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Background Detections of influenza A subtype specific antibody responses are often complicated by the presence of cross-reactive antibodies. We developed two novel multiplex platforms for antibody detection. The multiplexed magnetic fluorescence microsphere immunoassay (MAGPIX) is a high throughput laboratory-based assay. Chembio Dual Path Platform (DPP) is a portable and rapid test that could be used in the field. Methods Twelve recombinant globular head domain hemagglutinin (GH HA1) antigens from A(H1N1)pdm09 (pH1N1), A(H2N2), A(H3N2), A(H5N1), A(H7N9), A(H9N2), A(H13N9), B/Victoria lineage, B/Yamagata lineage viruses, and protein A control were used. Human sera from U.S. residents either vaccinated (with H5N1 or pH1N1) or infected with pH1N1 influenza viruses, and sera from live bird market workers in Bangladesh (BDPW) were evaluated. GH HA1 antigens and serum adsorption using full ectodomain recombinant hemagglutinins from A(H1N1) and A(H3N2) were introduced into the platforms to reduce cross-reactivity. Results Serum adsorption reduced cross-reactivity to novel subtype HAs. Compared to traditional hemagglutination inhibition or microneutralization assays, when serum adsorption and the highest fold rise in signals were used to determine positivity, the correct subtype-specific responses were identified in 86% to 100% of U.S. residents exposed to influenza antigens through vaccination or infection (N=49). For detection of H5N1 specific antibodies in sera collected from BDPW, H5 sensitivity was 100% (6/6) for MAGPIX, 83% (5/6) for DPP; H5 specificity was 100% (15/15) and cross-reactivity against other subtype was 0% (0/6) for both platforms. Conclusion MAGPIX and DPP platforms can be utilized for high-throughput and in-field detection of novel influenza virus infections. This article is protected by copyright. All rights reserved.

Background The development of serologic assays that can rapidly assess human exposure to novel influenza viruses remains a public health need. Previously, we developed an 11‐plex magnetic fluorescence microsphere immunoassay (MAGPIX) by using globular head domain recombinant hemagglutinins (rHAs) with serum adsorption using two ectodomain rHAs. Methods We compared sera collected from two cohorts with novel influenza exposures: animal shelter staff during an A(H7N2) outbreak in New York City in 2016‐2017 (n = 119 single sera) and poultry workers from a live bird market in Bangladesh in 2012‐2014 (n = 29 pairs). Sera were analyzed by microneutralization (MN) assay and a 20‐plex MAGPIX assay with rHAs from 19 influenza strains (11 subtypes) combined with serum adsorption using 8 rHAs from A(H1N1) and A(H3N2) viruses. Antibody responses were analyzed to determine the novel influenza virus exposure. Results Among persons with novel influenza virus exposures, the median fluorescence intensity (MFI) against the novel rHA from exposed influenza virus had the highest correlation with MN titers to the same viruses and could be confirmed by removal of cross‐reactivity from seasonal H1/H3 rHAs following serum adsorption. Interestingly, in persons with exposures to novel influenza viruses, age and MFIs against exposed novel HA were negatively correlated, whereas in persons without exposure to novel influenza viruses, age and MFI against novel HAs were positively correlated. Conclusions This 20‐plex high‐throughput assay with serum adsorption will be a useful tool to detect novel influenza virus infections during influenza outbreak investigations and surveillance, especially when well‐paired serum samples are not available.

Influenza hemagglutination inhibition (HI) and virus microneutralization assays (MN) are widely used for seroprevalence studies. However, these assays have limited field portability and are difficult to fully automate for high throughput laboratory testing. To address these issues, three multiplex influenza subtype-specific antibody detection assays were developed using recombinant hemagglutinin antigens in combination with Chembio, Luminex®, and ForteBio® platforms. Assay sensitivity, specificity, and subtype cross-reactivity were evaluated using a panel of well characterized human sera. Compared to the traditional HI, assay sensitivity ranged from 87% to 92% and assay specificity in sera collected from unexposed persons ranged from 65% to 100% across the platforms. High assay specificity (86–100%) for A(H5N1) rHA was achieved for sera from exposed or unexposed to hetorosubtype influenza HAs. In contrast, assay specificity for A(H1N1)pdm09 rHA using sera collected from A/Vietnam/1204/2004 (H5N1) vaccinees in 2008 was low (22–30%) in all platforms. Although cross-reactivity against rHA subtype proteins was observed in each assay platform, the correct subtype specific responses were identified 78% to 94% of the time when paired samples were available for analysis. These results show that high throughput and portable multiplex assays that incorporate rHA can be used to identify influenza subtype specific infections.

Much of what is known of the activities of polycystin-1 has been inferred from the effects of the isolated cytoplasmic COOH-terminal domain, but it is not clear whether the truncation acts like polycystin-1, as a dominant negative, or in unrelated pathways. To address this question, we have examined functional interactions between the intact and truncated forms of polycystin-1 in one cell system. In cells expressing only native polycystin-1, introduction of the truncation replicated the activity of the full-length protein. Conversely, when background levels of polycystin-1 were modestly elevated, the truncation acted as a dominant negative. Hence, the truncation acts in the polycystin pathway, but with effects that depend upon the background level of polycystin-1 expression. Our data raise the possibility that the cytoplasmic carboxyl terminus, either through cleavage products or intramolecular interactions, might feed back to modulate the activity of parent or intact polycystin-1.