Hepatitis A virus hemagglutination and a test for hemagglutination inhibition antibodies.

Like enteroviruses, hepatitis A virus (HAV) hemagglutinated various species of erythrocytes under similar conditions. HAV-specific antibodies in both acute- and convalescent-phase sera were found to inhibit hemagglutination. The HAV hemagglutination inhibition test can be used for diagnosis, epidemiological surveillance, and vaccine assessment.     

Comparison of 2 methods for determining antibodies to collagen: the ELISA test and passive hemagglutination

For the determination of antibodies against collagen in different rheumatic diseases the authors elaborated two serological techniques. It was particularly the passive haemagglutination, which proved to be little sensitive and insufficiently reproducible. Therefore, for the determination of antibodies against collagen the authors introduced the ELISA method as one of the varieties of enzyme immunoanalysis, giving more precise results. Both methods were compared and it has become apparent that the ELISA method is more reliable and more suitable for the determination of antibodies against collagen.     

Comparison of hemagglutination inhibition test and enzyme-linked immunosorbent assay for determining antibody to rubella virus.

The hemagglutination inhibition test (HAI) and the enzyme-linked immunosorbent assay (ELISA) for detecting antibody to rubella virus were compared by testing 25 sets of paired sera taken before and after infection and 10 sets of sera taken during acute and convalescent stages of the disease and by screening 700 serum samples from the Collaborative Perinatal Project, NIH/NINCDS. The tests were found to be comparable in their ability to detect positive and negative sera, rises in titers, and seroconversions. When a purified antigen and carefully prepared reagents were used, ELISA was found to be as accurate and reliable as HAI. ELISA required no pretreatment of serum, could easily be automated, and was less time-consuming than HAI.     

Comparison of the latex agglutination test with the hemagglutination inhibition test, enzyme-linked immunosorbent assay, and neutralization test for detection of antibodies to rubella virus.

The ability of a rapid, latex agglutination test to diagnose rubella infection and to measure immune status was evaluated by comparison with the hemagglutination-inhibition (HAI) test, enzyme-linked immunosorbent assay (ELISA), and the neutralization (NT) test. The latex agglutination test accurately detected serological conversions in 74 pairs of sera representing 21 natural infections and 53 immunizations. The antibody levels of 276 sera from the general population were determined by latex agglutination, HAI, and ELISA. The correlation coefficients between the titers obtained by HAI and latex agglutination and by ELISA and latex agglutination were statistically significant. Results on 12 sera did not agree when measured by the three tests. These sera were included among the 196 specimens tested by NT. The correlation coefficient between NT and latex agglutination titers was statistically significant. There was one serum positive by latex agglutination but negative by NT, and five sera were negative by latex agglutination but had titers of 4 to 8 in the NT. The relative sensitivity of detecting antibody was greater by latex agglutination than by HAI. An additional 49 sera containing residual nonspecific hemagglutinin inhibitors were evaluated by latex agglutination and NT. The untreated sera showed no false positive reactions, and 36 of 39 NT positive sera were positive in the latex agglutination test.     

Comparative serological studies with egg drop syndrome virus

The sensitivities of five serological tests (haemagglutination inhibition, ELISA, serum neutralisation, fluorescent antibody and agar gel immunodiffusion) for detection of Egg Drop Syndrome virus antibody were compared. In experimentally inoculated birds seroconversion was first detected at five days post-inoculation using haemagglutination inhibition, ELISA, serum neutralisation and fluorescent antibody test, and at seven days post-inoculation using agar gel immunodiffusion. Sera from birds experimentally infected with two or more different fowl adenovirus serotypes gave positive reactions in some of these tests, as did a small percentage of haemagglutination inhibition negative field sera. It is concluded that only haemagglutination inhibition or serum neutralisation should be used for detection of infection in commercial birds.     

