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PubMedDate not supplied

The use of ELISA and nonradioactive DNA hybridization assays for the detection of human cytomegalovirus.

A hybridization assay using a biotinylated DNA probe was compared to both ELISA and direct isolation methods for detecting human cytomegalovirus (HCMV). The biotin labeled HCMV AD 169 HindIII-O-DNA fragment was used in a dot-blot assay to screen for the presence of HCMV in 186 urine specimens obtained from kidney transplant patients. The biotinylated HCMV HindIII-O probe could detect 3 log10 TCID50 units of HCMV. Urine specimens were also examined for the presence of HCMV by either ELISA or direct isolation of virus in tissue culture. The HindIII-O fragment detected 12 of 20 culture positive samples (sensitivity, 60%). There were 5 samples which were probe positive and cell culture negative (specificity, 97%). The ELISA assay also detected 12 of 20 culture positive samples (sensitivity, 60%). Eight samples were ELISA positive, cell culture negative (specificity, 95%). Seven specimens were positive by all three criteria. Five specimens which were both ELISA positive and probe positive were cell culture negative. The ELISA positive, probe positive, culture negative specimens originated from patients who gave a culture positive specimen within 10 days of the original sample. The combination of probe and ELISA assays detected 16 of the 20 culture positive specimens (sensitivity, 80%). The combined use of biotinylated DNA probes and ELISA allows the detection of HCMV in urine specimens with good sensitivity and specificity.

Biotin↗
PubMed1989

An improved ELISA and serum neutralisation test for the detection of turkey rhinotracheitis virus antibodies.

The development of a double-well ELISA test for the detection of turkey rhinotracheitis (TRT) virus antibody in turkey and chicken sera, utilising a streptavidin-biotin detection protocol is described. This test was compared with a conventional ELISA, which detects antibody using a peroxidase-labelled anti-turkey IgG. The double-well streptavidin-biotin ELISA was more sensitive than the conventional ELISA, and, due to the consistently low background values obtained, was further modified to a single-well ELISA test. Antibody titres obtained using the single-well streptavidin-biotin ELISA were similar to those obtained using the conventional ELISA. A microtitre serum neutralisation test for TRT virus read using an indirect immunoperoxidase (IIP) detection protocol after 48 h was also developed and compared to the standard neutralisation test employing a 7 day incubation and read by CPE. The specificity of the single-well streptavidin-biotin ELISA was confirmed using the rapid (IIP) serum neutralisation test.

Animals↗
PubMed1991

Development and evaluation of an ELISA using secreted recombinant glycoprotein B for determination of IgG antibody to herpes simplex virus.

An ELISA for the determination of IgG antibody to herpes simplex virus (HSV) was developed using a secreted recombinant HSV-1 glycoprotein B (gB-1s) as a solid phase. The clinical validity of the ELISA was established by testing different groups of sera containing HSV-1, HSV-2, or mixed antibody, in parallel with gB-1s ELISA and conventional HSV-1/HSV-2 ELISA. The new gB-1s ELISA detected HSV-1/HSV-2 antibody in sera from 48 subjects with either HSV-1 or HSV-2 past infection as well as in sera from 20 patients with primary infections by either serotype, in complete agreement with the results obtained using conventional ELISA. In 7 patients with HSV-1 encephalitis the kinetics of the gB-1s serum/cerebrospinal fluid antibody-titre ratio paralleled that of conventional ELISA over a period of time of up to 4 years. Acute and convalescent-phase sera from 28 patients with acute infections by human herpesviruses other than HSV did not show a significant cross-reactivity with gB-1s. In conclusion, gB-1s ELISA is a reliable assay for determination of HSV immune status as well as for diagnosis of both primary HSV-1 and HSV-2 infections and for diagnosis of HSV-1 encephalitis.

Antibodies, Viral↗
PubMed1993

Blocking ELISA for detection of mumps virus antibodies in human sera.

