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Isolation and characterization of new infectious bronchitis virus variants in Hungary.

Two agents not agglutinating chicken erythrocytes were isolated, one in each of two flocks, from organ samples and tracheal swabs taken from 4- to 7-week-old chicks of 8 broiler flocks experiencing respiratory signs. Virus isolation was done in embryonated SPF hen's eggs. Morphological changes of the embryos, appearing as dwarfing or curling into a spherical form, usually occurred in the 3rd or 4th passage on postinoculation (PI) days 5-9. Some embryos had swollen kidneys covered with urate. Electron microscopy of ultrathin sections of these kidneys revealed the presence of virions reminiscent of coronaviruses. Similar viral particles were seen in resuspended pellets of isolates concentrated by ultracentrifugation. Based on embryo changes, cross-neutralization tests with type-specific antisera, physicochemical tests, results obtained in cell cultures, and electron microscopic findings the two isolates were identified as infectious bronchitis virus (IBV). By cross-neutralization tests the isolates differed from IBV reference strains M41 and H52 and can be considered distinct variants. Elucidation of their epizootiological role requires further investigations.

Animals↗

Serological investigation of an outbreak of simian varicella in Erythrocebus patas monkeys.

An epizootic of simian varicella occurring in a colony of Erythrocebus patas monkeys was studied serologically by using radioimmunoassay and neutralization tests against (i) a virus strain isolated from an animal that died during the epizootic, (ii) a simian varicella virus strain from an earlier outbreak of simian varicella-like disease at another facility, and (iii) human varicella-zoster virus. Serological tests detected more cases of infection among the animals exposed to virus during the epizootic than were evidenced by clinical findings; only 6 of the 26 animals with seroconversion developed a rash. Good correlation was seen between antibody responses demonstrated by radioimmunoassay and by the neutralization tests. Specificity of the radioimmunoassay was evidenced by the complete agreement with neutralization results for 17 animals which failed to show an antibody response over the course of the outbreak and were assumed not to have been infected. Thus radioimmunoassay is a reliable, rapid, and relatively economical method which could be used for serological screening of primates entering experimental colonies to identify those which might be potential sources of outbreaks through activation of latent simian varicella virus infection. Close correlation was seen between antibody responses to the virus strain from the current outbreak and the one from another epizootic, indicating that the two outbreaks were caused by antigenically similar viruses. Animals showing neutralizing antibody responses to the simian varicella viruses also showed responses to human varicella-zoster virus, which further substantiates the close antigenic relationship between human and simian varicella viruses.

Animals↗

Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus.

A competitive blocking enzyme-linked immunoassay (CELIA) was developed to detect bovine viral diarrhea virus (BVDV) antibodies in undiluted fetal bovine serum (FBS). The CELIA was based on competition of serum BVDV antibodies with biotin-labelled anti-BVDV immunoglobulins (Ig) for a limited quantity of solid-phase BVDV antigen. Antigen preparation was simple, FBS could be tested undiluted, and detergent-containing washes were unnecessary. A series of dilutions of postnatal bovine BVDV antiserum prepared in FBS and a set of 147 undiluted abbatoir FBS samples were tested by both CELIA and serum neutralization tests (SNT). CELIA results on both sets of specimens correlated positively with SNT titers (r = 0.99 and r = 0.85). Relative to the SNT, CELIA sensitivity was 100%; specificity was 76%. CELIA detected a level of BVDV antibody below the 1:2-titer threshold detectable with the SNT. Advantages, limitations, and theoretical differences between the CELIA and SNT are discussed. A similar comparison of CELIA with non-competitive enzyme-linked immunoassay approaches to BVDV serodiagnosis is made. It is concluded that the CELIA is valuable in selecting only BVDV-seronegative FBS for use in virologic cell culture media.

Animals↗

Hantavirus infection in laboratory and wild rodents in Argentina.

Serum samples from urban and laboratory rats, laboratory mice and wild and laboratory cricetids in Argentina were tested by immunofluorescence and plaque reduction neutralization tests to investigate prevalence of anti-Hantavirus antibodies. A total of 102 sera were obtained from laboratory rodents in 4 different animal-rooms, 31 from harbor rats and 30 from wild cricetids in 1985-1987. Anti-Hantavirus antibodies were detected in 22.5% of Rattus norvegicus in 3 of the animal-rooms but harbor rats were found to be free of Hantavirus infection. Previously, the presence of anti-Hantavirus antibodies had been demonstrated in the sera obtained from laboratory workers in these same 3 animal-rooms; it can be concluded that the laboratory rats were the source of this human infection. On the contrary, laboratory mice and cricetids failed to show Hantavirus infection while the wild vesper mouse Calomys musculinus (the main Junin virus reservoir) showed a prevalence of 23.5%. The presence of Hantavirus infection is hereby reported for the first time in wild C. musculinus and in laboratory R. norvegicus in Argentina.

