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A new diluent (ADGP) for the rubella virus hemagglutination (HA) and hemagglutination-inhibition (HI) tests is described. It was found that the HA and HI titers and the erythrocyte agglutination pattern were improved in ADGP compared to previously described diluents. The influence of the components of ADGP and of various test conditions on optimal HA and HI results were examined.
Groups of turkeys inoculated intravenously with hemagglutinating (HA) and nonhemagglutinating (NHA) strains of Mycoplasma meleagridis developed similar antibody titers as demonstrated by the tube agglutination (TA) test, but use of the hemagglutination-inhibition (HI) test indicated markedly higher titers for turkeys inoculated with the HA strain than for those inoculated with the NHA strain. Adsorption of serum antibody with NHA strain antigen removed most agglutination activity but had little effect on HI activity. These results indicate that the HI and TA tests demonstrated antibody against different antigens, with the HI test detecting antibody directed against the hemagglutinin and the TA test detecting antibody directed against membrane antigens common to both strains.
Hemagglutination inhibition (HI) tests against whole virus (WV) influenza B/Singapore/222/79 antigen detected prevaccination serum antibody in only 15 (20%) of 50 predominantly elderly volunteers and fourfold or greater titer rises in only three (6%) after they received 1981-1982 trivalent influenza vaccine containing antigens of this virus. HI titers against ether-treated (ET) B/Singapore/222/79 were about eightfold higher than those against WV antigen and were comparable to microneutralization titers against this virus. The ET HI detected prevaccination antibody in 84%, a postvaccination titer rise in 32%, and a final titer of 80 or higher in 66%. Among 51 additional persons with known or presumed influenza B virus infections early in 1982, ET B/Singapore/222/79 was also more sensitive than WV for serodiagnosis (69 versus 49%), but eight persons with both WV and ET B/Singapore/222/79 HI responses also had an HI titer rise to WV A/Brazil/11/78 (H1N1) antigen. Conversely, among 14 college students with febrile, culture-proven influenza A (H1N1) infections early in 1982, 6 (43%) developed HI titer rises to ET B/Singapore/222/79 with no other serological evidence of influenza B virus infection. Moreover, young adult volunteers with mild experimental influenza A (H1N1) infections also exhibited a 17% (3 of 18) incidence of ET B/Singapore/222/79 HI titer rises, versus none in matched, uninfected volunteers. These data indicate that ET B/Singapore/222/79 virus has increased sensitivity but reduced specificity compared to WV as an HI antigen and that caution is needed in interpretation of a single HI test for serodiagnosis, whether with WV or ET antigen.
The development of a hemagglutination inhibition test for urinary human follicle stimulating hormone (hFSHu) is described. Antisera were raised in rabbits and absorbed immunochromatographically with crude preparations of human chorionic gonadotrophin (hCG). hFSHu was coupled to erythrocytes by means of bis-diazobenzidine. The reagents, alone or in combination were subjected to various specificity tests such as reactions of hFSHu-coated erythrocytes with antisera against different antigens, reactions of anti-hFSHu with erythrocytes coated with different antigens, and reaction of "non-FSH" preparations on different test combinations of anti-hFSHu sera and hFSHu-coated erythrocytes. A comparative assay of an hFSHu preparation with different reagent combinations appeared to give the most stringent criterion for specificity. A modified hemagglutination inhibition test system was developed according to the principle reported earlier for urinary human luteninizing hormone (hLHu). Its sensitivity was fixed at 2 mIU FSH/ml which is sufficient for the estimation of hFSH in unconcentrated urine.
The dextran sulfate precipitation method for removal of nonspecific inhibitors from sera to be tested in the rubella hemagglutination-inhibition test was modified by shortening the dextran sulfate-serum mixture incubation period from 2 hr to 30 min. A comparative study using both treatment times showed no significant difference in rubella antibody titers. Reducing the treatment time even further also gave comparable results, indicating the select 30-min incubation period is not marginal for undertreatment. It was shown that 41.5% of the 159 serums tested had nonspecific agglutinins at levels from 1:8 to as high as 1:64. In each specimen, all demonstrable nonspecific agglutinin was removed by adsorption with 10%, 0- to 2-day-old chick red blood cells rather than the usually recommended 50% adult chicken erythrocytes.
