Detection of anti-gag antibodies of feline immunodeficiency virus in cat sera by enzyme-linked immunosorbent assay

Summary Usinggag protein of feline immunodeficiency virus (FIV) expressed inEscherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIVgag protein in cat sera. With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIVgag protein were observed in all cases by the ELISA at early stage of infection. When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9%) out of 70 IFA positive sera were judged as negative by the ELISA. However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gp130 but not to p26 by a radioimmunoprecipitation assay. The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.     

Expression of feline immunodeficiency virusgag gene inEscherichia coli

Summary Thegag gene of a Japanese feline immunodeficiency virus (FIV) isolate, designated as FIV TM 2, was expressed inEscherichia coli as a fusion protein with TrpE. Using this expressed protein, an enzyme-linked immunosorbent assay was developed for detection of antibodies to FIVgag protein in feline sera. With serum samples from a cat experimentally infected with FIV, it was demonstrated that the period of seroconversion detected by this method corresponded to that by Western blotting.     

Active immunization with feline leukemia virus envelope glycoprotein suppresses growth of virus-induced feline sarcoma

The efficiency was examined of immunization with feline leukemia virus glycoprotein complexes (gp85 rosettes) to protect cats against tumors induced by feline sarcoma virus (FeSV). The glycoprotein was isolated from feline leukemia virus (FeLV). Young cats were vaccinated with the purified viral glycoprotein and challenged with FeSV (FeLV). FeLV gp85 antibody levels were measured by enzyme-linked immunosorbent assay and tumor volumes were determined. In immunized animals tumor development was reduced. Gp85 antibody levels before challenge were correlated inversely with tumor size (r2 = 0.79). This method appears to be suitable for fast and efficient testing of future FeLV vaccines.     

Monoclonal blocking ELISA detecting serum antibodies to the glycoprotein gII of Aujeszky's disease virus

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Aujeszky's disease virus (ADV) in porcine serum was developed. This ELISA is based on the reaction between virus antigen immobilized in a microdilution plate and a monoclonal antibody (MAb) reactive with a highly stable epitope on a glycoprotein complex, gII, of ADV. The viral epitope was expressed by 18 European field, laboratory and vaccine strains of ADV. The MAb used in the test was selected among 15 MAbs all reactive with viral epitopes apparently recognized by the porcine immune system as well. Good agreement was found when serum samples from 375 pigs were tested in both a polyclonal and the monoclonal blocking ELISA.     

Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis.

The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence cleavage occurred in Sf9 cells expressing the envelope precursor, but further processing was not observed. Crude lysates of insect cells infected with the wild-type baculovirus or with the recombinant viruses were used to develop an enzyme-linked immunosorbent assay for the detection of feline immunodeficiency virus-specific antibodies in cat sera. The assay showed a higher sensitivity and specificity than immunofluorescence and Western blotting (immunoblotting).     

Sensitivity and specificity of pooled versus individual sera in a human immunodeficiency virus antibody prevalence study.

We evaluated the efficacy of testing pooled versus individual sera for the detection of human immunodeficiency virus antibody. A total of 5,000 individual specimens and 500 pools of 10 specimens each were assayed by an enzyme-linked immunosorbent assay. There was complete agreement in human immunodeficiency virus enzyme-linked immunosorbent assay reactivity for pooled versus individual specimens. An estimated savings of 60 to 80% (labor and supplies) can be realized dependent upon pooling and assay format.     

Monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of p27

Three different monoclonal antibodies were developed against the major core protein (p27) of feline leukemia virus (FeLV). Each antibody was directed against a different epitope of the species-specific portion of FeLV-p27. The 3 antibodies reacted with 5 different isolates of FeLV but not with 7 other retroviruses (MuLV (Rauscher), MuLV (AKR), MPMV, MMTV, SMRV, BAEV, RD 114). These monoclonal antibodies could readily be adapted to an enzyme-linked immunosorbent assay (ELISA) for the specific measurement of FeLV-p27. When compared in an ELISA with conventional reagents, the battery of monoclonal antibodies proved to be as sensitive as conventional polyclonal antibodies.     

Detection of Specific Antibodies to an Antigenic Mannoprotein for Diagnosis of Penicillium marneffei Penicilliosis

The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.     

Evaluation of a 2-minute anti-human immunodeficiency virus (HIV) test using the autologous erythrocyte agglutination technique with populations differing in HIV prevalence.

A total of 1,800 blood specimens (1,000 from healthy blood donors, 300 from patients with sexually transmitted disease, and 500 from intravenous drug users) were simultaneously tested with anti-human immunodeficiency virus enzyme-linked immunosorbent assay (ELISA) kits and a newly developed 2-min test for anti-human immunodeficiency virus based on the principle of autologous erythrocyte agglutination (AGEN Biomedical Limited). We found that AGEN's rapid test was as sensitive and specific as the other ELISA kits.     

Determination of subgroup-specific feline oncornavirus-neutralizing antibody.