Animals↗

Serological survey of immune response to tetanus toxoid using antitoxin enzyme immunoassay.

A method for testing serum tetanus immunoglobulin (TIG) was set up using enzyme immunoassay kit (EIA kit) and prepared by competitive principle in this laboratory. Forty-eight human sera tested by both EIA and toxin neutralization test (NT) in mice were in good agreement. Determination coefficient (r2) of the EIA and NT was 0.922. The lowest detectable dose was 0.1 IU/mL ELISA value. Many authors believe this ELISA value to be a safe protection TIG level. For serological survey of immune response to tetanus toxoid, sera from two groups of subjects were examined. Among 83 subjects, each over 40 years of age, only 47.0% were positive to TIG EIA level. Forty-six subjects with undetectable EIA titres to tetanus toxoid (TT) were immunized against two doses of TT at an interval of four weeks; 44 (95.6%) demonstrated TIG concentration of greater than or equal to 0.2 IU/mL within two weeks to one month following the last dose vaccination. The TIG positive rates of 725 school students' sera, selected by multistage sampling from 2395 specimens, were reported. Those were collected by simple random sampling from geographical area in the North, South, Central, Central Mountain, and East areas of Taiwan according to age groups. They were 80.8%, 65.1%, 56.0%, and 42.0% to the age groups of 0-4, 5-9, 10-14, and 15-17 years respectively. The probabilities of each near by two groups were p less than 0.1; p less than 0.05; p less than 0.05. The differences of positive rates in the last three groups were significant.

Adolescent↗

Microneutralization test for influenza A and B and parainfluenza 1 and 2 viruses that uses continuous cell lines and fresh serum enhancement.

A modified microneutralization test for influenza A and B and parainfluenza 1 and 2 viruses was developed. Use of continuous cell lines (Madin-Darby canine kidney and LLC-MK2), hemagglutination for virus detection, and transfer plates and other microtiter equipment resulted in a highly mechanized technique suitable for titrating large numbers of sera easily and relatively inexpensively. Titers of heat-inactivated human sera were enhanced 0.5 to 3.5 log2 by addition of fresh hamster or human serum to the test. Seroconversion rates and identification of seronegative persons were not changed by fresh serum enhancement, but the magnitude of seroconversion was often increased and the distribution of positive titers was broadened. For influenza A and B, seroconversion rates in the microneutralization test were equivalent to those obtained in rhesus monkey kidney tube neutralization tests. For influenza B, seroconversion rates by micro-neutralization were almost double those obtained with the hemagglutination inhibition test, but the rates were similar for influenza A/USSR (H1N1). Preexisting microneutralization titers correlated well with resistance of infection with influenza B. Limited experience with parainfluenza 1 and 2 was similar to previously reported findings with the tube neutralization test.

Adult↗

A new subgroup 2 bovine adenovirus proposed as the prototype strain 10.

A slowly growing subgroup 2 bovine adenovirus (BAV) strain designated Ruakura 78-5371 was isolated from a yearling heifer with systemic adenovirus infection. Cross neutralization tests and restriction endonuclease analysis of the viral DNA showed the virus to be distinct from the other 9 recognised types of BAV. It is proposed that this strain should be regarded as the prototype strain of the new type BAV-10.

Adenoviridae↗

Assessment of neutralizing antibodies elicited by a vaccine (Nakayama) strain of Japanese encephalitis virus in Taiwan.

A total of 368 blood specimens were resampled from a serum collection containing 2914 blood samples which were collected by a random sampling in Taiwan in 1991. The plaque reduction neutralization test was applied to evaluate the neutralizing ability to two strains of Japanese encephalitis viruses, i.e. Nakayama (the present vaccine strain) and JE5 (a Taiwan isolate). The result revealed that antibodies against JE virus were present in each stratified age group. Antibody positive rates were both highest in the group older than 70 years although the lowest rates were located in different groups. In addition, the result showed that the immunogenicity potency of the antibody induced by the vaccine strain did not have a good coverage against JE5. The rate of neutralizing antibodies above the level of protective efficacy of the present vaccine was limited as low as 37.93%. Efficacy of the vaccine used at present was apparently not efficient. Consideration of a more promising vaccine may be necessary.

