Serological diagnosis of influenza A and B infections by enzyme immunoassay. Comparison with the complement fixation test

Paired sera from 784 patients with symptoms of acute respiratory disease were examined for antibodies against influenza A, B and parainfluenza (1 and 3) viruses by complement fixation (CF) and enzyme immunoassay (EIA). The internal variation of the EIA test results was low and an increase of 0.250 in absorbance values which corresponded to a two-fold increase in end-point titres was considered a diagnostic antibody rise. EIA detected significantly more diagnostic rises than the CF test in the case of influenza A (222 vs. 162, P less than 0.001) and parainfluenza virus antibodies (29 vs. 16, P less than 0.01). More diagnostic rises in influenza B antibodies were also observed by EIA compared to the CF test (104 vs. 99, not significant). There were only two patients who showed a diagnostic rise in CF antibodies (both influenza B) but not in EIA. Most often patients with a diagnostic antibody rise only by the EIA method had a two-fold rise in the respective CF antibodies (68% of cases). EIA was found to be a sensitive and reliable method for the serological diagnosis of influenza A, B and parainfluenza infections.     

Serological diagnosis of parainfluenza virus infections by enzyme immunoassay with special emphasis on purity of viral antigens

Enzyme immunoassay (EIA) for parainfluenza virus type 1, 2, and 3 antibodies was compared with the complement fixation test (CF) as a diagnostic method in 180 patients with respiratory symptoms. The CF test detected rises in parainfluenza virus antibodies in 30 cases, whereas EIA detected 47 rises. Patients with antibody rises in parainfluenza or mumps virus antibodies were studied for cross-reactions by CF, hemagglutination inhibition (HAI), and EIA using purified viral envelope glycoprotein and nucleocapsid preparations. All methods showed marked cross-reactivity between parainfluenza virus type 1 and 3 antibodies. Mumps virus infection often raised heterologous antibodies even against purified viral antigens. Rabbit antisera produced against viral envelope glycoproteins showed heterologous antibody responses between parainfluenza 1 and 3 antibodies and between parainfluenza 1 and mumps antibodies by HAI, EIA, and immunoprecipitation. The cross-reactive antibodies were usually directed against both of the envelope glycoproteins, HN and F proteins, of the viruses.     

H1亚型流感病毒HA蛋白单克隆抗体的制备及部分特性鉴定

目的:制备针对H1亚型流感病毒HA蛋白的单克隆抗体(mAb),并分析其反应特性。方法:分别以2009年甲型H1N1、季节性A1流感病毒裂解疫苗为免疫原,常规法免疫、融合、克隆化,获得各抗原特异性mAb。应用ELISA、HI试验和Western blot等技术研究mAb的反应性和特异性。结果:获得稳定分泌抗H1亚型流感病毒HA蛋白的杂交瘤细胞97株。其中株特异性mAb39株,29株具有HI活性;亚型特异性mAb7株,5株具有HI活性;2009年流行株与季节性A1、A3流行株共同抗原的mAb16株,9株具有HI活性;针对流感病毒共同抗原mAb35株,22株具有HI活性。结论:两种疫苗均具有较好的免疫原性和免疫保护活性,这些mAb的获得为流感病毒株特异、亚型特异性诊断试剂盒及流感病毒通用诊断试剂盒的制备提供了实验资料,为进一步研究H1N1流感病毒HA的抗原表位奠定了基础。     

Profiling of humoral immune responses to influenza viruses by using protein microarray

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.     

Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes

The hemagglutination inhibition (HI) assay is a widely used serological method to measure the levels of protective antibody responses against influenza viruses. However, the traditional HI assay which uses chicken erythrocytes is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes was able to increase the sensitivity of HI assay. The current report describes further optimization of this modified HI assay. It was shown that this method was able to increase detection of HI activities in rabbit sera immunized with H5 HA antigens, and proved that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, the modified HI assay using horse erythrocytes increased the sensitivity of detecting HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people who may have asymptomatic infection with avian influenza viruses. Based on these results, the optimized use of horse erythrocytes should be standard practice for detecting HI activities against avian influenza viruses.     

