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The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners. Additionally, sandwich assays that are reliant on commercial antibodies can suffer from changes to reagent quality outside the researchers’ control. This report presents a reimagined and simplified phage display selection protocol that directly identifies sandwich binding peptides and Fabs. The approach yielded two sandwich pairs, one peptide–peptide and one Fab–peptide sandwich for the cancer and Parkinson’s disease biomarker DJ-1. Requiring just a few weeks to identify, the sandwich pairs delivered apparent affinity that is comparable to other commercial peptide and antibody sandwiches. The results reported here could expand the availability of sandwich binding partners for a wide range of clinical biomarker assays.

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic interference (AIN), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a prior infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous putative antigen, a sequence derived from the neuraminidase protein of H3N2 influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIN from a previous infection could underlie some cases of COVID-19 disease severity.