Adolescent↗

Parvovirus B19 empty capsids as antigen carriers for presentation of antigenic determinants of dengue 2 virus.

For the production of dengue-vaccine candidates, empty capsids, or virus-like particles (VLPs), of parvovirus B19 that carry dengue 2-specific epitopes were employed as antigen carriers. Two epitopes (comprising amino acids 352-368 and 386-397) of domain BIII of the envelope glycoprotein were chosen to produce recombinant B19 VLPs for immunization of BALB/c mice. Serum samples from immunized mice revealed that recombinant B19 VLPs elicited strong humoral immune responses. In summary, this B19 VLP-vaccine platform produced high (> or =2.0 x 10(5)) anti-dengue 2 titers and robust (< or =1 120) 50%-plaque-reduction neutralization test (PRNT(50)) titers, which effectively neutralized live dengue 2 virus in PRNT(50) assays.

Animals↗

The neutralization of interferons by antibody. II. Neutralizing antibody unitage and its relationship to bioassay sensitivity: the tenfold reduction unit.

The importance of establishing a common method of reporting neutralizing antibody levels is emphasized by the fact that patients injected repeatedly with a human interferon (HuIFN) may develop such antibodies that can abrogate the beneficial effects of the treatment. The earlier experimental and theoretical constructs of Kawade led to certain recommendations by the World Health Organization (WHO) concerning the methodology of neutralization tests and how to report the resultant data. A WHO international collaborative study on two human sera with antibodies against HuIFN-alpha and HuIFN-beta provided the opportunity not only to test the theoretical concepts concerning the neutralization reaction with data obtained in different bioassay systems in different laboratories but also to obtain enough data points for statistical evaluation with bioassays having a great range of sensitivity to IFN. The analyses substantiate and extend the original conclusions of Kawade that the neutralization follows the reaction mode of low-affinity antibody, in accord with the constant proportion hypothesis by which antibody reduces IFN activity in a set ratio of added/residual biologically active IFN, a consequence of the low molar concentration of free IFN at the neutralization end point. The present results support the recommendation that the preferred way to state the index of neutralization of antibodies is a titer (t), calculated by the formula t = f(n - 1)/9, where f is the reciprocal of the antibody dilution achieving the end point, and n is the IFN concentration measured in that day's titration. The tenfold reduction unit (TRU) of neutralization is proposed for use in expressing the quantity, or unitage, of IFN neutralizing antibody. The utility of its application is explained. The use of the index of neutralization described and the proposed derivative term of antibody unitage, TRU, should help make the results from different laboratories employing different bioassay systems more readily comparable and interpretable, provided the bioassays are sufficiently sensitive to IFN.

Antibodies↗

New Minto virus: a new rhabdovirus from ticks in Alaska.

Three strains of a virus were isolated from Haemaphysalis leporis-palustris (Packard) ticks removed from snowshoe hares (Lepus americanus Erxleben) in east central Alaska. We suggest that the virus be named New Minto for the location in which the ticks were collected. Prototype New Minto virus is sensitive to the action of sodium deoxycholate and kills suckling mice by the intracerebral but not intraperitoneal route; weaned mice do not die after intracerebral, intraperitoneal, or subcutaneous inoculation. The virus produces plaque in serially propagated Vero but not in primary Pekin duck embryo cells. By complement-fixation and neutralization tests New Minto is related to Sawgrass Virus, a hitherto ungrouped virus from Florida. The establishment of a Sawgrass group is suggested. In addition, Sawgrass virus was found by electron microscopy to belong to the Family Rhabdoviridae.

Alaska↗

Characterization of eight new phlebotomus fever serogroup arboviruses (Bunyaviridae: Phlebovirus) from the Amazon region of Brazil.

Eight new members of the phlebotomus fever arbovirus serogroup (family Bunyaviridae; genus Phlebovirus) from the Amazon region of Brazil are described. One serotype was recovered from a febrile patient, three from small wild animals and four from sand flies. A small serum survey carried out with the human isolate, Alenquer virus, suggests that it rarely infects man. Complement-fixation and plaque reduction neutralization tests were done, comparing the eight new viruses with other members of the phlebotomus fever serogroup. A close antigenic relationship was demonstrated between one of the new agents (Belterra) and Rift Valley fever virus. This finding is of considerable interest and deserves further investigation. Addition of these eight new viruses to the genus Phlebovirus brings to 14 the number of serotypes known to occur in the Amazon region and to 36 the total number reported worldwide. More detailed clinical and epidemiological studies should be conducted in Amazonia in order to define the public health impact caused by phleboviruses.