Evaluation of the Single Radial Hemolysis Test for Measuring Hemagglutinin- and Neuraminidase-Specific Antibodies to H3N2 Influenza Strains and Antibodies to Influenza B

Antibodies to the H3 hemagglutinin of influenza A virus could be specifically measured by single radial hemolysis (SRH) when test antigens were recombinant viruses containing the relevant H3 hemagglutinin antigen and irrelevant Neq1 neuraminidase of A/equine/Prague/1/56 virus. Antibodies to influenza B virus could also be measured by the SRH technique. Antibody rises to influenza A or B virus measured by SRH agreed with results of hemagglutination inhibition (HI) tests for about 80% of the sera tested, including sera from volunteers receiving killed influenza vaccine and sera from patients naturally infected with influenza. Correlation between antibody titers measured by SRH and HI was also good. Antibodies to the N2 neuraminidase of influenza A virus could be specifically measured by SRH when test antigens were recombinant viruses containing the relevant N2 neuraminidase antigen and irrelevant Heq1 hemagglutinin of A/equine/Prague/1/56 virus. The SRH test for neuraminidase antibodies was more strain specific than was the SRH test for hemagglutinin antibodies. Probably for this reason, agreement between neuraminidase antibody determinations in human sera by the SRH test and by the neuraminidase inhibition test was poorer than agreement between the SRH test for hemagglutinin antibodies and the HI test.     

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.     

Detection of cells producing surface antigen-specific antibody to influenza viruses.

B cells producing antibodies to influenza virus antigens were detected and quantitated by a hemolytic plaque assay. Responses of mice after primary infection and immunization with influenza viruses were measured and compared with responses after secondary immunization. The B-cell responses were specific and differentiated between A and B influenza viruses and between different subtypes of A influenza viruses. Responses to closely related influenza A virus strains of the H3N2 subtype cross-reacted but could also be differentiated. Cells secreting antibody to either of the virus surface antigens (hemagglutinin and neuraminidase) could be separately enumerated. Evidence that immunoglobulin G- secreting cells are detected in the assay without the use of facilitating anti-immunoglobulin G sera is presented.     

Genetic and Antigenic Typing of Seasonal Influenza Virus Breakthrough Cases from a 2008-2009 Vaccine Efficacy Trial

Estimations of the effectiveness of vaccines against seasonal influenza virus are guided by comparisons of the antigenicities between influenza virus isolates from clinical breakthrough cases with strains included in a vaccine. This study examined whether the prediction of antigenicity using a sequence analysis of the hemagglutinin (HA) gene-encoded HA1 domain is a simpler alternative to using the conventional hemagglutination inhibition (HI) assay, which requires influenza virus culturing. Specimens were taken from breakthrough cases that occurred in a trivalent influenza virus vaccine efficacy trial involving >43,000 participants during the 2008-2009 season. A total of 498 influenza viruses were successfully subtyped as A(H3N2) (380 viruses), A(H1N1) (29 viruses), B(Yamagata) (23 viruses), and B(Victoria) (66 viruses) from 603 PCR- or culture-confirmed specimens. Unlike the B strains, most A(H3N2) (377 viruses) and all A(H1N1) viruses were classified as homologous to the respective vaccine strains based on their HA1 domain nucleic acid sequence. HI titers relative to the respective vaccine strains and PCR subtyping were determined for 48% (182/380) of A(H3N2) and 86% (25/29) of A(H1N1) viruses. Eighty-four percent of the A(H3N2) and A(H1N1) viruses classified as homologous by sequence were matched to the respective vaccine strains by HI testing. However, these homologous A(H3N2) and A(H1N1) viruses displayed a wide range of relative HI titers. Therefore, although PCR is a sensitive diagnostic method for confirming influenza virus cases, HA1 sequence analysis appeared to be of limited value in accurately predicting antigenicity; hence, it may be inappropriate to classify clinical specimens as homologous or heterologous to the vaccine strain for estimating vaccine efficacy in a prospective clinical trial.     