Serological survey for the prevalence of antibodies to egg drop syndrome 1976 virus in domesticated and wild birds in Israel

A serological survey of chicken, turkey, goose and duck flocks for the presence of antibodies to egg drop syndrome virus 1976 (EDS 76) has been carried out in Israel. In most of the chicken flocks sampled egg production was not normal, but no antibodies against this virus were detected. Likewise, turkey breeding flocks were similarly negative. All Pekin duck flocks tested showed some serological activity. Muscovy ducks that were reared in direct or indirect contact with Pekin ducks had haemagglutination-inhibition antibodies to EDS 76 virus. Seven cattle egrets trapped on a duck farm were serologically positive. No antibodies were detected in a collection of water fowl raised in a zoological garden.     

Detection of BK virus IgM antibodies by two enzyme-linked immunosorbent assays (ELISA) and a hemagglutination inhibition method

We have used an antigen solid-phase enzyme-linked immunosorbent assay (SP-ELISA) and an IgM antibody capture ELISA (MACELISA) for detecting IgM antibodies to human polyomavirus BK (BKV). These tests were compared with the standard hemagglutination inhibition test (HAI) of IgM serum fractions following sucrose density gradient fractionation. The SP- and MACELISA were not influenced by concomitant BKV-IgG, but high levels of both BKV-IgG and rheumatoid factor could cause false positive results by SPELISA, but not by MACELISA. The MACELISA gave much higher positive to negative ratios than the SPELISA. The sensitivity and specificity of the two tests were high compared to the IgM-HAI method. The sera could be tested in a single dilution (1:160), and thus the ELISA-tests are useful for testing large numbers of sera.     

Purification and hemagglutinating properties of egg drop syndrome 1976 virus

Summary We purified three populations of virus particles, F7, F9 and F17, with buoyant densities of 1.34, 1.33 and 1.29 g/ml, respectively, in CsCl equilibrium density gradients from cultures of chick embryo liver cells infected with the H-162 strain of the virus of egg drop syndrome 1976. F9 particles were infectious complete virions and most F17 particles were empty particles. F7 particles were less infectious, and had little capacity of hemagglutination (HA). HA titers were the same at 4° and 37° C and maximal between pH 6.4 and 8.4 and ionic strength from 0.14 to 0.54m of NaCl. HA titer was inversely proportional to erythrocyte concentration. Potassium periodate destroyed markedly the infectivity of the virus and partially its HA activity at 37° C. HA activity was stable at 56° C or lower temperatures and destroyed at 80° C. Trypsin, -chymotrypsin, papain, ficin and neuraminidase had no effect on HA activity. Alpha-chymotrypsin destroyed the receptor for the virus on chicken erythrocytes, whereas trypsin and neuraminidase did not affect the receptor.With 2 Figures     

Comparison of a simple latex agglutination test with hemolysis-in-gel, hemagglutination inhibition, and radioimmunoassay for detection of rubella virus antibodies.

Rubella virus antibodies were measured in 300 sera from pregnant women visiting a maternity center by using a new, simple latex test, Rubalex. The results were compared with those obtained by using hemolysis in gel, hemagglutination inhibition, and radioimmunoassay. The sensitivity of the latex test was 100, 98.0, and 99.6% when compared with hemolysis in gel, hemagglutination inhibition, and radioimmunoassay, respectively. Its comparative specificity was 96.2, 95.7, and 90.7%, and the predictive value of a positive result was 99.2, 99.2, and 98%, respectively. When assayed with the British standard anti-rubella serum its sensitivity was 11 IU/ml. The latex test gave a positive result within 2 min, and 87% of the positive samples had already reacted after 1 min. The negative results remained as such for at least 8 min. No prozone effect was observed for sera with hemagglutination inhibition titers from 256 to 2,048. We concluded that the latex test, Rubalex, was readily applicable for measuring rubella immunity with a reaction time of 2 min in undiluted samples.     

