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A test for the titration of B. pertussis toxin with antisera on Chinese hamster ovary (CHO) cells has been worked out. B. pertussis protective antigenic cell-free complex containing 48-54% of B. pertussis toxin has been used as antigen. The specificity of the effect of this complex on CHO cells has been confirmed in the toxicity neutralization test with antisera. CHO cells have been adapted to reagents and culture media made in the USSR. The titration of B. pertussis toxin and antisera on CHO cells did not require the use of highly purified antigen.
Statistical analysis of the results of tumor-cell neutralization (Winn) tests has been presented in a refined manner using three variables: tumor incidence, time to palpable tumor, and growth rate. The concept of relative risk and the Z statistic were used to describe the latter two variables. This analysis is then applied to a particular experiment. Lymphocytes sensitized in vitro were compared with specifically immune lymphocytes and were found to facilitate the growth rate. Further applications of this analysis are discussed.
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The correlation between skin reaction, exhibited by vaccinees immunized against smallpox, and antibody titer determined by plaque neutralization and ELISA, was evaluated. Twenty eight out of 35 young adults (vaccinated at infancy and at the age of 8 years), who were injected with vaccinia virus, displayed a major skin reaction a week later. An increase of four-folds and more, in antibody titer against vaccinia virus, is generally considered positive immunization take-up against smallpox. According to this criterion, only 17 of the vaccinees were found positive by plaque neutralization, while 25 by the indirect micro-ELISA. Thus, there were eight vaccinees who were considered immunized by the ELISA, (seven of them also according to the skin reaction), but not by the plaque neutralization test.
An improved micromethod for infectivity assays and neutralization (N) tests of dengue (DEN) type 1-4 viruses was developed, using 96-well plates and the PAP (peroxidase-antiperoxidase) staining technique. The foci formed on BHK-21 cell monolayers in wells of the plate were readily countable under an ordinary stereomicroscope. This micromethod has the advantages over the micromethod of the Lab-Tek 8 chamber slide system of lower cost, requirement for smaller volumes of test sera and applicability to larger number of serum specimens for N tests of DEN viruses.
Weekly oral chloroquine prophylaxis for malaria has been associated with impaired antibody response to intradermal rabies vaccination. Experimental data indicate that chloroquine may inhibit yellow fever virus in vitro, yet there has been no clinical evidence to suggest that antibody response to yellow fever vaccine is impaired by concomitant oral administration of chloroquine. A prospective trial was undertaken to evaluate the antibody response to yellow fever 17D vaccine (Connaught Laboratories) of volunteers who were randomized to taking either chloroquine or no drug. Of fifty subjects, 28 were randomized to taking chloroquine, 22 were randomized to taking no drug. Yellow fever 17D vaccine was administered on day 0 and blood sampled on days 0, 14, 35 and 210. Chloroquine was administered weekly for four weeks. There was no significant difference in peak antibody titer by plaque reduction neutralization testing (PRNT) between the group that took chloroquine (mean log peak of reciprocal titer 1.43 +/- SD 0.60) with vaccine subcutaneously compared to vaccine-only group (mean log peak of reciprocal titer = 1.21 +/- 0.55). All fifty subjects seroconverted to yellow fever vaccine by day 210. ELISA testing was also performed on all subjects. The two tests showed good correlation (Spearman r = 0.675), although ELISA readings were positive by day 14 in significantly more subjects (p = .01). We conclude that routine anti-malarial doses of chloroquine do not affect antibody response to yellow fever 17D vaccine. ELISA testing, a less complex and less time-consuming test, correlates well with PRNT and is proposed for additional trials to measure yellow fever 17D vaccine response in flavivirus non-immune subjects.