Hemagglutinin and neuraminidase specific cell-mediated immune responses to influenza A virus following immunization in guinea pigs

Groups of guinea pigs were immunized with different inactivated recombinant influenza A viruses including H3ChN2Ch, Heq1N2Ch, H3ChNeq1 or uninfected allantoic fluid. Employing hemagglutination and neuraminidase inhibition tests, an in-vitro lymphocyte transformation (LTF) assay, and rosetting techniques for the separation of lymphocytes, influenza hemagglutinin (HA) and neuraminidase (NA) specific antibody and cell-mediated immune (CMI) responses were evaluated. Inactivated H3ChN2Ch, H3ChNeq1, HavN2Ch, and Heq1Neq1 recombinant influenza viruses were used as test antigens. Following immunization the CMI and antibody responses to influenza were characterized by the induction of specific LTF and antibody activity to homotypic HA or NA antigens but not to heterotypic HA or NA antigens. The temporal kinetics of the antibody response to influenza antigens was characterized by a prompt onset being initially detected at 1-2 weeks and reaching peak titers 3-4 weeks after immunization. Influenza specific LTF responses were first detected one week after immunization and declined to minimal responses at eight weeks. T-lymphocytes but not B-lymphocytes were capable of in-vitro recognition of the HA and NA antigens. After recognition the subsequent in-vitro lymphoproliferation was shown to involve both T and B lymphocytes.     

Impaired serum antibody response to inactivated influenza A and B vaccine in renal transplant recipients.

Before and after vaccination with a commercial inactivated influenza vaccine containing A/Aichi/2/68 (H3N2) and B/Massachusetts/1/71 antigens, the serum hemagglutination inhibition antibody titers to homologous and heterologous strains of A and B influenza viruses were measured in 45 renal transplant patients and 66 healthy controls (62 for the B strains). At least a fourfold titer rise to the homologous A strain occurred in 14 of 45 transplant patients (31%) versus 37 of 66 controls (56%). Fourfold or greater heterologous A rises occurred in only 8 of 45 transplant patients (18%) compared with 40 of 60 controls (61%). In both the homologous and heterologous B responses, at least fourfold hemagglutination inhibition titer rises were seen in significantly fewer transplant patients than control subjects. In the transplant group, no correlation was observed between degree of antibody response and age, previous influenza vaccination, percentage of patients initially seronegative, time since transplantation, dose of immunosuppressive drugs, level of renal function, or nature of original renal disease.     

Antigen-specific H1N1 influenza antibody responses in acute respiratory tract infections and their relation to influenza infection and disease course

BackgroundEarly antibody responses to influenza infection are important in both clearance of virus and fighting the disease. Acute influenza antibody titers directed toward H1-antigens and their relation to infection type and patient outcomes have not been well investigated.ObjectiveUsing hemagglutination inhibition (HI) assays, we aimed to characterize the H1-specific antibody titers in patients with influenza infection or another respiratory infection before and after the H1N1-pandemic influenza outbreak. Among patients with acute influenza infection we related duration of illness, severity of symptoms, and need for hospitalization to antibody titers.MethodsThere were 134 adult patients (average age 34.7) who presented to an urban academic emergency department (ED) from October through March during the 2008–2011 influenza seasons with symptoms of fever and a cough. Nasal aspirates were tested by viral culture, and peripheral blood serum was run in seven H1-subtype HI assays.ResultsAcutely infected influenza patients had markedly lower antibody titers for six of the seven pseudotype viruses. For the average over the seven titers (log units, base 2) their mean was 7.24 (95% CI 6.88, 7.61) compared with 8.60 (95% CI 8.27, 8.92) among patients who had a non-influenza respiratory illness, p < 0.0001. Among patients with seasonal influenza infection, titers of some antibodies correlated with severity of symptoms and with total duration of illness (p < 0.02).ConclusionIn patients with acute respiratory infections, lower concentrations of H1-influenza-specific antibodies were associated with influenza infection. Among influenza-infected patients, higher antibody titers were present in patients with a longer duration of illness and with higher severity-of-symptom scores.     