A microneutralization assay was developed for antibody-to-subgroup-specific feline oncornaviruses. This study combines the economic advantage of a microtiter system and the quantitative focus reduction method which permits contruction of multiplicity curves for determination of virus-neutralizing titers. A twofold increase in Synder-Theilen feline sarcoma virus (ST-FeSV) on feline embryo cells decreased by approximately twofold the titer of reference goat serum prepared against Kawakami-Theilen feline leukemia virus. Similar dose effects with FeLV serotype virus preparations were not observed. An assay system utilizing FeLV serotypes on sarcoma-positive leukemia-negative cells demonstrated slightly greater sensitivity than one employing ST-FeSV on FE cells. Differential antibody responses to the three subgroup-specific feline oncornaviruses (A, B ,and C) were observed in reference goat sera. This test demonstrated good reproducibility as well as sensitivity and constitutes a significant improvement over end point dilution assay systems.     

Competitive enzyme immunoassays for the rapid detection of antibodies to feline infectious peritonitis virus polypeptides.

Monoclonal antibodies specific for the envelope (E1), peplomer (E2), and nucleocapsid (N) polypeptides of feline infectious peritonitis virus (FIPV) were used in rapid, competitive enzyme-linked immunosorbent assays (ELISA) to study the humoral immune response of cats to FIPV infection. Results from the competitive ELISAs were correlated with those from immunofluorescent antibody assays (IFAs) on 203 samples obtained from 64 individual cats. The IFA results correlated best with those obtained with the anti-E1 specific competitive ELISA (85.7%). In contrast, anti-N and anti-E2 competitive ELISA results correlated with IFA results only 65.5 and 2.4% of the time, respectively. The results of the anti-E1 specific competitive ELISA were not influenced by the total immunoglobulin concentration or the possible presence of free viral antigens in the serum. These results suggest that a competitive ELISA involving the use of enzyme-conjugated monoclonal antibody to the E1 glycoprotein of FIPV is a simple and rapid replacement for the more cumbersome IFA.     

Evaluation of a computer-assisted, kinetics-based enzyme-linked immunosorbent assay for detection of coronavirus antibodies in cats.

A computer-assisted, kinetics-based enzyme-linked immunosorbent assay was adapted for the detection of coronavirus antibodies in feline serum. An alkaline antigen diluent (carbonate-bicarbonate buffer, pH 9.6) used in initial experiments produced diffuse, nonspecific color reactions in both viral and control antigen cuvettes which were correlated, paradoxically, with coronavirus antibody levels in test sera. These interfering reactions were minimized by use of lower-pH antigen diluents such as water and phosphate-buffered saline. Background kinetics-based enzyme-linked immunosorbent assay reactivity directed against a noncoronaviral component of antigen tissue culture fluids could then detected in numerous sera, particularly in samples with lower titers. Much of this reactivity was shown to be associated with bovine gamma globulins in cell culture fluid. It was not serum lot or species specific, since a variety of bovine serum lots as well as individual lots of serum from other mammalian and avian species reacted. Reactivity was markedly reduced when cells for antigen preparation were grown in gamma globulin-free bovine serum. Generation of corrected slope values from the kinetics-based enzyme-linked immunosorbent assay made it possible to correct for residual background reactivity in individual test sera and thus eliminate a potentially major source of false-positive reactions. Collectively, these studies indicated that the control of nonspecific reactivity in feline coronavirus serology is absolutely essential to obtain useful estimates of specific antibody responses.     

Saliva as a source of feline leukemia virus antigen for diagnosis of disease.

An enzyme-linked immunosorbent assay (ELISA) for detection of feline leukemia virus (FeLV) p27 in saliva was tested for its accuracy and sensitivity in diagnosing FeLV infections. Saliva and serum samples from 564 clinical cases were tested with a 99.2% specificity. The overall accuracy of the saliva ELISA reactive to the serum ELISA was 97.9%. Experimentally, the ELISA saliva was the least sensitive in diagnosing early FeLV infections. However, the overall accuracy, ease of use, and simplicity of the test support its use as a screening procedure in clinical practice.     

Detection and differentiation by sandwich enzyme-linked immunosorbent assay of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus- and acquired immunodeficiency syndrome-associated retroviruslike clinical isolates.

Monoclonal antibodies can be used in sandwich enzyme-linked immunosorbent assays to measure viral antigens. Such an assay was developed to detect the core protein, p24, of human T-cell lymphotropic virus type III and lymphadenopathy-associated virus, etiologic agents of the acquired immunodeficiency syndrome (AIDS). Another AIDS-associated virus, AIDS-associated retrovirus type 2 (ARV-2) could not be detected in this assay because of the low affinity of one of the monoclonal antibodies to ARV-2 p24. Detection of ARV-2 was accomplished with a monoclonal antibody-rabbit polyclonal antibody sandwich enzyme-linked immunosorbent assay. These two assays were used to efficiently detect AIDS-related viruses in lymphocyte cell cultures and to distinguish strains of the viruses.     

