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Neutralization of vaccinia virus with immune rabbit serum occurred optimally when incubated at 37 C for 16 to 24 hr in the plaque neutralization test employing the MA-104 embryonic rhesus monkey kidney cell line.
Sera were collected from 238 high-school students in Prince Edward Island for the determination of immune status before an anticipated measles outbreak. In addition, history of vaccination status and measles infection was obtained. In the subsequent outbreak, 28 students did contract measles. Specificity for hemagglutination inhibition (HI), antihemolysin (AH), and enzyme-linked immunosorbent assay (ELISA) was 100%, compared with the neutralization test. Corresponding sensitivity values for the tests were 66.0% (HI), 99.5% (AH), and 99.0% (ELISA). Predictive values for susceptibility were 26.9% (HI), 77.8% (AH), 75.7% (ELISA), 80% (neutralization), and 41.4% as determined by history of infection or vaccination. The predictive value for immunity as determined by history of previous infection or vaccination was 91.8%, compared with 100% for the four serological tests. No false-positive results were seen with any of these tests. Compared with the neutralization test, the HI test had 69 false-negative results, the AH had 1, and the ELISA test had 2. The AH and ELISA tests provided sensitive and specific alternatives to the commonly used HI test for immune status determination.
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At our institute the mouse neutralization test (MNT) was replaced by the rapid fluorescent focus inhibition test (RFFIT). Therefore, RFFIT results were statistically analyzed for their percentage limits at the 95% confidence level and were compared with those of the MNT. An anti-rabies immunoglobulin of human origin and the WHO standard serum were assayed on 20 consecutive days to determine the reproducibility of the RFFIT. Sera of 60 vaccinees were assayed for rabies antibodies by the RFFIT and MNT. The titers as well as the international units per ml showed a correlation of 0.95 and 0.87 respectively. In accordance with previous publications by other authors it was found that the RFFIT is a significantly better reproducticible test system than the MNT and excellently correlates with the latter method.
The dynamics of the immune response to streptococcal extracellular antigens as measured by the streptozyme agglutination test have been defined in experimental animals andcompared with the dynamics of the immune response as determined by three established neutralization tests for streptococcal antibodies (ASO, anti-DNase B,and anti-NADase). In rabbits immunized with streptococcal extracellular Streptozyme-measured antibodies rose more quickly and peaked earlier than did antibodies to streptolysin O, streptococcal DNase-B, and streptococcal NADase. Additional studiesrevealed that the early peak was primarily due to 2-mercaptoethanol-sensitive (19S) antibody and that the later rise in Streptozyme-measured antibodies was due to 2-mercaptoethanol-resistant (7S) antibody. Preliminary observations using sucrose gradient ultracentrifugation tended to confirm this interpretation. These data provide a possible explanation for the early detection of a streptococcal antibody response by the Streptozyme test which has also been described in humans. The data suggest a theoretical advantage for the Streptozyme agglutination test in patients with streptococcal infections and their sequealae. However, the complexity of the Streptozyme reagent suggests that more extensive studies are needed to assure the consistent reproductivity of results with different lots of the test reagent.
A microculture neutralization test is described for measuring specific antibody levels to the 35 human adenovirus serotypes in Vero cells. The test is read at 5 days by macroscopic observation after staining the uninfected cells with crystal violet. The test is performed with a minimum of manipulations and gives serum titers comparable with those obtained in tube macrocultures of monkey kidney, human embryonic kidney, and Vero cells. The Vero microculture neutralization test measures inhibition of adenovirus toxicity, although selected human adenoviruses serially subpassaged in Vero cells were shown to successfully adapt and replicate in the absence of detectable helper viruses.
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Serum samples from 42 groups of mice immunized for different immunization periods with various doses of Adsorbed Diphtheria-Tetanus Vaccine, Adsorbed Diphtheria-Tetanus and Pertussis Vaccine and a standard diphtheria toxoid were assayed for their diphtheria antitoxin content by indirect haemagglutination (IHA) and by toxin neutralization (TN) tests. A very good correlation of 0.91 was obtained between the results of the two methods. There was no statistically significant difference between the IHA and the TN titres obtained. Adsorption with sheep red cells and treatment of the sera with 2-mercaptoethanol had no effect on the IHA titres. The minimum level of antitoxin detectable by the IHA test was 0.00039 IU ml-1. IHA proved to be a sensitive, specific and reproducible method which can be used reliably for the assay of diphtheria antitoxin in mouse sera.