Animals↗

Newcastle disease virus antigens and strain variations.

Study of antigenic differences among strains of Newcastle disease virus is complicated by the presence in most field isolates and strains of several genetically distinct plaque populations, and by differences in avidity (reactions to antibody) among Newcastle disease viruses. Kinetic neutralization tests were used to demonstrate antigenic differences among 3 plaque clones of velogenic viscerotropic Newcastle disease viruses recently isolated from 3 avian species. The 3 viruses could be distinguished on the basis of plaque structure and hemagglutinin and neuraminidase activity. Antigenic differences among these viruses distinguished them from older, well characterized Newcastle disease viruses.

Animals↗

Isolation and characterization of a novel antigenic subtype of infectious bronchitis virus serotype DE072.

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.

Amino Acid Sequence↗

The classification of seven serotypes of equine encephalosis virus and the prevalence of homologous antibody in horses in South Africa.

Selected isolates of equine encephalosis virus were shown to have comparable viral protein profiles and to represent seven distinct serotypes, based on cross-neutralization tests. Serotype-specific virus-neutralizing antibody in serum samples from horses confirmed the widespread occurrence of infection. The distribution and prevalence of individual serotypes however, varied considerably. Localised foci with an increased seasonal seroconversion in groups of horses to a specific serotype and the detection of an ongoing low level of infection from other serotypes within the population, confirmed the independent persistence of the viruses in a maintenance cycle. The identification of donors with antibody resulting from infection with multiple serotypes indicated a low level of cross-protection in horses to natural reinfection.

Animals↗

[Isolation of the West Nile fever virus from the great cormorant Phalacrocorax carbo, the crow Corvus corone, and Hyalomma marginatum ticks associated with them in natural and synanthroic biocenosis in the Volga delta (Astrakhan region, 2001)].

Four strains identified as West Nile fever virus by inhibited hemagglutination and neutralization tests, enzyme immunoassay, and reverse transcription polymerase chain reaction were isolated during a virological examination of birds and their collected ticks in the natural and synanthropic biocenoses of the Volga delta. The strains were isolated from the great cormorant (Phalacrocorax carbo), the crow (Corvus corone) and its collected Hyalomma marginatum nymphs. The types of interpopulational relations in the ecological system wild-birds-virus-mosquitoes-synanthroic birds-ticks are discussed.

Animals↗

Contamination of some avian species with viruses of the leukosis-sarcoma complex.

Contamination of 8 avian species with leukosis viruses was studied. An insignificant portion of sera from 4 and 24 months old chickens contained neutralizing antibody to 3 sero-types (A, B and D) of Rous sarcoma virus (RSV). Sera from guinea-fowl reacted positively in the neutralization test only with the RSV-RAV-1 strain. None of the Peking duck and semi-domestic Maran fowl sera was found to contain antibody to RSV strains. Twenty-five and 6.6% of embryos derived from 7-8 and 24 to 30 months old chickens respectively, 30% of liver specimens from 3-4 months old chickens 3.3% of liver specimens from adult guinea-fowl and 72% of liver specimens from Maran fowl contained the group-specific leukosis complement-fixing antigen (gs-antigen). Duck embryos and livers from 24-30 months old chickens, crows, sparrows, rooks or jackdaws contained no gs-antigen. Duck embryos did not react and ducklings reacted poorly to inoculation with RSV whereas guinea-fowl embryos and chickens proved to be highly susceptible to RSV.

Age Factors↗

[Isolation and characteristics of Shigella antigens during the course of dysentery].

In 2,436 fecal samples and 1,272 urinary samples taken from 633 patients with dysentery caused by S. flexneri, S. newcastle and S. sonnei, Shigella antigens were detected by means of the passive hemagglutination test with antibody diagnostic agents and the antibody neutralization test. The antigen-binding activity of shigellae in urine dynamically increased in the course of dysentery. The comparison of the parallel results of both serological tests made it possible to evaluate the dispersion of the released antigen: it dynamically increased in the course of the disease, this increase being particularly high in feces. The dispersion of Shigella antigens in urine was greater than in feces over the entire course of the disease. These regularities in the release of the antigens and especially the specific features of the serological tests determined the scheme of the serological diagnosis of dysentery by the indication of Shigella antigens.

Antigens, Bacterial↗