Establishment of a feline astrocyte-derived cell line (G355-5 cells) expressing feline CD134 and a rapid quantitative assay for T-lymphotropic feline immunodeficiency viruses

Few laboratory strains of feline immunodeficiency virus (FIV) can infect Crandell feline kidney cells (an epithelial-type of cells), however, most primary isolates are T-lymphotropic. T-lymphotropic FIV requires both feline CD134 (an activation marker of helper T-lymphocytes) and CXCR4 (a chemokine receptor) in infection as primary and secondary receptors, respectively. Using feline T-lymphoblastoid cell lines, titration of primary FIV isolates was carried out, however the titration assay was laborious and time-consuming. In this study, using G355-5 cells (a feline astrocyte-derived cell line) transduced with a cDNA of feline CD134 as target cells, an assay system was developed to quantitate primary FIV isolates. With a previous method using a feline T-lymphoblastoid cell line (MYA-1 cells) highly sensitive to FIV, it took 12 days to complete the assay, however, it took only 2 days with the new method. The FIV-infected cells became in a state of persistent infection, producing a large amount of FIV, indicating that the cells will be useful for propagation of T-lymphotropic FIV strains.     

Antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses.

Transmissible gastroenteritis virus of swine (TGEV), feline infectious peritonitis virus (FIPV), and canine coronavirus were studied with respect to their serological cross-reactivity in homologous and heterologous virus neutralization, immune precipitation of radiolabeled TGEV, electroblotting, and enzyme-linked immunosorbent assay using individual virion polypeptides prepared by polyacrylamide gel electrophoresis. TGEV was neutralized by feline anti-FIPV serum, and the reaction was potentiated by complement; heterologous neutralization involved antibody reacting with the peplomer protein (P), the envelope protein (E), and cellular (glycolipid) components incorporated into the TGEV membrane. Electrophoretic analysis of immune precipitates containing [35S]methionine-labeled disrupted TGEV and feline anti-FIPV antibody confirmed the reaction with the P and E polypeptides and showed the nucleocapsid protein (N) in addition. Electroblotting, followed by incubation with antibody, 125I-labeled protein A, and fluorography, disclosed cross-reactions between the three viruses at the N and E levels and revealed differences in the apparent molecular weights of the latter. Enzyme immunoassays performed with standard amounts of immobilized P, N, and E polypeptides of the three viruses showed recognition of the antigens by homologous and heterologous antibody to comparable degrees. These results indicate a close antigenic relationship between TGEV, FIPV, and canine coronavirus due to common determinants on the three major virion proteins. The taxonomic implications of these findings are discussed.     

Development of a blocking enzyme-linked immunosorbent assay for the serological diagnosis of chicken anaemia virus

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibody to chicken anaemia virus (CAV) are described. This test depends on the abilities of CAV-specific antibodies present in convalescent chicken serum to block the reaction between virus antigen, adsorbed to the ELISA plate. and a CAV-specific mouse monoclonal antibody (MAb), 2A9, that has been conjugated to horseradish peroxidase. The 2A9 MAb has been shown to react with 10 geographically different field isolates of CAV, a finding which indicates that the test will find worldwide application. In comparative experiments involving 525 serum samples from specific pathogen free and commercial breeder flocks, there was 98.5% agreement between the results obtained with the blocking ELISA and those obtained with an indirect ELISA developed previously in this laboratory. The blocking ELISA was found to have advantages in terms of speed and cost compared with the indirect ELISA format.     

A rapid immune plaque assay for the detection of Hendra and Nipah viruses and anti-virus antibodies.

Rapid immune plaque assays have been developed to quantify biohazard level 4 agents Hendra and Nipah viruses and detect neutralising antibodies to both viruses. The methods rely on the fact that both viruses rapidly generate large syncytia in monolayers of Vero cells within 24 h and that monospecific antiserum to the Hendra virus phosphoprotein (P) detects that protein in both Hendra and Nipah virus-induced syncytia after methanol fixation of virus-infected cells. The P protein is a constituent of the ribonucleoprotein core of the viruses and a component of the viral RNA-dependent RNA polymerase and is made in significant amounts in infected cells. In the immune plaque assay, anti-P antibody is localised by an alkaline phosphatase-linked second antibody and the Western blot substrates 5-bromo-4-chloro-3-indolyl phosphate and p-nitro blue tetrazolium. A modification of the rapid immune plaque assay was also used to detect antibodies to Nipah virus in a panel of porcine field sera from Malaysia and the results showed good agreement between the immune plaque assay and a traditional serum neutralisation test. After methanol fixation, plates can be stored for up to 7 months and may be used in the immune plaque assay to complement the enzyme-linked immunosorbent assay screening of sera for antibodies to Nipah virus. At present, all enzyme-linked immunosorbent assay positive sera are subject to confirmatory serum neutralisation tests. Use of the immune plaque assay may reduce the number of sera requiring confirmatory neutralisation testing for Nipah virus antibodies under biohazard level 4 conditions by identifying those that generate false positive in the enzyme-linked immunosorbent assay.