A new blocking enzyme-linked immunosorbent assay (ELISA) for detection of mumps virus (MuV) specific antibodies in large numbers of human serum samples was developed. The blocking ELISA is based on the reaction of MuV-specific, conjugated monoclonal antibodies (mAbs) with immobilized virus antigen, that has previously been incubated with a two-fold dilution of human serum. Mouse hybridomas that produce antibodies against MuV proteins were generated. They could be divided into 4 groups according to their hemagglutination inhibiting- and virus neutralizing capacities and their reaction in the blocking ELISA with MuV strain Enders. Ascites material from 22 mAbs derived from the 4 groups was further characterized with the MuV strains Enders and Jeryl Lynn. When mAbs from different groups were mixed in the blocking ELISA, an additional increase in absorbance could be observed. A mixture of 2 MuV neutralizing mAbs that were directed against HN and F protein, was used to assay 3 consecutive pre-, early post- and late postvaccination serum samples of 138 children, vaccinated at the age of 1.5 yr. A correlation of 94% was found between the blocking ELISA and the normal indirect ELISA, and of 98% between the blocking ELISA and the neutralization enzyme immunoassay (N50-EIA). The specificity and rapidity of the blocking ELISA makes it suitable for routine use in the determination of MuV neutralizing antibodies in large quantities of serum samples.

Antibodies, Monoclonal↗
PubMed1995

Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus.

A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1%, respectively, compared with EAV neutralization test and the recombinant antigen did not crossreact in ELISA with equine sera directed against other common equine respiratory viruses. Three post-EAV infection equine sera raised against different EAV isolates reacted strongly in the ELISA, as did two equine sera raised against EAV vaccines, indicating that the viral epitope was conserved between the viruses tested. Following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen ELISA preceded the development of a virus-neutralizing response. The study demonstrates the potential application of rGL 55-98 as a diagnostic antigen.

Animals↗
PubMed1992

Infectivity of bovine leukaemia virus infected cattle: an ELISA for detecting antigens expressed in in vitro cultured lymphocytes.

A simple ELISA is described for quantifying expression of bovine leukaemia virus (BLV) antigens in short-term cultures of peripheral blood lymphocytes (PBL) isolated from infected cattle. The PBL-ELISA demonstrated that antigen expression levels in infected cattle could vary by more than 50-fold. Inoculation of sheep with dilutions of lymphocytes from two BLV-infected cattle, differentiated in the PBL-ELISA by 50 to 100-fold, suggested that antigen expression levels were correlated with infectivity. Haematological data indicated that increased antigen expression in PBL cultures was associated with an increased number of circulating B-lymphocytes, irrespective of whether or not an animal had lymphocytosis. This supported the hypothesis that BLV-infected cattle that are PBL-ELISA positive are more infectious and may present a greater risk of transmitting the disease. The applicability of the PBL-ELISA to a field situation was assessed with 98 BLV-infected cattle from three commercial dairy herds with infection prevalences of 11%, 23% and 47%. Similar percentages (49%, 50% and 52%) of PBL-ELISA positive cattle were identified among those infected cattle available for testing in the three herds. An additional 22 infected cattle from an experimental herd were tested to assess the stability of antigen expression levels over an 8 month period. Fewer (27%) of these cattle were identified as PBL-ELISA positive and antigen expression levels were generally lower than those observed in the commercial herds. Antigen expression levels in the experimental herd remained stable over the period of the study. The potential of the PBL-ELISA to assist in BLV eradication programs by identifying those seropositive cattle with the greatest potential to transmit infection is discussed.

Animals↗
PubMed1996

Detection of antibodies against Streptococcus suis capsular type 2 using a purified capsular polysaccharide antigen-based indirect ELISA.

In the present study a purified capsular polysaccharide antigen-based indirect ELISA (CPS-ELISA) to detect antibodies against Streptococcus suis capsular type 2 was developed and compared with a whole cell antigen-based ELISA (WCA-ELISA). The WCA-ELISA presented a very low specificity when rabbit antisera to other capsular types were tested. Most of these cross-reactions were due to common proteins. The standardized CPS-ELISA gave satisfactory results using a concentration of 0.1 micrograms/well; most cross-reactions decreased significantly, with some exceptions, such as those shared by capsular types 1/2, 12 and 17. These cross-reactions were mainly due to common epitopes present in the capsule, as shown by immunoblotting. In a second experiment, the CPS-ELISA was used to detect antibodies in experimentally infected piglets. Despite the fact that capsular type 2 S. suis could be reisolated from all infected animals during and/or after the trial, antibody titers against a second infection. Sera from piglets experimentally infected were completely protected against a second infection. Sera from piglets experimentally infected with S. suis capsular types 1/2 or 12 presented cross-reactions at low dilutions, confirming data previously obtained with rabbit sera. Finally, sera of animals from herds with clinical signs associated with S. suis capsular type 2 did not present titers significantly different from those of disease free herds. From our results we concluded that the CPS-ELISA developed in this study can not be used as a diagnostic tool to identify infected animals.

