Utility of Pandemic H1N1 2009 Influenza Virus Recombinant Hemagglutinin Protein-Based Enzyme-Linked Immunosorbent Assay for Serosurveillance

Abstract

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against the pandemic H1N1 2009 influenza A virus, employing a recombinant hemagglutinin protein of the virus, was compared to the hemagglutination inhibition (HI) test using 783 serum samples. The results showed a concordance of 98.4%, suggesting the utility of the ELISA in serosurveillance. Two hundred sixty-nine (100%) serum samples with an HI titer of >20 were ELISA reactive.Influenza viruses are negative-strand RNA viruses that belong to the family Orthomyxoviridae, which includes 4 genera, Influenzavirus A, B, and C and Thogotovirus. Influenza A viruses are widely distributed in nature and can infect a wide variety of mammals and birds. Based on the antigenicity of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza A viruses have been classified into 16 HA and 9 NA subtypes. Of these, H1, H2, and H3 HA subtypes and N1 and N2 NA subtypes have circulated in human populations. In recent years, H5N1 virus of avian origin was expected to be a pandemic-causing pathogen (4). However, the first pandemic of this century was caused by a novel H1N1 influenza A virus of swine origin that emerged in 2009 (hereinafter called p-H1N1-09) (3, 5).Due to the circulation of several influenza A virus subtypes, cross-reactivity is a major problem in influenza virus serology. The inhibition of hemagglutination (HI) caused by antibodies to the HA of the virus is routinely used for assessing the prevalence of a specific virus in a community or an animal population.India was affected by the pandemic H1N1 influenza during the latter half of 2009 (2). In order to understand the degree of exposure of different populations to the virus, an extensive serosurvey was undertaken by the National Institute of Virology, Pune, India. The test of choice, HI, was performed as described earlier (1). Considering the requirement of fresh red blood cells and time and the cumbersome protocol, it was thought important to evaluate the utility of a recombinant HA protein enzyme-linked immunosorbent assay (ELISA) for the detection of p-H1N1-09 IgG antibodies as evidence of exposure to this novel virus.The HA gene of the p-H1N1-09 influenza virus isolated at the National Institute of Virology (A/India-Blore/NIV310/ 2009, GenBank accession no. GU292347) was PCR amplified, cloned into the pFastBac1 vector (Invitrogen) within the EcoRI and XhoI restriction sites, and expressed with a baculovirus expression system (Invitrogen) in an insect cell line. The sequence of the cloned HA was identical to that of the original isolate. The HA protein was purified by lentil lectin affinity chromatography (GE Healthcare) and used for ELISA.An indirect sandwich ELISA was performed. Briefly, a Maxisorb microtiter plate (Nunc) was coated with p-H1N1-09 HA protein, 2 g/well, and incubated at 37°C for 2 h. The plate was blocked with phosphate-buffered saline (PBS) containing 10% donor calf serum, 0.5% Tween 20, 0.5% gelatin (blocking solution) at 37°C for 30 min. A...

Rituximab impairs immunoglobulin (Ig)M and IgG (subclass) responses after influenza vaccination in rheumatoid arthritis patients

“…Our study shows that influenza‐specific IgG and IgM antibodies can be measured by ELISA, which has advantages over the HI method. Commercially available IgG and IgM anti‐influenza type A or B ELISAs have been used in literature, but not compared to HI . Another study reported on the use of an IgG ELISA using the pandemic H1N1 HA protein as a coating antigen, and they found a concordance of 98·4% with HI .…”

Two Years after Pandemic Influenza A/2009/H1N1: What Have We Learned?

“…However, this testing cannot differentiate different lineages of the same subtype of the influenza A virus. The majority of individuals have already been exposed to the seasonal influenza A virus, which has almost identical NP and matrix protein sequences; therefore, a 4-fold rise in the complement fixation antibody titer with these antigens indicates a recent influenza A virus infection without differentiating between seasonal H1N1 or H3N2 infection and the pandemic 2009 H1N1 infection (14). Although recombinant H has been found to be sensitive and specific for antibody detection by EIA, cross-reactivity between seasonal H1N1 and pandemic H1N1 may still occur, because their overall amino acid identity was 80.1% for H (440 out of 549 amino acids), 72% for HA1, and 92% for HA2 ( Fig.…”

Expression of recombinant HA1 protein for specific detection of influenza A/H1N1/2009 antibodies in human serum

“…When highly pathogenic or novel influenza strains are involved, specialized laboratories with stronger containment facilities and specially trained personnel are required for safe implementation of virus culture and testing . In contrast, ELISA is a suitable alternative for seroepidemiological surveillance testing because it is highly sensitive and easy to perform , . In this study, we describe the development and use of an indirect ELISA method that is based on the use of recombinant HA1 proteins of the 2009 H1N1 viruses isolated from humans in Mexico and from swine in Canada and that specifically recognizes A(H1N1)pdm09 virus antibodies in human serum samples.…”