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PubMed1979

A vesicular stomatitis virus (cytomegalovirus) pseudotype and its use in neutralization tests.

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.

Adult↗
PubMed1991

Comparative evaluation of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies directed against glycoprotein I of pseudorabies virus and a conventional ELISA and neutralization tests.

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.

Animals↗
PubMed1989

Comparison of a liquid-phase blocking sandwich ELISA and a serum neutralization test to evaluate immunity in potency tests of foot-and-mouth disease vaccines.

Sera from cattle vaccinated against either foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, or C1 Detmold were tested in a serum neutralization test (SNT) and a liquid-phase blocking sandwich ELISA (LBE), and the titers were compared with the results of intradermolingual challenge tests. The LBE test results were significantly more reproducible (P less than 0.005) than the SNT results. The correlation coefficients between SNT and LBE were 0.91 for FMDV strains A10 Holland and O1 BFS, and 0.82 for FMDV strain C1 Detmold (P less than 0.0005). The regression coefficient for strain A10 Holland was 0.80, for strain O1 BFS the value was 0.87, and for strain C1 Detmold it was 0.64. In probit analysis, titers at which 95% of the cattle were protected against challenge with the homologous strain were determined for the SNT and the LBE. In the SNT the 95% protection levels for strains A10 Holland were greater than or equal to 0.84, for O1 BFS greater than or equal to 1.59, and for C1 Detmold greater than or equal to 0.83. In the LBE they were greater than or equal to 1.28, greater than or equal to 1.71, and greater than or equal to 1.74, respectively. Because the SNT and the LBE are highly significantly correlated, and the LBE is more reproducible, the LBE is likely to predict protection more reliably than the SNT.

Animals↗
PubMed2004

Real-time quantitative PCR-based serum neutralization test for detection and titration of neutralizing antibodies to chicken anemia virus.

Detection and titration of chicken anemia virus (CAV)-neutralizing antibodies has relied on tedious, time-consuming passaging of infected cells, or subjective recognition of cytopathic effect in individual cells, because CAV replicates in culture only in lymphoblastoid cell lines, and thus generates no plaques. This paper describes a rapid method, in which CAV genomes in infected cells are quantitated by qPCR 3-4 days postinfection (p.i.), without passaging cells. Three sera, weakly positive with a commercial CAV ELISA kit, from broiler chickens immunized with a commercial CAV vaccine, were used to develop the assay. Virus neutralization titers of these sera were determined using two different CAV-susceptible cell lines (MDCC-MSB1 and MDCC-CU147) by the conventional method of passaging cells infected with 10,000 TCID(50) CAV per well, and by qPCR-based methods using cells infected with 100 or 10,000 TCID(50) per well in 24-well or 96-well plates. The method was also adapted to conventional PCR. The positive sera exhibited virus neutralization activity at dilutions ranging from 1:10 to 1:320 by the various assays. Although virus neutralization titers differed somewhat depending on the assay conditions used, the relative order of the titers of the three positive sera was the same for all assays. The qPCR-based assays are as sensitive and more rapid for detection of neutralizing antibody than the conventional assay based on passaging infected cells, and more sensitive for detection of low-level CAV antibodies than a commercial blocking ELISA.

Animals↗
PubMed1983

A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.

Animals↗
PubMed1999

Measurement of measles virus-specific neutralizing antibodies: evaluation of the syncytium inhibition assay in comparison with the plaque reduction neutralization test.

Plaque reduction neutralization (PRN) is the "gold-standard" for the measurement of measles-specific neutralizing antibodies. However, it is a complicated assay and tends to be operator-dependent. It has been suggested that the simpler syncytium inhibition assay (SIA) can give results comparable to the PRN test. We compared these two assays using 594 serum or plasma samples obtained from children at various times after natural infection, primary measles immunization, and measles revaccination. The results of the two assays correlated well overall (r = .86; p < 0.0001). The strain of challenge virus (wild-type versus vaccine strain) did not significantly influence SIA titers and the assay performed equally well with serum and plasma. PRN titers > or = 120 and > 800 are thought to indicate protection against clinical illness and infection respectively. The equivalent SIA cut-off values using 125 plaque-forming units as the challenge inoculum were > or = 16 and > 128 respectively. At low PRN titers (< 200), the correlation between PRN and SIA values was reasonable (r = 0.60; p < 0.001) when a challenge inoculum of 12.5 plaque-forming units was used. At the lowest PRN titers (< 100), 15% of the samples gave divergent results. These data confirm the utility of the SIA in the determination of measles-specific neutralizing antibodies when antibody titers are high. However, the PRN assay remains the test of choice when maximum sensitivity at low titers is required.

