Standard Agar Gel Immunodiffusion Test Research Articles

Comparative efficacy of standard AGID and precipitinogen inhibition test with monoclonal antibodies based competitive ELISA for the serology of Peste des Petits Ruminants in sheep and goats

This project was conducted to investigate the comparative efficiency of competitive ELISA (cELISA), standard Agar Gel Immunodiffusion Test (AGID) and Precipitinogen Inhibition Test (PIT) for the diagnosis of Peste des Petits Ruminants (PPR) in Pakistan. To deal with this, serum samples from 198 sheep and 82 goats were collected from three different government livestock farms and all the samples were run simultaneously with the three serological tests. The samples found positive for PPR antibodies through cELISA, AGID and PIT were 96 (34.2%), 60 (21.4%) and 72 (25.7%), respectively. Kappa statistics were applied to evaluate the concordance between the laboratory-based test (cELISA) and field-based tests (AGID and PIT). Kappa statistics scores for cELISA versus AGID and PIT were 0.6343 (95% Confidence Interval CI 0.5231-0.7456) and 0.7134 (95% Confidence Interval CI 0.5987-0.8281), respectively, which indicate a "substantial" agreement between cELISA and AGID and "significant" agreement between cELISA and PIT. AGID and PIT revealed relative diagnostic sensitivities with cELISA of 59.3% and 69.7% and relative diagnostic specificities of 98.3% and 97.2%, respectively. The data suggested that for mass screening and control of PPR, these serological tests proved practical in the absence of cELISA since they have high relative diagnostic specificities and a satisfactory relative diagnostic sensitivities.

Sensitive and specific detection of proviral bovine leukemia virus by 5′ Taq nuclease PCR using a 3′ minor groove binder fluorogenic probe

Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5′ Taq nuclease assays using 3′ minor groove binder DNA probes (TaqMan ®MGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log 10 dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan ®MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5′ Taq nuclease assay.

A new sensitive serological assay for detection of lentivirus infections in small ruminants.

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99. 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Detection of foot-and-mouth disease virus infection-associated antigen antibodies: comparison of the enzyme-linked immunosorbent assay and agar gel immunodiffusion tests

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was compared with the standard agar gel immunodiffusion test (AGID) to identify and quantify antibodies against foot-and-mouth disease (FMD) virus infection-associated (VIA) antigen. A total of 3181 cattle sera were tested. Of these sera, 1885 were from cattle which had not been exposed to FMD. A total of 1296 sera were either from cattle which were experimentally exposed to FMD virus or from cattle involved in field outbreaks. The results indicate that the ELISA has the same specificity as the AGID test, but is more efficient in detecting cattle exposed to FMD virus. The ELISA technique will probably prove to be a more satisfactory test in support of the prevention, control and eradication programs for the disease.

Development of an enzyme-linked immunosorbent assay for equine infectious anemia virus detection using recombinant Pr55gag

To provide more sensitive and convenient methods for the detection of equine infectious anemia virus (EIAV), we developed an enzyme-linked immunosorbent assay (ELISA) employing the EIAV gag precursor (Pr55gag) produced by using recombinant DNA techniques. The antigenic reactivity of the recombinant EIAV Pr55gag was found to be equivalent to that of the virion p24gag and elicited high-titered antiserum in rabbits. When a large number of horse sera were analyzed for the presence of antibodies to EIAV by this ELISA, a radioimmunoassay for EIAV p15gag, or the standard agar gel immunodiffusion test, there was 98.7% concordance among the assays. By using the ELISA it was possible to specifically detect antibodies earlier after experimental infection of horses with EIAV than with the other two tests. A competition ELISA developed in order to detect EIAV gag antigens was found to be approximately 15 times more sensitive than the radioimmunoassay for EIAV p15gag. Antigens of other animal lentiviruses as well as those of the prototype oncovirus failed to compete in this assay.

Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody.

A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in tissue culture fluids.