Serum antibody responses in naturally occurring influenza A virus infection determined by enzyme-linked immunosorbent assay, hemagglutination inhibition, and complement fixation

Serum antibody responses to influenza A virus infection were examined in 388 normal subjects during a trial of chemoprophylaxis in an outbreak of influenza A in 1980-1981 in which both A/H1N1 and A/H3N2 viruses circulated. Paired serum specimens obtained over a 6-week period were tested for antibodies to both A/H1N1 and A/H3N2 viruses by conventional hemagglutination inhibition, complement fixation, and an enzyme-linked immunosorbent assay (ELISA). Antibody responses detected by ELISA were determined by calculation of the area generated between titration curves of paired sera (area method), as well as by a conventional endpoint dilution method (endpoint method). Forty-two significant antibody rises were detected; 42 by ELISA (area method), 33 by ELISA (endpoint method), 32 by hemagglutination inhibition, and 13 by complement fixation. ELISA (area method) detected rises more frequently than either ELISA (endpoint method) (P less than 0.01), hemagglutination inhibition (P less than 0.005), or complement fixation (P less than 0.001). Another sensitive assay, the microneutralization test, detected significantly fewer rises (33, P less than 0.025) than the ELISA (area method). In the 42 subjects with ELISA (area method) rises, corroborating evidence of influenza A infection by other techniques (virus isolation, microneutralization, hemagglutination inhibition, or complement fixation tests) were available for 39 (93%). ELISA (area method) rises were subtype specific in all serum pairs in which other documentation of subtype-specific infection was available (38 of 38). Thus, ELISA (area method) was the single most sensitive assay for detection of serum antibody rises in this setting and possessed a high degree of subtype specificity.     

IgM,IgG, and IgA Antibody Responses to Influenza A(H1N1)pdm09 Hemagglutinin in Infected Persons during the First Wave of the 2009 Pandemic in the United States

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.     

Simultaneous determination of the level of antibodies to influenza virus surface and internal proteins by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) has been adopted for simultaneous determination of the levels of antibodies to different influenza virus proteins in human sera with known haemagglutination-inhibition (HI) titre. Whole virus of serotypes H1N1 and H3N2, haemagglutinin (HA), matrix (M) and nucleoprotein (NP) proteins have been used as antigens. For detection of antibodies bound to the antigen, peroxidase labelled Staphylococcus protein A conjugate has been used. Correlation of the ELISA and HI titres of anti-HA antibody has been demonstrated. The use of isolated HA as antigen increased the specificity of ELISA. The analysis of human reconvalescent sera has shown that increase in the titre of antibodies to internal proteins does not always coincide with the increase of antibody level to HA. Out of 8 sera with significant increase of the HI titre to the H3 subtype 5 specimens showed 4-fold increase of antibody titre to NP protein. The antibody titre to M protein was elevated in 2 sera only, while 1 serum showed no rise of antibody response to the tested viral proteins.     

Analysis of antigenic relationships among influenza virus strains using a taxonomic cluster procedure. Comparison of three kinds of antibody preparations

Hemagglutination inhibiting (HI) monoclonal antibody preparations (MA) were raised against six influenza A (H3N2) strains from the period 1977-1982. Twenty-three hybridomas were selected and titrated in HI assays against these strains and against 18 influenza A (H3N2) viruses isolated in The Netherlands during the seasons 1981-1982 and 1982-1983. Similar HI tests were performed with conventional post-infection ferret antisera and with ferret antisera adsorbed with heterologous strains of influenza A (H3N2) virus. The resulting serological data were subjected to a computerized taxonomic cluster procedure based on the Euclidean distance between viruses. With respect to the degree of separation between clusters the unadsorbed ferret antisera were inferior to the adsorbed antisera whereas the MA were superior to both. Our results demonstrate that computer programs based on numerical taxonomy can be helpful in processing large numbers of serological data and that MA are indispensable in epidemiological and diagnostic influenza studies.     

