Phage vs. Phage: Direct Selections of Sandwich Binding Pairs.

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.

Evidence for deleterious effects of immunological history in SARS-CoV-2.

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.

Under-5-Minute Immunoblot Assays by Vortex Fluidic Device Acceleration.

Unlocking the potential of personalized medicine in point-of-care settings requires a new generation of biomarker and proteomic assays. Ideally, assays could inexpensively perform hundreds of quantitative protein measurements in parallel at the bedsides of patients. This goal greatly exceeds current capabilities. Furthermore, biomarker assays are often challenging to translate from benchtop to clinic due to difficulties achieving and assessing the necessary selectivity, sensitivity, and reproducibility. To address these challenges, we developed an efficient (<5 min), robust (comparatively lower CVs), and inexpensive (decreasing reagent use and cost by >70 %) immunoassay method. Specifically, the immunoblot membrane is dotted with the sample and then developed in a vortex fluidic device (VFD) reactor. All assay steps-blocking, binding, and washing-leverage the unique thin-film microfluidics of the VFD. The approach can accelerate direct, indirect, and sandwich immunoblot assays. The applications demonstrated include assays relevant to both the laboratory and the clinic.

Evidence for Deleterious Antigenic Imprinting in SARS-CoV-2 Immune Response.

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 imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous 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 potential 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, AIM from a previous infection could underlie some cases of COVID-19 disease severity. IMPORTANCE: Infections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples’ immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.