Development of highly sensitive DENV NS1 antigen detection Immunoassay

Abstract

Purpose: Commercial dengue virus (DENV) NS1 antigen detection immunoassays perform poorly, particularly in secondary DENV infection. The objective of this work was to generate monoclonal antibodies (mAbs) targeting diverse epitopes on DENV NS1 and to develop an ELISA with enhanced sensitivity to detect DENV NS1 antigen. Methods & Materials: Mouse hybridoma technique was used to generate anti-DENV NS1 monoclonal antibodies (mAbs), and the selected mAb pair was used to develop NS1 detection ELISA. Sera from the febrile adult population (n=161), febrile pediatric population (n=193), and non-febrile (n=100) subjects were tested on the developed assay. The performance of the in-house developed NS1 ELISA was assessed in comparison to Bio-Rad DENV NS1 ELISA and clinical diagnosis. Results: 95 anti-DENV NS1 mAbs were generated and characterized as DENV complex specific (29 mAbs), subcomplex specific (31 mAbs), and serotype-specific (35 mAbs) binders. 11 mAbs were shortlisted, for further work in ELISA format, based on the results from multiparametric interaction studies. All the shortlisted antibodies were found to have affinities (KD) lying in the sub-nanomolar range. After extensive optimization studies, we developed a stable NS1 antigen ELISA with a total run time of 100 minutes. The developed stabilized ELISA was found to be suitable for all the three sample matrices (serum, citrate plasma and EDTA plasma) tested. The overall positivity rate in the developed ELISA was 97.92% (95%CI: 88.93%-99.95%) and 100.00% (95%CI: 75.29%-100%) for febrile adult and pediatric population respectively when cross-referenced with Bio-Rad ELISA. Additionally, four samples from pediatric and 21 samples from the adult sera panel scored positive in the developed NS1 ELISA but were negative in Bio-Rad ELISA. These discrepant samples were suggestive of DENV infection based on DENV IgM/ DENV IgG capture positivity and clinical findings. The developed immunoassay was found to be 100% (95% CI: 96.38%-100.00%) specific. Conclusion: The large mAb repertoire, generated against DENV NS1, and extensive characterization studies, allowed us to generate a stabilized ELISA to detect DENV NS1 antigen with sensitivity higher than well-established commercial ELISA.