Increasing numbers of reports of clinical falcon herpesvirus infection (Falconid herpesvirus-1; FHV-1) have been seen in the UK since 1996. The aim of this epidemiological study was to investigate the seroprevalence of FHV-1 and owl herpesvirus (Strigid herpesvirus-1; StHV-1) infection in the UK, using virus neutralization tests, and to evaluate the prevalence of herpesvirus infection in captive and wild raptor populations. The results, using the English FHV-1 CVL 32/93 isolate, revealed a seroprevalence of 3.97% (10/252). The seroprevalence for StHV-1 was 12.3% (8/65). Analysis of the data by captivity status, age and species revealed that the family Falconidae showed the highest seroprevalence with 6.7% (5/75), while only one of 104 captive Accipitridae was positive for FHV-1 (0.96%). The incidence of FHV-1 neutralizing antibodies in owls was 5.5% (4/73), representing only wild individuals. Eighty-nine serum samples were additionally tested using two other FHV-1 isolates, the German isolate Merlin 1869/92 and the Dutch isolate Peregrine Z100. The seroprevalences of FHV-1 were 28.1% (25/89) and 32.6% (29/89), respectively. All these samples, however, were negative using the CVL 32/92 isolate.
When assessed by a conventional plaque neutralization (NT) test, about one third of recipients of live mumps vaccine were found to have failed to seroconvert, although the majority of them became seropositive by the complement-mediated neutralization (CNT) test. The discrepancy between the conventional NT and CNT was found to result from two factors. First, the predominant production of antibodies to the fusion (F) protein during the early phase of antibody response, and second, a low efficiency of NT of the early antibodies in the absence of complement. These features of antibody response were also seen in natural mumps infection and in experimental infection of monkeys, but were particularly prominent in vaccinees because of the limited extent of antibody response in the latter. The discrepancy between conventional NT and CNT diminished with time after vaccination, after natural infection, and also after experimental infection of monkeys. The CNT test was therefore considered better suited than the conventional NT test for assessment of the outcome of vaccination, at least until 6 to 9 weeks after vaccination. The protective role of the antibody in vivo, which requires the addition of complement for neutralization, was inferred from the observation that the antibody incorporated in the agar overlay significantly reduced the size of plaques formed by mumps virus.
The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.
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The RAST, originally developed for the assay of reaginic IgE antibodies, was applied for the detection of blocking antibodies. Sera taken ante- or post-hyposensitization were incubated with solid phase coupled allergen which neutralized reaginic IgE antibodies in the serum. The remaining activity of the reaginic IgE antibodies was measured by the RAST. When blocking antibodies were present in the serum they competed with the reaginic IgE antibodies and the amount of reaginic antibodies that was neutralized by the solid phase coupled allergen was decreased. The blocking antibodies were found in the IgG fraction of the serum taken post-hyposensitization. Blocking antibodies to various allergens were detected in 25 out of 26 tests on sera taken post-hyposensitization. The results were compared with those of Prusnitz-Küstner neutralization tests and/or patient self-neutralization tests. There was no correlation between semi-quantitative results of these in vivo tests and those of the RAST neutralization tests. However, cases with subjectively a strong improvement had a significantly higher mean value for RAST neutralization index than those with subjectively a weak improvement. The method is suggested as a supplement to clinical tests for evaluation of hyposensitization treatments.
Seroepidemiological surveys were performed on neutralizing antibody to bovine herpesvirus 2 (BoHV-2) among cattle in Japan. A total of 1,819 sera were collected from cattle in 27 prefectures from 1997 to 1998. Antibodies were detected in only 18 sera collected from 4 prefectures. However, the most prevalent areas of the infection were found in two islands located in the subtropical zone. Additional 353 sera were collected in three including these islands in 1999-2001. The antibody-positive rates in the farms in these islands ranged from 10% to 81.1%. It was confirmed that BoHV-2 was prevalent in these areas. However, the infection seemed to be latent, because no diseases have been noticed. This is the first report showing the presence of BoHV-2 infection among cattle in Japan.
The antigenic relationships among the neuraminidases of influenza A strains from 1957 to 1973 were examined by postinfection application of neuraminidase antisera. This procedure causes inhibition of virus spread and apparent neutralization. Neuraminidase (apparent) neutralization and neuraminidase inhibition tests with chicken antisera gave similar results. Neuraminidase inhibition tests were more discriminating than neuraminidase neutralization tests when rabbit and goat antisera were used. Antibody absorption studies revealed that the neuraminidase, like the hemagglutinin, may possess two kinds of antigenic determinants, which can give rise to "common," or "cross-reacting," and "specific" antibodies. "Specific" antibody appears to be more effective in the inhibition of enzyme activity than in the inhibition of virus spread.
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