An improved membrane-filtration enzyme immunoassay for the rapid serological diagnosis of viral infections

A one-step modification of the membrane-filtration enzyme immunoassay (MF EIA) (Barnett et al. J. Clin. Microbiol., 23:385–399, 1987), for estimation of virus-specific antibody is described. The modified MF EIA allowed serum, antigen and enzyme-conjugated anti-globulin to be incubated together in membrane-based 96-well plates to enable the formation of immune complexes in solution at 37°C. The assay required only 45 min for completion and polyethylene glycol was shown to be an essential component in reaction mixtures for IgG assays to enhance immune complex formation. The modified MF EIA was as sensitive as the previous two-step method for monitoring responses to influenza vaccin, and control antigen backgrounds were significantly reduced. The one-step procedure was also shown to be suitable for the rapid serodiagnosis of naturally acquired influenza A and B infections. However, MF EIA detected cross-reactive H1N1 responses in 57.7% of naturally-acquired H3N2 infections, suggesting that responses to common internal antigens were being measured. Cross-reactive responses to influenza A viruses could not be detected in volunteers receiving subunit vaccines.     

Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: Unmasking of cross-reactive HA2 determinants

Acid treatment of influenza A and B virus preparations followed by addition of dithiothreitol (DTT) and centrifugation through a sucrose cushion removes the HA1 subunit of hemagglutinin from virus. Rabbit sera made against these subviral particles and untreated virus were tested in a radioimmune precipitation assay using [³⁵S]cysteine-labeled virus. Conditions of the assay permitted discrimination of discrete HA1- and HA2-specific antibody populations. It was found that (a) sera raised to intact influenza A virus preparations contained both HA1- and HA-2 specific antibodies, (b) sera made to subviral particles of influenza A virus contained HA2-specific antibody but had little or no detectable HA1-specific antibody. (c) the HA2-specific antibodies were partially cross-reactive with the HA2 of an influenza A virus of a different subtype, and (d) sera raised against two strains of untreated influenza B viruses contained antibodies which were cross-reactive with the HA2 as well as the NP of influenza A viruses.     

Analysis of antigenic drift in recently isolated influenza A (H1N1) viruses using monoclonal antibody preparations.

Monoclonal antibodies to the hemagglutinin (HA) and neuraminidase (NA) of A/USSR/ 90/77(HlN1) were prepared and used to study antigenic drift in the H1N1 subtype of influenza viruses. The results obtained with five different monoclones to each molecule were compared with the results obtained with postinfection ferret sera. Monoclonal antibodies and postinfection ferret sera detected antigenic drift in the hemagglutinin and neuraminidase molecules of H1N1 viruses and monoclonal antibodies showed that the A/USSR/90/77, A/ Roma/1/49, and A/Fort Warren/1/50 viruses were identical at five sites on both the hemagglutinin and neuraminidase molecules. To further evaluate monoclonal antibodies, they were used in hemagglutination inhibition tests with recently isolated influenza A(HlNl) viruses, including some shown in tests with postinfection ferret sera to have undergone antigenic drift. Three patterns of reactivity with the monoclonal antibodies were detected with the variants: two variants differed at two of the five sites identified by the monoclonal antibodies and a third variant differed at only one of the five sites. Two of these variant groupings had not been distinguished from each other in HI tests with ferret sera, whereas ferret sera were capable of distinguishing between two groups of variants that had similar reaction patterns with the five monoclonal antibody preparations. Monoclonal antibodies failed to detect antigenic drift in the NA molecules of the recent isolates of H1N1 influenza viruses, suggesting that antigenic changes occur less frequently in this molecule. The observation that antigenic differences could be detected between the recent variants with such a small panel of monoclonal antibodies (less than 10% of the number obtained to the HA of A/PR/8/34) suggests that monoclonal antibodies may provide an exquisitely sensitive method for antigenic mapping of influenza viruses.     

Serological survey of antibodies to influenza A viruses in a group of people without a history of influenza vaccination

A serological survey for antibodies to influenza viruses was performed in China on a group of people without a history of influenza vaccination. Using the haemagglutination inhibition (HI) assay, we found seropositivity rates for seasonal H3N2 to be significantly higher than those for seasonal H1N1. Samples positive for antibodies to the pandemic (H1N1) 2009 virus increased from 0.6% pre-outbreak to 4.5% (p <0.01) at 1 year post-outbreak. Interestingly, HI and neutralization tests showed that 1.4% of people in the group have antibodies recognizing H9N2 avian influenza viruses, suggesting that infection with this subtype may be more common than previously thought.