Adolescent↗
PubMed1993

[Comparison of two neutralization tests, used in vesiculovirus Piry serodiagnosis].

A serological survey of Piry was undertaken of the sera of inhabitants of Catolância-Bahia State, Brazil. Serum results obtained by a vesiculovirus neutralization test of C6/36 cells read by ELISA are compared with those obtained by the classic technique, carried out on newborn mice. The agreement between the results was as high as 98.7% of the 204 sera tested and the neutralization test of C6/36 cells was chosen as the most suitable technique for the sero-survey testing.

Animals↗
PubMed1991

Comparison of European isolates of viruses causing hemorrhagic fever with renal syndrome by a neutralization test.

Different virus isolates causing hemorrhagic fever with renal syndrome (HFRS) were compared using a neutralization test. Patient convalescent sera and antisera prepared in rabbits were used to compare Puumala-related Hantavirus isolates from Finland, Sweden, Belgium, and the USSR. The majority of European isolates were indistinguishable from each other using both homologous rabbit antisera and patient convalescent sera. The European isolates of HFRS were also compared with prototype Hantaan (the etiologic agent of Korean hemorrhagic fever). The one-way cross-reaction between the Hantaan and Puumala viruses, previously described using human convalescent sera tested by indirect immunofluorescence and immunoprecipitation, was also seen by the neutralization test.

Europe↗
PubMedDate not supplied

Neutrality tests on mtDNA: unusual results from nematodes.

McDonald-Kreitman tests of neutrality on mitochondrial DNA (mtDNA) of butterflies, Drosophila, and a variety of vertebrates usually show excess (over the neutral expectation) intraspecific polymorphism at nonsilent sites. These results are of great interest because they are the opposite of what is usually found for nuclear genes, in which the neutral pattern or evidence of adaptive divergence between species is usually observed. However, only vertebrates and insects have been tested so far, so it is not clear whether this intriguing pattern is typical for mtDNA in all taxa. Here I tested three pairs of nematode species and found that they all show a deficit of replacement polymorphism. Taken at face value, this result suggests that adaptive evolution proceeds more efficiently in nematode mtDNA than in mtDNA of vertebrates or insects. An alternate explanation is that the nematode pattern is an artifact of silent-site saturation that results from the rapid and composition-biased way in which nematode mtDNA evolves. Further studies are needed to distinguish between these two hypotheses.

Animals↗
PubMed1980

A micro-neutralization test for flavivirus antibodies.

A micro-neutralization test for the detection of flavivirus antibodies is described. The test utilizes the PS line of cells in flat bottomed microtiter plates to detect the neutralization of cytopathic effect by specific antibodies. Cross-neutralization of flaviviruses found in Southeast Asia with homologous and heterologous hyperimmune mouse ascitic fluids revealed the test to be specific. The test is simple and inexpensive to perform.

Antibodies, Viral↗
PubMed1965

FLUORESCENT CELL-COUNTING NEUTRALIZATION TEST FOR PSITTACOSIS.

Hahon, Nicholas (Fort Detrick, Frederick, Md.), and Kenneth O. Cooke. Fluorescent cell-counting neutralization test for psittacosis. J. Bacteriol. 89:1465-1471. 1965.-A sensitive, precise, and specific serological procedure, the fluorescent cell-counting neutralization test, was developed to detect and to measure quantitatively psittacosis serum-neutralizing antibodies within 24 hr. The test is based on the reduction of fluorescent cells in McCoy cell monolayers resulting from the neutralization of infective agent particles by specific antiserum. Small but significant rises in neutralizing titers were measured in serum specimens from monkeys previously exposed to the psittacosis agent and from humans with diagnoses of subclinical or established psittacosis infections.

Animals↗
PubMed1985

Micro-neutralization test for mumps virus using the 96-well tissue culture plate and PAP (peroxidase-antiperoxidase) staining technique.