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.
Four different assays for detection of rubella IgG antibodies, two latex agglutination (Rubalex and Rubascan), one hemagglutination inhibition (HAI) and one enzyme-linked immunosorbent assay (ELISA IgG), were used to test 372 human serum samples. All samples were also tested with a rubella ELISA IgM test and all of them were found negative. The results obtained in 358 (96.2%) out of 372 samples tested were identical with all procedures. Inconclusive results were obtained in 14 (3.8%) specimens, in that there were positive and negative results for the different assays. These 14 specimens were sent to the Institute of Virology in Turku (Finland) and tested by using a hemolysis in gel and a non commercially-available ELISA IgG test. Results obtained with the two last tests were considered definitive. The two latex tests were found equal or better than HAI and ELISA IgG for sensitivity and specificity. Furthermore they were also cost effective and more simple to perform.
Single radial hemolysis (SRH), neutralization (NT), and hemagglutination inhibition (HI) tests were carried out on sera from horses immunized against the Prague and Miami strains of equine influenza virus. The HI and NT tests demonstrated good sensitivity; the sensitivity of the SRH test was somewhat lower. The NT titers of individual sera were correlated very closely with the HI titers, although the NT titers were higher. SRH zone diameters of individual sera also showed significant correlation with the NT and NI titers. The SRH test appears to be suitable for large-scale serological surveys and offers the advantages of rapidity and simplicity.
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Modification of the pH of kaolin-serum treatment produces hemagglutination-inhibition test results comparable with the plaque neutralization test in duck embryo tissue culture; the standard treatment does not.
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.
The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to influenza B virus was compared with that of the hemagglutination inhibition test on acute- and convalescence-phase sera obtained from adults and children infected with influenza B virus. Two whole virus, tissue culture-grown antigen preparations were used in the ELISA, influenza B/West Virginia/81 and influenza B/Hong Kong/72. Four antigens were used in the hemagglutination inhibition test. These included the tissue culture-grown whole virus antigens that were used in the ELISA. In addition the standard egg-grown antigens, influenza B/Singapore/79 and influenza B/Hong Kong/72, were included for comparison. The ELISA antibody titer was significantly correlated to the hemagglutination inhibition antibody titer, and 10 of 10 adults and 17 of 21 children infected with influenza B had fourfold antibody increases as detected by ELISA with either antigenic type of tissue culture-grown whole virus. Increases in geometric mean antibody titers of 16- to 71-fold were detected by ELISA. Increases in geometric mean antibody titers of 3- to 10-fold were detected by hemagglutination inhibition depending on the type of antigen utilized. We found that ELISA with whole virus antigens was a sensitive and specific test for the detection of antibody to influenza B virus.
The sensitivity of ELISA and the hemagglutination-inhibition test was comparatively studied in the demonstration of Newcastle disease antibodies in the sera of 57 vaccinated birds. Differences were established in the antibody titers as demonstrated by the two methods. The sensitivity of ELISA was found to be 100 times higher than that of the HI-test. Discussed is the possible use of ELISA in cases where no antibodies or low-titer hemagglutinating ones are demonstrated via the HI-test.
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A 21% incidence of false-positive hemagglutination inhibition tests for pregnancy associated with tubo-ovarian abscess is described. Five illustrated cases and the causes for such false-positive reactions are discussed. Substances generated by pelvic inflammatory masses may interfere with immunologic assays for pregnancy, resulting in misdiagnosis or inappropriate management.
Formalinized chick erythrocytes agglutinated with Japanese encephalitis virus antigens over a wider range of pH (6.0-7.2) than fresh erythrocytes (pH 6.4-6.8). The virus antigens were unstable below pH 7.0 and therefore the use of the formalinized erythrocytes made it possible for a one-diluent system of the hemagglutination-inhibition tests to be used. In these tests phosphate-buffered saline at pH 7.0 was used throughout as a diluent for serum, antigen and erythrocytes, although the conventional system, using fresh erythrocytes, requires two buffers of different pHs, one an alkaline diluent for serum and antigen and the other an acidic diluent for erythrocytes.