Generation of a Large Repertoire of Monoclonal Antibodies against Dengue Virus NS1 Antigen and the Development of a Novel NS1 Detection Enzyme-Linked Immunosorbent Assay.

Commercial dengue virus (DENV) nonstructural-1 (NS1) Ag detection immunoassays often perform poorly, particularly in secondary DENV infection. To develop a highly sensitive NS1 ELISA, we generated a large repertoire of anti-DENV NS1 mouse mAbs (n = 95) that falls into 36 mAb classes based on binding specificities. The identified mAb pair, capable of efficiently detecting NS1 from four DENV serotypes in an immunoassay, was selected based on multiparametric analysis. The selected mAbs have subnanomolar affinities for NS1 with recognition sites outside the immunodominant wing domain. The assay was converted to an ELISA kit, which showed higher analytical sensitivity (3-fold to 83-fold) for NS1 from four DENV serotypes than commercial Platelia NS1 ELISA (Bio-Rad Laboratories). Compared to RT-PCR, the developed NS1 ELISA showed 78.57% (66 of 84) sensitivity, whereas Platelia NS1 ELISA showed a sensitivity of 60.71% (51 of 84). In a subgroup of RT-PCR-positive secondary dengue samples, our ELISA showed a sensitivity of 70.18% (40 of 57), whereas Platelia ELISA detected only 47.37% (27 of 57) samples. Furthermore, unlike Platelia ELISA, our test equally detected NS1 from four serotypes; Platelia ELISA performed poorly for the DENV-2 serotype, in which only 8 of 21 (38.10%) samples were detected compared with 17 of 21 (80.95%) in our ELISA. Moreover, our ELISA showed 100% specificity in 342 challenging dengue-negative samples. The large and diverse mAb repertoire generated against DENV NS1 and the appropriate selection of mAbs allowed us to establish an ELISA that can efficiently detect NS1 Ag even in secondary dengue and without serotype level bias.

Development of a Fast SARS-CoV-2 IgG ELISA, Based on Receptor-Binding Domain, and Its Comparative Evaluation Using Temporally Segregated Samples From RT-PCR Positive Individuals.

SARS-CoV-2 antibody detection assays are crucial for gathering seroepidemiological information and monitoring the sustainability of antibody response against the virus. The SARS-CoV-2 Spike protein's receptor-binding domain (RBD) is a very specific target for anti-SARS-CoV-2 antibodies detection. Moreover, many neutralizing antibodies are mapped to this domain, linking antibody response to RBD with neutralizing potential. Detection of IgG antibodies, rather than IgM or total antibodies, against RBD is likely to play a larger role in understanding antibody-mediated protection and vaccine response. Here we describe a rapid and stable RBD-based IgG ELISA test obtained through extensive optimization of the assay components and conditions. The test showed a specificity of 99.79% (95% CI: 98.82-99.99%) in a panel of pre-pandemic samples (n = 470) from different groups, i.e., pregnancy, fever, HCV, HBV, and autoantibodies positive. Test sensitivity was evaluated using sera from SARS-CoV-2 RT-PCR positive individuals (n = 312) and found to be 53.33% (95% CI: 37.87-68.34%), 80.47% (95% CI: 72.53-86.94%), and 88.24% (95% CI: 82.05-92.88%) in panel 1 (days 0-13), panel 2 (days 14-20) and panel 3 (days 21-27), respectively. Higher sensitivity was achieved in symptomatic individuals and reached 92.14% (95% CI: 86.38-96.01%) for panel 3. Our test, with a shorter runtime, showed higher sensitivity than parallelly tested commercial ELISAs for SARS-CoV-2-IgG, i.e., Euroimmun and Zydus, even when equivocal results in the commercial ELISAs were considered positive. None of the tests, which are using different antigens, could detect anti-SARS-CoV-2 IgGs in 10.5% RT-PCR positive individuals by the fourth week, suggesting the lack of IgG response.