Rongqing Zhao1, Pengcheng Yu2, Yi Shan3, Nagarajan Thirumeni4, Maohua Li4, Yanli Lv5, Jianli Li4, Wenlin Ren4, Lisong Huang3, Jingshuang Wei6, Yufei Sun7, Wuyang Zhu8, Le Sun9. 1. MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, PR China; AnyGo Technology, D1117 New China International Square, 89 Dayangfang Rd, Chaoyang District, Beijing, PR China. 2. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.155 Changbai Rd, Changping District,Beijing, PR China. 3. Department of Emergency, The Sixth Medical Center of the General Hospital of the People's Liberation Army, Beijing 100048, PR China. 4. AbMax Biotechnology Co., LTD, 99 Kechuang 14th Street, Building 18-2-201, Beijing, PR China. 5. College of Veterinary Medicine, China Agricultural University, 2 Yuanmingyuan Xilu, Haidian District, Beijing, PR China. 6. New Drug R&D Center, State Key Laboratory of Antibody Research & Development, North China Pharmaceutical Corporation, 388 Heping East Road, Shijiazhuang, PR China. 7. AnyGo Technology, D1117 New China International Square, 89 Dayangfang Rd, Chaoyang District, Beijing, PR China. 8. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.155 Changbai Rd, Changping District,Beijing, PR China. Electronic address: Zhuwuyang1971@sina.com. 9. AnyGo Technology, D1117 New China International Square, 89 Dayangfang Rd, Chaoyang District, Beijing, PR China; AbMax Biotechnology Co., LTD, 99 Kechuang 14th Street, Building 18-2-201, Beijing, PR China. Electronic address: Lsun@antibodychina.com.
Abstract
BACKGROUND: Vaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects. METHODS: Using a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy. RESULTS: RABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02-0.06 IU/mL and the linear range was 0.2-10.0 IU/ mL. The inter-assay CVs were in the range of 6.60-10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively. CONCLUSIONS: The validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.
BACKGROUND: Vaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects. METHODS: Using a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy. RESULTS: RABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02-0.06 IU/mL and the linear range was 0.2-10.0 IU/ mL. The inter-assay CVs were in the range of 6.60-10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively. CONCLUSIONS: The validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.
Authors: Sergey V Generalov; Pavel S Erokhin; Oleg S Kuznetsov; Elena G Abramova; Ivan M Zhulidov; Natalya A Osina Journal: Avicenna J Med Biotechnol Date: 2021 Jul-Sep