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A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure.
A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.
A virus-neutralization test for infectious laryngotracheitis virus was performed in microtiter plates using standard techniques. To assess the reproducibility of the test, 11 sera were each titrated repeatedly once a week for 8 weeks, and the results were compared to a standard. The standard used for reproducibility was that the 95% logarithmic confidence intervals of the mean calculated from three titrations of the same serum had to be smaller than the logarithmic distance "within" two microtiter plate wells. For the virus neutralization test to give reproducible results, such confidence intervals had to fall "within" two wells at least 75% of the time. Over the 8 weeks, percent reproducibility varied from 43.5% to 81.5%. The infectious laryngotracheitis virus-neutralization test did not meet our defined standard of reproducibility with positive antisera. Results with negative control sera were reproducible, however. Percent reproducibility varied from 31.8% to 93.8% for different sera tested, but it was not related to the titer of the sera.
Foci of cells infected with human cytomegalovirus were noted to stain more intensely than uninfected cells with neutral red, and this provided the basis for development of a plaque assay and plaque reduction neutralization test for cytomegalovirus. Plaques demonstrable by neutral red staining could be counted at 8 days after infection; thus, results could be obtained earlier than for plaque assay systems based upon the viral cytopathic effect, a fewer manipulations were required for staining cell monolayers to demonstrate plaques. Certain variables affecting plaque size and numbers and antibody titers were defined. Addition of fresh guinea pig complement to the reaction mixtures markedly enhanced cytomegalovirus-neutralizing antibody titers of hyperimmune animal sera, but titers of human sera were enhanced only two-or fourfold.
The serum dilution neutralization test was evaluated for serological diagnosis of California group arbovirus infections and identification of virus isolates. The technical advantages and the degree of subtype specificity of the serum dilution neutralization test over the hemagglutination inhibition test and the complement fixation test were demonstrated with paired specimens from human cases, single human survey sera, and sentinel rabbit sera. Twenty-one virus isolates from various geographical areas of the United States were also used to evaluate the efficacy of the serum dilution neutralization test for specific virus identification.
A sensitive mumps virus plaque neutralization test has been developed based on the potentiation of virus-antibody complexes by heterologous anti-immunoglobulins (AIG). The enhanced neutralization test was approximately 100 times more sensitive than the conventional neutralization test or the hemagglutination-inhibition test. Using AIG against human immunoglobulin G (IgG) or human IgM permitted determination of the relative titers of the two classes of mumps antibody. The test does not require special equipment or expertise and can be readily introduced in virological laboratories.
Infection with vesicular stomatitis virus (VSV) of human diploid cells preinfected with the AD-169 strain of human cytomegalovirus (CMV) resulted in the formation of a VSV (CMV) pseudotype. Its formation was favored by increasing the bicarbonate content in doubly-infected cultures. The pseudotype was capable of infecting not only human but also rabbit cells. Pseudotype particles formed after infection with the tl 17 mutant of VSV, which carries a thermolabile lesion in its neutralization antigen, were more stable at 45 degrees than the original tl 17 virus. The pseudotype was used in the neutralization test with human sera. All sera positive for CMV antibody in the complement-fixation (CF) test were also reactive in the neutralization test. In addition, numerous sera negative for CMV antibody in the CF test neutralized the pseudotype.
To determine whether a newly developed enzyme-linked immunosorbent assay (ELISA), which detects antibodies directed against glycoprotein I (gI) of pseudorabies virus, is suitable for serodiagnosis, it was compared with a conventional ELISA and two neutralization tests. The gI ELISA was 99.2% as sensitive and 100% as specific as the conventional ELISA. Antibody titers measured by the gI ELISA were lower than those measured by the conventional ELISA, comparable to those measured by the 24-h neutralization test, and higher than those measured by the 1-h neutralization test. After experimental infections in pigs, antibodies were first detected by the gI ELISA on postinoculation day (PID) 14, by both neutralization tests on PID 7, and by the ELISA on PID 10. All four tests showed the same rate of decline of maternal antibodies in pigs born to sows that had been vaccinated with inactivated vaccine. Thus, not only is the gI ELISA unique in being able to differentiate infected pigs from those vaccinated with gI-negative vaccines, but it is also as useful as a conventional ELISA and as neutralization tests for serodiagnosis.