Development of nanobody-based DAS-ELISA and Au nanoparticle-based immunochromatographic test strip for highly sensitive detection of Chikungunya virus

Mengmeng Guo¹Liyuan Song²Jingliang Liu¹Yating Hu¹Yihui Chen¹Guangcheng Fu²Hongyu Yuan^1,2*Jianmin Li^1,2*

• ¹Laboratory of Advanced Biotechnology, Zhejiang University-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China
• ²Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing, China

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that can cause severe diseases in both humans and animals, and it continues to pose a significant global public health threat. Current diagnostic methods primarily rely on the detection of CHIKV-specific IgM and IgG antibodies; however, these methods are limited to the phase of humoral immunity following viral infection. To address the diagnostic gap during the window period of antibody response, we developed two antigen detection assays that can directly detect CHIKV Envelope protein (CHIKV-E), enabling early and rapid viral detection. In this work, twenty nanobodies (Nbs) specifically binding to CHIKV-E protein were screened and identified from a constructed immune nanobody library via phage display technology. These Nbs were fused to human IgG1-Fc and expressed as recombinant Nb-Fc antibodies in Expi293F cells. Subsequently, the preferred Nb-Fc (N055-Fc/N055-mIgG1-Fc and 10G4-Fc) was used to establish DAS-ELISA and Au nanoparticle-based immunochromatographic test strip (AuNP-ICTS) detection methods, which demonstrated high sensitivity for CHIKV-E. In addition, the two established assays demonstrated exclusive specificity for CHIKV-E protein, showing no cross-reactivity with envelope proteins from four related alphaviruses or two co-circulating flaviviruses. Moreover, epitope mapping showed that Nanobody N055 and 10G4 recognized different linear epitopes, respectively. N055 targets E1 Domain II, while 10G4 recognizes the E1 fusion loop with additional interactions in E1 Domain II and E2 Domain B. Quantitative sensitivity analysis reveals that both DAS-ELISA and AuNP-ICTS assays effectively detect CHIKV-E in serum, with detection limits (LoD) of 49 pg/mL and 1.56 ng/mL, respectively. These results demonstrate superior sensitivity compared to existing similar antigen detection methods. Notably, the AuNP-ICTS method can complete the detection within 10 min, which significantly improves detection efficiency. Collectively, the developed DAS-ELISA and AuNP-ICTS methods overcome the limitations of conventional IgM/IgG serological assays, demonstrating superior sensitivity and rapid detection capabilities that make them promising diagnostic tools for early CHIKV detection.