Exploring the Standardized Detection and Sampling Methods of Human Nasal SARS-CoV-2 RBD IgA

Xuanxuan Zhang¹Jizong Jia²Chaoying Hu¹Yulong Fu³Guanxing Liu⁴Yajing Li⁵Qian He¹Fan Gao¹Na Li⁶Lina Wang²Jianping Chu²Henggang Xu²Zhihao Fu¹Hui Zhao¹Zhenglun Liang¹Jingxin Li^7*Miao Xu^1*Qunying Mao^1*

• ¹National Institutes for Food and Drug Control (China), Beijing, China
• ²Beijing Wantai Biological Pharmacy (China), Beijing, Beijing Municipality, China
• ³Shenyang Pharmaceutical University, Shenyang, Liaoning Province, China
• ⁴Changchun Institute of Biological Products Co. Ltd, Changchun, Hebei Province, China
• ⁵Shanghai Institute of Biological Products (SIBP), Shanghai, Shanghai Municipality, China
• ⁶Beijing Minhai Biological Technology Co., Ltd., Beijing, China
• ⁷Jiangsu Provincial Center for Disease Control And Prevention, Nanjing, Jiangsu Province, China

Vaccines capable of effectively inducing mucosal immunity, particularly specific IgA antibodies, represent an ideal strategy for preventing infections and the transmission of pathogens such as SARS-CoV-2 and influenza viruses that rapidly replicate in the upper respiratory tract and cause clinical symptoms. However, a lack of standardized nasal antibody detection and sampling methods has hindered cross-study comparability and vaccine development. To address this issue, this study uses SARS-CoV-2 as a model pathogen to standardize nasal antibody detection methods and sampling methods. Following the scientific guidelines (Q14 and Q2(R2)) for analytical procedure development and validation released by International Council for Harmonization (ICH), the first validated ELISA for SARS-CoV-2 RBD-specific IgA detection in the nasal mucosa was established through analytical target profiling (ATP), risk assessment, and design of experiment optimization. Systematic validation demonstrated exclusive specificity for the target antigen, with intermediate precision of <17% and relative bias of <±4%, meeting ATP requirements. Analysis of 154 clinical samples demonstrated strong concordance between the novel assay and electrochemiluminescence assays, with a concordance correlation coefficient of 0.87 for quantitative results and a kappa coefficient of 0.85 for results above and below the dilution-adjusted limit of quantification (LOQ). Applying this novel assay, a clinical comparison revealed that expanded sponge sampling achieved superior performance in terms of the single-day detection rate (above dilution-adjusted LOQ 95.5%), 5-day consecutive detection rate (above dilution-adjusted LOQ 88.9%), and median SARS-CoV-2 RBD IgA concentration (171.2 U/mL), significantly outperforming nasopharyngeal swabs (68.8%; 48.7%; 28.7 U/mL, p<0.0001) and nasal swabs (88.3%; 77.3%; 93.7 U/mL, p<0.05). The standardized detection system established in this study can be adapted with appropriate modifications for the clinical evaluation of other respiratory mucosal vaccines, thereby advancing the development of mucosal vaccines.