To develop nanobodies with broad-spectrum reactivity, specificity, and high sensitivity that can be used for detecting multiple subtypes of influenza A virus, and to establish a nanobody-based enzyme-linked immunosorbent assay (ELISA) method.

Gene sequences of twelve nanobodies against influenza A virus were retrieved from the National Center for Biotechnology Information (NCBI) and nanobody databases. The nanoantibodies were prepared using molecular biological techniques including gene synthesis and recombinant expression. The binding activity, specificity, sensitivity, and affinity of these nanobodies were determined by ELISA screening and Gator affinity analysis. A double-antibody sandwich ELISA assay was established by combining the selected nanobody with a traditional mouse monoclonal antibody.

Twelve nanobodies were expressed and purified. Two nanobodies capable of binding to multiple subtypes of influenza virus including H1, H3, H5, H7, and H9 were obtained and designated as VHH54 and KV108. Both nanobodies showed no cross-reactivity with other respiratory virus antigens. Furthermore, the KV108 nanobody exhibited the highest binding affinity, with a dissociation constant of 5.94×10^-9 mol/L for the influenza virus nucleoprotein (NP), and the lowest detection concentration for the NP antigen reached 0.00064 μg/mL. The double-antibody sandwich ELISA, using a combination of KV108 and a mouse monoclonal antibody, could sensitively detect the five common subtypes of influenza A virus (H1N1, H3N2, H5N1, H7N9, and H9N2). The lowest detection limit reached 110-403 PFU/mL, which was higher than that of the commercial colloidal gold kitfor influenza virus detection.

This study has identified a nanobody KV108, which is capable of binding to multiple subtypes of influenza virus, and established a nanobody-based ELISA method that can detect multiple subtypes of influenza A virus. This study can facilitate the development of nanobody-based influenza detection technologies.

Since the outbreak of coronavirus disease (COVID-19), the high infection rate and mutation frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent, have contributed to the ongoing global pandemic. Vaccination has become the most effective means of controlling COVID-19. Traditional neutralizing tests of sera are complex and labor-intensive, therefore, a rapid test for detecting neutralizing antibodies and antibody status post-immunization is needed.

Based on the fact that antibodies exhibit neutralizing activity by blocking the binding of the S protein receptor-binding domain (S-RBD) to ACE2, we developed a rapid neutralizing antibody test, ACE2-Block-ELISA. To evaluate the sensitivity and specificity, we used 54 positive and 84 negative serum samples. We also tested the neutralizing activities of monoclonal antibodies (mAbs) and 214 sera samples from healthy individuals immunized with the inactivated SARS-CoV-2 vaccine.

The sensitivity and specificity of the ACE2-Block ELISA were 96.3% and 100%, respectively. For neutralizing mAb screening, ch-2C5 was selected for its ability to block the ACE2–S-RBD interaction. A plaque assay confirmed that ch-2C5 neutralized SARS-CoV-2, with NT₅₀ values of 4.19, 10.63, and 1.074 µg/mL against the SARS-CoV-2 original strain, and the Beta and Delta variants, respectively. For the immunized sera samples, the neutralizing positive rate dropped from 82.14% to 32.16% within 4 months post-vaccination.

This study developed and validated an ACE2-Block-ELISA to test the neutralizing activities of antibodies. As a rapid, inexpensive and easy-to-perform method, this ACE2-Block-ELISA has potential applications in rapid neutralizing mAb screening and SARS-CoV-2 vaccine evaluation.