Animals↗
PubMed1988

Development of urease conjugated enzyme-linked immunosorbent assays (ELISA) for the detection of IgM and IgG antibodies against Mycoplasma pneumoniae in human sera.

Urease conjugated enzyme linked immunosorbent assays (ELISA) were developed for the detection of human IgM and IgG antibodies against Mycoplasma pneumoniae. Results obtained by ELISA were compared with complement fixation test (CFT); which showed that of the 214 serum specimens tested, 80 were found to have antibody against M. pneumoniae. ELISA revealed that 70 of these specimens were IgG antibody, and 27 of them also contain IgM antibody. CFT failed to detect the presence of antibody against M. pneumoniae in five serum specimens tested. However, by using ELISA, three of them were found to have IgG and IgM antibodies. and the other two sera have IgG antibody only. Four out of the five specimens tested were the first serum specimens collected from patients with clinical and serological evidence of M. pneumoniae infection. In addition, 28 serum specimens, including 10 sera containing IgM rheumatoid factors and sera known to contain IgM antibody to other infectious organisms, were also tested for IgM antibody against M. pneumoniae by ELISA. None of these specimens showed a nonspecific reaction. ELISA had a sensitivity of 87.5% and a specificity of 96.3% when compared with CFT. Thus, ELISA developed in our laboratory is a specific test, and the results indicated that IgM ELISA might be used as a rapid diagnosis for M. pneumoniae infection.

Antibodies, Bacterial↗
PubMed1996

Diagnosis of hepatitis C virus (HCV) infection by antigen-capturing ELISA.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis. Detection of circulating antibodies against HCV by enzyme-linked immunosorbent assay (ELISA) has provided the main approach for the diagnosis of HCV infection. Most ELISA kits use a mixture of core, NS3, NS4 and NS5 antigen as capture antigens and enzyme-labeled goat anti-human IgG as conjugate. OBJECTIVES: To establish an ELISA system based on the antigen-capturing principle, using a recombinant chimeric polyprotein containing four HCV antigenic components as antigen. STUDY DESIGN: HCV antigens were expressed in Escherichia coli as chimeric polyprotein either in inclusion bodies or in soluble form. Protein expressed in inclusion bodies was used as solid-phase antigen, and the antigen expressed in a soluble form was used as enzyme conjugate after being labeled with horseradish peroxidase (HRP). RESULTS: Genes coding HCV antigens were cloned and sequenced, chimeric polyproteins containing four immunodominant components (core, NS3, NS4 and NS5) were expressed in E. coli both in soluble and in inclusion body form. These two chimeric proteins retained the antigenicity of HCV antigens. Antibody-capturing ELISA using the chimeric antigens showed a sensitivity of 97% (97/100) and a specificity of 98% (97/99) using the reference panel from the National Institute for the Control of Pharmaceutic and Biological Products of China (NICPBC); the same assay showed a sensitivity of 97.9% (48/49) and a specificity of 100% (43/43) using the self-established reference panel. Antigen-capturing ELISA was set up using the antigen labeled with horseradish peroxidase as conjugate, and was shown to be as sensitive as (97.9%) and more specific than (100%) antibody-capturing ELISA using the reference panel in this work. The antigen-capturing ELISA also showed a high accordance (98.9%) with UBI HCV enzyme immunoassay (EIA) 4.0 kits (United Biomedical Inc. USA). CONCLUSION: Antigen-capturing ELISA provided a convenient, sensitive and more specific approach for the diagnosis of hepatitis C virus infection.

Journal Article↗
PubMed2006

Application of an ELISA for PCB 118 to the screening of dioxin-like PCBs in retail fish.

A commercially available enzyme-linked immunosorbent assay (ELISA) kit was evaluated for the determination of toxic equivalents (TEQs) of dioxin-like polychlorinated biphenyls (PCBs) in retail fish. The ELISA was highly specific for 2,3',4,4',5-pentachlorobiphenyl (PCB 118), which is generally the most abundant dioxin-like PCB isomer found in fish. The quantitative limit of the ELISA (using 3,3',4'-trichloro-4-methoxybiphenyl as a surrogate standard for PCB 118) was 10 ng ml(-1) (125 pg assay(-1)) in the standard curve, corresponding to 50 pg PCB 118 g(-1) in the tested sample. Good recoveries of PCB 118 (78.7-112.3%) were obtained for spiked purified fish extracts according to the ELISA. Good linearity was also obtained in dilution tests using purified fish extracts. No significant interference of the matrix was observed in the ELISA when this purification procedure was used. Recovery tests in which PCB 118 was added to fish samples also resulted in acceptable recoveries (60.2-82.3%) in the ELISA following purification. The ELISA results for fish samples correlated well with the TEQ concentrations of dioxin-like PCBs obtained by high-resolution gas chromatography/high-resolution mass spectrometry (r = 0.92, n = 26). These data indicate that the ELISA kit is suitable for screening retail fish for the TEQs of dioxin-like PCBs.