A rapid micro-test method for mumps virus neutralization was developed. In this method, a 96-well tissue culture plate was used for preparation of cell monolayers and the PAP staining technique was used for visualization of mumps virus infected cells. Clusters of infected cells were observed as a focus and the numbers of foci could be counted by the naked eye 2 days after the infection. A linear relationship between virus dilutions and focus numbers was observed. When neutralizing antibodies in sera from cases of natural mumps infection were assayed, a good correlation was observed between those obtained by the focus reduction method applying the micromethod and those obtained by the ordinary plaque method. Our results indicate that this micromethod is useful in mumps virus neutralization tests and it has many advantages over other methods previously reported.

Animals↗
PubMed1978

[Detection of antibodies to Rickketsia prowazekii by using the antigen neutralization test].

The authors studied a possibility of using the antigen neutralization test with dry immunoglobulin typhus erythrocytic diagnostic agent for the purpose of detection of Rickettsia prowazeki antibodies. Blood sera of 315 healthy persons, 24 patients with sporadic typhus, and 18 laboratory animals immunized with R. sibirica and R. burneti, as well as with Proteus OX19 were examined. The results obtained pointed to the high specificity and sensitivity of the given serological test. A possibility of its use for antibody detection both in the typhus patients and in persons who sustained this infection in the past was demonstrated. In difference from the complement fixation test it permits to study anticomplementary sera.

Antibodies, Bacterial↗
PubMed1999

Comparison of commercial ELISA test kits from Australia and the USA with the serum neutralization test in cell cultures for the detection of antibodies to the infectious laryngotracheitis virus of chickens.

The objective of this work was the evaluation under our conditions of two commercial ELISA kits for the detection of antibodies to avian infectious laryngotracheitis (ILT) virus, one from Australia (Trop-ELISA, TropBio) and one from the USA (ProFLOK-ELISA, KPL), and to compare their performance with the conventional serum neutralization test (SNT) in chick embryo liver cells. Repeatability varied considerably, particularly when using the Trop-ELISA. Therefore, if individual results are important, at least two parallel measurements per serum sample are recommended. In 89.3% of the sera tested by SNT, results of two parallel measurements did not vary by more than one 2-fold dilution step. There was good linear correlation between both ELISAs and the SNT, the correlation coefficient for the Trop-ELISA being r = 0.758 and for the ProFLOK-ELISA r = 0.867. The negative/positive cut-off was redefined to suit our conditions. Sera with a SN titre of > or = 1:4 were considered positive. Sera with < or = 15% absorption in the ProFLOK-ELISA were considered clearly negative. For the Trop-ELISA, extinctions of > or = 0.477 were considered positive, < or = 0.168 clearly negative. Values in between were regarded as doubtful for young chickens and as possibly due to non-specific reactions in older chickens. The sensitivity and specificity of the ELISAs relative to the SNT were 87 and 77% for the Trop-ELISA, and 95 and 60%, respectively, for the ProFLOK-ELISA. However, the results indicate that the sensitivity of the ELISA is higher than that of the SNT, because most sera showed similar deviations from SNT results with both ELISAs. Generally, both ELISAs were a suitable alternative to the SNT under our conditions, as long as only negative/positive results are required.

Journal Article↗
PubMed1981

Role of virus strain in conventional and enhanced measles plaque neutralization test.

A low-passage Edmonston strain of measles virus gave on the average four-fold higher antibody titers than two high-passage strains of Edmonston virus when used in a virus plaque neutralization test. Heterologous anti-human immunoglobulin did not affect antibody titers obtained with the low-passage virus but enhanced antibody titers obtained with the high-passage viruses on the average eight-fold. These differences in virus sensitivity to neutralization and neutralization potentiation by anti-immunoglobulin were interpreted as being possibly due to differences in density or accessibility of the functional antigens of measles virus. The conventional plaque neutralization test utilizing the low-passage Edmonston virus was about 10 times more sensitive than the virus CPE-neutralization test, 60 times more sensitive than the measles hemagglutination-inhibition test and 220 times more sensitive than the measles complement-fixation antibody test.

Animals↗
PubMed2003

Use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test.

A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4 degrees C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

Animals↗
PubMed1990

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.

Animals↗