Measurement of antibodies to avian influenza virus A(H7N7) in humans by hemagglutination inhibition test

During the epizootic of highly pathogenic avian influenza A(H7N7) in 2003 in The Netherlands, RT-PCR and culture confirmed infection was detected in 89 persons who were ill. A modified hemagglutination inhibition (HI) test using horse erythrocytes and 2 hemagglutinating units of virus was applied to assess retrospectively the extent of human (subclinical) infection. Validation of the HI-test with sera from 34 RT-PCR and culture confirmed A(H7) infected persons and sera from 100 persons from a human influenza vaccine trial in autumn 2002 showed that this HI-test had a sensitivity of 85% and a specificity of 100% when using a cut-off titer of > or =10. Using this cut-off value, A(H7) specific antibodies were detected in 49% of 508 persons exposed to poultry and in 64% of 63 persons exposed to A(H7) infected persons. Correlation of seropositivity with the occurrence of eye symptoms in exposed persons who had not received antiviral prophylaxis and of reduced seropositivity with taking antiviral prophylaxis provided further evidence that the A(H7) HI antibody titers were real. In conclusion, by applying an HI-test using horse erythrocytes human antibodies against the avian A(H7N7) virus were detected with high sensitivity and specificity in an unexpectedly high proportion of exposed persons.     

Nonspecific reactions in the hemagglutination inhibition test for detection of rubella antibodies.

Approximately 7% of the sera tested to determine the presence of rubella-specific antibodies by the hemagglutination inhibition test demonstrated abnormal patterns of reactivity, rendering the test unreadable. Another 3% of sera were shown to have false-positive titers as high as 1:128. When these abnormally reacting and false-positive sera were heated at 56 degrees C for 30 min after chemical treatment they always converted to negative, indicating the absence of specific rubella hemagglutination-inhibiting antibody. These results were confirmed by fractionation of the sera after sucrose gradient centrifugation. It was established that manifestation of these nonspecific results was dependent on the concentration of Ca2+ or Mn2+. The heat-labile inhibitor(s) responsible for abnormal and false-positive reactions was found not to be complement. This inhibitor(s) was detected in the light fractions of sera and when added to negative sera was capable of reproducing the abnormal patterns of reactivity. These results emphasize the necessity of heating sera for the rubella hemagglutination inhibition test after the chemical removal of nonspecific inhibitors.     

A hybridoma secreting a neutralising monoclonal antibody to egg drop syndrome 1976 virus

A hybridoma (HPRS/AM/1) secreting neutralising antibody to Egg drop syndrome 1976 virus strain D61 was established. This monoclonal antibody also neutralised two other strains tested thereby showing that all the strains possess at least one common antigenic determinant which is important in infection. However, this epitope was not involved in haemagglutination. The antigen was identified in the nuclei of infected chick kidney cells by an indirect fluorescent antibody test.     

Genomic characterization of Indian isolates of egg drop syndrome 1976 virus

Five Indian isolates of egg drop syndrome (EDS) 1976 virus and the reference strain 127 were compared by restriction enzyme analysis of viral DNA, and the hexon gene amplified by polymerase chain reaction. Using these techniques, no differences were seen among these viruses. However, partial sequencing of the hexon gene revealed major differences (4.6%) in one of the isolates sequenced, EDS Kerala. Phylogenetic analysis also placed this isolate in a different lineage compared with the other isolates. The need for constant monitoring of the genetic nature of the field isolates of EDS viruses is emphasized.     

Preparation of noninfectious hepatitis A virus hemagglutinin for detecting hemagglutination inhibition antibodies

Hepatitis A virus (HAV) harvested from infected MRC-5 cells can hemagglutinate various species of erythrocytes at acid pH (Eckels et al., 1989). Further studies revealed that the majority of the hemagglutinin (HA) in MRC-5 and BS-C-1 cells was cell-associated. A simplified procedure for preparing HAV-HA consisted of collecting infected cells in phosphate-buffered saline followed by three cycles of freeze-thawing and sonication. The fluids were clarified and stored at 4 degrees C. The analysis of HA by rate-zonal sucrose gradient centrifugation indicated that the majority of HA co-migrated with infectious virus. Complete inactivation of infectious HAV with 0.03% beta-propiolactone (BPL) did not affect HA activity, while inactivation with 0.05% formalin caused a 16-fold reduction in titer. There was no difference in HAI antibody titers when BPL-treated HA was compared to untreated HA in the hemagglutination inhibition (HAI) test.     

