institute of India Pvt. Ltd, Hadapsar, Pune 410028, Maharashtra, India.
Research and Development Department, Serum Institute of India Pvt. Ltd, Hadapsar, Pune 410028, Maharashtra, India.
J Immunol Methods. 2021 May;492:112939. doi: 10.1016/j.jim.2020.112939. Epub 2020 Dec 9.
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 r > 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均基于美国国立卫生研究院（NIH）效力测试来衡量，该测试费力、耗时，涉及大量测试变化，并且需要牺牲大量动物。为了规避这些限制，一些研究人员和世界卫生组织专家工作组讨论了开发替代的体外方法来替代 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定量狂犬病糖蛋白（G 蛋白），但我们报告了一种基于使用中和狂犬病糖蛋白位点 III 的新型治疗性人狂犬病单克隆抗体（RAB1）的体外竞争抑制 ELISA（CIA）方法，该方法显示与小鼠 NIH 效力测试等效，可识别糖蛋白的中和位点。使用 CIA 方法通过 CIA 对世界卫生组织第 7 次狂犬病疫苗国际标准（IS）进行体外效力测试，使用 RAB1 和内部参考标准（IHRS）作为标准来评估其用于评估验证参数的适用性，结果表明具有<15％的变异系数，具有准确而精确的值。该方法使用 5PL 标准曲线进行验证，线性 r>0.98，LLOQ 为 0.125IU/mL，表明该方法具有灵敏度。该方法被发现可精确，稳健且准确地定量最终疫苗中的完整狂犬病糖蛋白，并与许可的 Vero 细胞狂犬病疫苗的 NIH 效力值显示出很强的相关性（Pearson r=0.81）。RAB1 的 CIA 测试能够准确地定量狂犬病疫苗的降解，并评估冻干和重新配制的液体狂犬病疫苗在热应激条件下的抗原性损失。该方法能够区分效力强和效力降低的疫苗样品。因此，使用 RAB1 的新的体外竞争抑制 ELISA 方法可以替代 NIH 测试。
