Novel multiplex assay platforms to detect influenza A hemagglutinin subtype‐specific antibody responses for high‐throughput and in‐field applications

Abstract

BackgroundDetections 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.MethodsTwelve 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(pH1N1) and A(H3N2) were introduced into the platforms to reduce cross‐reactivity.ResultsSerum 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%‐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% (six of six) for MAGPIX, 83% (five of six) for DPP, H5 specificity was 100% (15/15), and cross‐reactivity against other subtype was 0% (zero of six) for both platforms.ConclusionMAGPIX and DPP platforms can be utilized for high‐throughput and in‐field detection of novel influenza virus infections.

Identification of novel influenza A virus exposures by an improved high‐throughput multiplex MAGPIX platform and serum adsorption

“…S2 samples were selected for adsorption based on MFI values (Figure ). S2 samples that showed MFIs ≥3500 against H7.NY.16.Ecto in A(H7N2) study (except one serum showed MN ≥40 and MFI = 2239) (18 out of 119) and MFIs ≥1300 against H5.Ind.05 GH in A(H5N1) study (14 out of 29) in the initial test were first adsorbed with two ectodomain rHAs (2‐Ads) from CA/09 and Perth/09 conjugated to latex beads as described previously (Figure and Table ). Selected S2 samples for 2‐Ads showed a range of MN titers against homologous viruses from 5 to 80 (Figure ).…”

“…Bio‐Plex Pro Magnetic COOH beads (Bio‐Rad, CA) were conjugated with nineteen trimeric ectodomain and/or GH HA1 antigens from influenza A(H1N1), A(H2N2), A(H3N2), A(H5N1), A(H7N2), A(H7N7), A(H7N9), A(H9N2), A(H13N9), B Victoria lineage (B/Brisbane/60/2008), B Yamagata lineage (B/Wisconsin/1/2010), and a protein A control as described previously (Table ) . The antigens were either in‐house made at CDC or obtained from International Reagent Resource and ThermoFisher Scientific (PA, USA) (Table ); the purity, trimerization, and receptor‐binding activity of in‐house made rHAs were confirmed as previously described .…”

“…The serum samples were tested at 1:40 dilution in duplicate by using in‐house made phycoerythrin‐conjugated protein A (RPE‐PA) and read by a Bio‐Plex MAGPIX Multiplex Reader. Median fluorescence intensity (MFI) was obtained and analyzed with Bio‐Plex Manager Software …”

“…Seroconversion with a ≥ 4‐fold rise in HI or MN antibody titers is indicative of recent influenza virus infection or vaccination . In recent years, magnetic multiplex fluorescence microsphere immunoassays (MAGPIX) have been developed to investigate antibody profiles following influenza vaccination/infection and aid in the diagnosis of influenza . Previously, we developed a high‐throughput MAGPIX assay with serum adsorption using two ectodomain recombinant HAs (rHAs) from A(H1N1)pdm09 (A/California/7/2009, CA/09) and A/Perth/16/2009 (Perth/09, A(H3N2)) to identify novel influenza exposures when well‐paired human serum samples were available .…”

“…In this study, we improved the MAGPIX assay by expanding the multiplex to 20 antigens using 19 globular head domain (GH) HA1 and/or ectodomain (Ecto) rHAs from 9 subtypes of influenza A viruses (H1N1, H2N2, H3N2, H5N1, H7N2, H7N7, H7N9, H9N2, and H13N9), 2 lineages of influenza B viruses (Yamagata and Victoria lineages), and a protein A control (Table ). Furthermore, given that past exposures to seasonal influenza viruses through infections or vaccinations in the human populations can often cause cross‐reactive antibody responses to novel influenza viruses and complicate the interpretation of serologic data, we also expanded serum adsorption from two to eight ectodomain rHAs to reduce cross‐reactivity. This multiplex MAGPIX platform with serum adsorption can be a valuable tool to detect novel influenza virus infections during influenza surveillance and outbreak investigations.…”