Comparison of hemagglutination inhibition,serum neutralization and ELISA-PVM tests for the detection of antibodies to pneumonia virus of mice in rat sera

Recently, the seroneutralization, hemagglutination inhibition, and the ELISA procedures were used to determine the presence of serum antibody to the pneumonia virus of mice in rat sera. This study was initiated to evaluate the reliability of these procedures to detect specific antibodies in our laboratory animal species.The results obtained indicate that: 1) the seroneutralization test is not reliable since numerous false-positive sera were detected; 2) the ELISA is more sensitive than both the seroneutralization and hemagglutination inhibition procedures, and 3) the presence of low hemagglutination inhibition titers should not be considered significant.     

Differentiation of egg drop syndrome virus isolates by restriction endonuclease analysis of virus DNA

Thirteen isolates of egg drop syndrome (EDS) virus were compared by restriction endonuclease analysis of the virus DNA. One virus, an Australian chicken isolate, was distinguished from the others using the endonucleases EcoRl, BamUl, Kpnl, Hindlll, Pstl and Pvull, all of which recognise six base pair DNA sequences. Polyacrylamide gel restriction fragment patterns generated by Haelll, Hhall and TaqI, which recognise four base pairs, allowed further differentiation of the virus isolates. Nine chicken viruses isolated in the United Kingdom and Belgium in the period 1976 to 1987 were identical and could be distinguished from three United Kingdom duck isolates. The Australian isolate, in addition to possessing a DNA deletion (0.4 kbp) at one end of the genome (32.6 kbp) also differed from the European isolates at base sequence level. Genome maps for EcoRl, BamHl, Kpnl and Pstl are reported for the 127 isolate of EDS virus.     

Serodiagnosis of rubella: comparison of ELISA and hemagglutination inhibition

Serodiagnosis in current practice by the method ELISA. Utilization of commercial kits. I. - Rubella. Comparative study with hemagglutination inhibition test. A comparative study of the hemagglutination inhibition test and the method ELISA for the serodiagnosis of rubella in current practice has been achieved on 1 000 sera using commercial products in kits. The conditions of utilization, the principal factors of the reaction and the expression of results in ELISA titres are defined. The results show a higher sensibility and a higher precision for ELISA and an uncertain interpretation concerning the low titres for IHA. The use of ELISA for the serodiagnosis of rubella in routine with the kit Rubelisa does not present any particular difficulty and raises the quality of the results.     

Recombinant egg drop syndrome subunit vaccine offers an alternative to virus propagation in duck eggs

Egg drop syndrome (EDS) virus vaccines are routinely produced in embryonated duck eggs (Solyom et al., 1982). This procedure poses the risk of dissemination of pathogens, such as avian influenza virus, as the eggs used are not from specific pathogen free birds. To address this problem, the knob and part of the shaft domain of the fibre protein of the EDS virus (termed knob-s) were expressed in Escherichia coli and assessed as a subunit vaccine. A single vaccination with the recombinant protein induced the production of anti-EDS virus antibodies, as detected by haemagglutination inhibition, enzyme-linked immunosorbent assay and virus neutralization tests, for at least 20 weeks. A positive correlation was demonstrated between these three assays. A dose-response assessment showed that the vaccine was effective over the range of 2 to 64 microg protein per dose. Two vaccinations with the recombinant protein, administered before the onset of lay, induced high haemagglutination inhibition antibody titres, comparable with those induced by an inactivated whole-virus vaccine. The vaccine did not have any adverse effects on egg production, quality or weight. The present study has shown that two vaccinations with the recombinant knob-s protein elicited high neutralizing antibody titres that persisted for more than 50 weeks of lay.