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Currently, the standard assay employed to diagnose human orthopneumovirus infection is real-time reverse transcriptase PCR assay (rRT-PCR), a costly and time-consuming procedure that requires the manipulation of infectious viruses. In addition to RT-PCR, serological tests can complement the molecular diagnostic methods and have proven to be important tools in sero-surveillance. In this study, we report the development, optimization, and validation of a novel and rapid in-house diagnostic ELISA kit to detect human orthopneumovirus in clinical samples. We developed three sensitive ELISA formats through the immunization of rats with novel recombinant pPOE-F or pPOE-TF vectors. The two vectors expressed either the full-length (pPOE-F) or the truncated form (pPOE-TF) of the fusion (F) protein. The developed ELISA kits were optimized for coating buffer, capture antibody, blocking buffer, sample antigen, detection antibodies, and peroxidase-conjugated antibody, and validated using 75 rRT-PCR-confirmed nasopharyngeal aspirate (NPA) human orthopneumovirus samples and 25 negative samples collected from hospitalized children during different epidemic seasons between 2014 and 2017. Our results indicate that rats immunized with pPOE-F or pPOE-TF showed significant induction of high levels of MPAs. Validation of the ELISA method was compared to the rRT-PCR and the sensitivity hierarchy of these developed ELISA assays was considered from highest to lowest: indirect competitive inhibition ELISA (93.3%) &gt; indirect antigen-capture ELISA (90.6%) &gt; direct antigen-capture ELISA (86.6%). The development of the rapid in-house diagnostic ELISA kits described in this study demonstrates that a specific, rapid and sensitive test for human orthopneumovirus antigens could be successfully applied to samples collected from hospitalized children during different epidemics and can help in the efficient diagnosis of respiratory syncytial viral infections.

Development and Validation of Rapid In-House Diagnostic ELISA Kits for Detection of Human Orthopneumovirus in Clinical Samples