F. exposed influenza disease had the highest correlation with MN titers to the same viruses and could become confirmed by removal of crossreactivity from seasonal H1/H3 rHAs following serum adsorption. Interestingly, in individuals with exposures to novel influenza viruses, age and MFIs against revealed novel HA were negatively correlated, whereas in individuals without exposure to novel influenza viruses, age and MFI against novel HAs were positively correlated. == Conclusions == This 20plex highthroughput assay with serum adsorption will be a useful tool to detect novel influenza virus infections during influenza outbreak investigations and monitoring, especially when wellpaired serum samples are not available. Keywords:hemagglutinin, influenza, MAGPIX, serum adsorption, subtype == 1. Intro == Influenza viruses constantly undergo antigenic drift and shift leading to the periodic emergence of novel viruses that can cause human infections. Humans also have complex immune history to influenza because of repeated infections and vaccinations to seasonal influenza. Hemagglutination inhibition (HI) and disease microneutralization (MN) assays are platinum requirements in influenza serologic studies, and both primarily detect antibodies to hemagglutinin (HA) of influenza viruses. Seroconversion having a 4folder rise in HI or MN antibody titers is definitely indicative of recent influenza virus illness or vaccination.1,2In recent years, magnetic multiplex fluorescence microsphere immunoassays (MAGPIX) have been developed to investigate antibody profiles following influenza vaccination/infection and aid in Cefiderocol the diagnosis of influenza.3,4,5Previously, we Cefiderocol developed a highthroughput 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 wellpaired human serum samples were available.4However, during novel influenza disease outbreak investigations, paired serum collection may not always be feasible, posing difficulties to determine influenza disease infections through serology. Recently, we identified the second case of A(H7N2) human illness from pet cats using solitary serum samples collected from workers/volunteers in New York City Cefiderocol animal shelters where A(H7N2) influenza A viruses caused the 1st documented cattohuman transmission.6,7,8For novel influenza surveillance in the animalhuman interface, we also analyzed combined sera collected from workers inside a live poultry Cefiderocol market in Bangladesh where multiple avian influenza viruses cocirculate among poultry and may cause sporadic A(H5N1) influenza disease infections in human beings. In this study, we improved the MAGPIX assay by expanding the multiplex to 20 antigens using 19 globular head website (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 (Table1). Furthermore, given that past exposures to seasonal influenza viruses through infections or vaccinations in the human being populations can often cause Epha1 crossreactive antibody reactions to novel influenza viruses and complicate the interpretation of serologic data,4we also expanded serum adsorption from two to eight ectodomain rHAs to reduce crossreactivity. This multiplex MAGPIX platform with serum adsorption can be a important tool to detect novel influenza virus infections during influenza monitoring and outbreak investigations. == Table 1. == Correlations between MN titer and Cefiderocol MFI value against recombinant ectodomain (Ecto) and/or globular head website HA1 (GH HA1) Pearson’srbetween microneutralization titer against A/New York/108/2016 (H7N2) and MFI ideals against recombinant ectodomain (Ecto) and/or globular head website HA1 (GH HA1). Pearson’srbetween microneutralization titer against A/duck/Bangladesh/19097/2013 (H5N1, 2.3.2.1a) and MFI ideals against recombinant ectodomain (Ecto) and/or globular head website HA1 (GH HA1). The highest Pearson’srwas demonstrated in daring. == 2. MATERIALS AND METHODS == == 2.1. Human being sera == Three units of human being serum samples were used in this study (Furniture2andS1). In the A(H7N2) study, solitary convalescent serum specimens (S2) were collected from 119 shelter workers and volunteers (1873 years, median age 31 years) during an A(H7N2) animal shelter outbreak in New York City in 20162017, and the median days from last exposure to serum collection was 36 days (Table2). One seropositive participant (MN titers 40 and HI titers 40), 5 indeterminate participants (MN titer 40 and HI titer < 40), and 113 seronegative participants (MN < 40 and HI < 40) were recognized in the seroepidemiology study.7 == Table 2. == Serum samples tested by Mock, 2Ads, AND 8Ads with this study and four rHAs from H3N2 (A/Bangkok/1/79, A/Beijing/32/92, A/Perth/16/2009, and A/Maryland/26/2014)..