Validation of Adapted Neutralization Assays Developed to Discriminate Anti-Rabies Virus Activity of Two Different Anti-Rabies Virus Monoclonal Antibodies Administered as a Combination

A. Companjen, S. Moore, B. Boulanger, S. Kostense, W. Marissen
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Abstract

Assessment of rabies virus (RABV) neutralizing antibodies in subjects vaccinated or injected with anti-RABV immunoglobulins is central in determination of rabies protection. The rapid fluorescent focus inhibition test (RFFIT) is used for assessment of anti-RABV activity in serum. The current anti-RABV polyclonal preparations on the market pose difficulties in production and vary in quality. RABV neutralizing monoclonal antibodies (MAbs) are being evaluated as replacements. Different anti-RABV MAbs may neutralize different RABV isolates, thus two or more MAbs directed against different epitopes on the RABV glycoprotein are needed. It is therefore important to ensure neutralizing activity against all RABV isolates in sera of subjects injected with an anti-RABV MAb product consisting of two or more MAbs. The RFFIT, utilizing CVS-11 as challenge virus, cannot discriminate between the activities of different anti-RABV MAbs. We developed and validated two RFFIT methods enabling specific assessment of two different anti-RABV MAbs (CR57 and CR4098) in using two mutant CVS-11 strains resistant to either CR57 or CR4098 neutralization. The validation results demonstrate that both RFFIT assays using MAb resistant RABV are precise, accurate, linear, specific, and stable within the linear range of 0.025 IU/mL to 1.0 IU/mL. This method design can, therefore, be used to determine MAb specific anti-RABV activity in human serum samples.
两种不同的抗狂犬病毒单克隆抗体联合使用的抗狂犬病毒活性鉴定方法的验证
评估接种或注射抗RABV免疫球蛋白的受试者的狂犬病病毒(RABV)中和抗体是确定狂犬病保护的核心。采用快速荧光焦点抑制试验(RFFIT)评价血清抗rabv活性。目前市场上抗rabv多克隆制剂生产困难,质量参差不齐。RABV中和单克隆抗体(mab)正在被评估作为替代品。不同的抗RABV单抗可以中和不同的RABV分离株,因此需要针对RABV糖蛋白上不同表位的两种或两种以上的单抗。因此,确保注射由两种或两种以上单克隆抗体组成的抗RABV单抗产品的受试者血清中对所有RABV分离株的中和活性是很重要的。利用CVS-11作为攻击病毒的RFFIT不能区分不同抗rabv单克隆抗体的活性。我们开发并验证了两种RFFIT方法,使用两种抗CR57或CR4098中和的突变CVS-11菌株,对两种不同的抗rabv单克隆抗体(CR57和CR4098)进行特异性评估。验证结果表明,在0.025 IU/mL ~ 1.0 IU/mL的线性范围内,两种抗单抗RABV RFFIT检测方法均具有精密度、准确度、线性、特异性和稳定性。因此,该方法设计可用于测定人血清样品中单克隆抗体特异性抗rabv活性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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