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

Abstract

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(pH1N1) 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%-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.

Conclusion: MAGPIX and DPP platforms can be utilized for high-throughput and in-field detection of novel influenza virus infections.

Keywords: MAGPIX; Chembio Dual Path Platform; antibody; hemagglutinin; influenza.

Figure 1

Increases in MFI and DPP values following vaccination and natural infection. Paired serum samples from ASO3‐adjuvanted split H5N1 vaccine study were tested by MAGPIX (A) and DPP (B). The paired serum samples from pH1N1‐exposed persons were tested by MAGPIX (C) and DPP (D). For each group, the mean MFI/DPP value and ± the standard deviation are shown.*P<.05

Figure 2

Cross‐reactive antibodies can be removed by serum adsorption without loss of subtype‐specific antibodies. Serum samples from persons who received ASO3‐adjuvanted split H5N1 vaccine were either mock or pH1/H3 rHA‐conjugated latex beads adsorbed. Pre‐ and post‐adsorption S2 sera were tested by MAGPIX (A) and DPP (B); post‐adsorption S1 and S2 sera were tested by MAGPIX (2C) and DPP (2D). For each group, the mean MFI/DPP value and ± the standard deviation are shown. *P<.05

Figure 3

Higher anti‐H2 GH HA1 antibodies were detected in elderly population. The S1 serum samples were divided into two groups (born before 1967 and born after 1970), the S1 sera were adsorbed with pH1/H3 rHA‐conjugated latex beads, and mock‐treated or adsorbed sera were tested by MAGPIX (A and B) and DPP (C and D). For each group, the mean MFI and ± the standard deviation are shown. *P<.05. (A) <1967, the binding antibodies to H2 GH HA1 are consistent, P=.57. (B) >1970, the binding antibodies to H2 GH HA1 are reduced significantly, P<.05. (C) <1967, the binding antibodies to H2 GH HA1 are consistent, P=.60. (D) >1970, the binding antibodies to H2 GH HA1 are reduced significantly, P<.05