Animals↗
PubMed2006

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.

Africa↗
PubMed2004

Comparison of three commercially available thrombomodulin ELISA kits.

Thrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of soluble serum thrombomodulin are regarded as parameters of disease activity in vasculitides and vasculopathies. However, the mean thrombomodulin values of different studies show marked concentration differences of the control values. The purpose of this study was to further investigate these differences. We examined 60 sera of patients with systemic lupus erythematosus (SLE) and 10 of healthy controls with three commercially available thrombomodulin ELISA kits for determination of their thrombomodulin concentration and correlation to disease activity. The disease activity of the SLE patients was determined with the SLAM-score. Raised thrombomodulin values were found in 58% (test A), 55% (test B) and 61.6% (test C). The thrombomodulin values significantly correlated with the SLE disease activity independently of the ELISA kit used (correlation coefficients: r=0.84 (test A), r=0.80 (test B), and r=0.65 (test C)). In addition, the correlation coefficients between the respective thrombomodulin values of the three tests were r=0.86 (test A to B), r=0.73 (test A to C) and r=0.79 (test B to C). However, significant differences between the results of the three ELISA kits were found between the detected thrombomodulin concentrations. The mean thrombomodulin concentrations of the controls were 25.6 ng/ml (test A), 3.53 ng/ml (test B), and 2.52 ng/ml (test C). Our results reveal that the soluble thrombomodulin values of all three commercially available ELISA kits significantly correlate with the disease activity of SLE patients. However, the results show significant differences in the determined thrombomodulin concentrations. A calibration would be required of the different ELISA kits in order to permit a direct comparison of the results of these thrombomodulin ELISAs. A general reference standard would be desirable for this calibration of all thrombomodulin ELISAs. However, this general reference standard has to be adapted to the distinct test conditions of all test kits as well as including all epitopes of thrombomodulin which are recognised by the different antibodies used in the respective test kits. At present, only ELISA kits from the same manufacturer should be used during a single study including any follow-up investigations.

Adolescent↗
PubMed2005

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.

Animals↗
PubMed2005

Discriminating between serological responses to European-genotype live vaccine and European-genotype field strains of porcine reproductive and respiratory syndrome virus (PRRSV) by peptide ELISA.

A peptide ELISA was developed based on an immunodominant and hypervariable epitope in the ORF4 envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). The peptide sequence was derived from the Porcilis live-attenuated PRRSV vaccine strain (genotype 1, European). Antibodies induced by the field PRRSVs currently circulating in Poland were not detected by the Porcilis ORF4 peptide ELISA. In contrast, Porcilis-vaccinated animals seroconverted in the ORF4 peptide ELISA at 21 days post-vaccination. Maximal titers were seen 30-92 days post-vaccination; most sera had endpoint titers between 1:1000 and 1:100,000. In a paired format, where sera were assayed in two separate ELISAs using ORF4 peptides derived from the genetically very closely related Porcilis and Lelystad PRRSV strains, it was possible to differentiate between antibodies induced by these two viruses. The Porcilis and Lelystad ORF4 peptide ELISAs had sensitivities of 89 and 100%, respectively. Thus, ORF4 peptide ELISA afforded specific detection of antibodies induced by an European-genotype live-attenuated vaccine PRRSV strain (Porcilis). The results suggest that specific ORF4 peptide ELISAs can be custom-made for European-genotype PRRSV strains, using general peptide design criteria described in this work. Thus, ORF4 ELISAs may be generally useful, to monitor safety and operational aspects of European-genotype live-attenuated PRRSV vaccine virus use in populations with circulating field European-genotype PRRSVs.

Amino Acid Sequence↗
PubMed2005

[Antihistones antibodies in systemic lupus erythematosus, comparison of three assays: Elisa, dot blot and immunoblot].

AIMS: The purpose of our study is to determine and compare the sensitivity of an enzyme linked immunosorbent assay (ELISA), a dot blot assay and an immunoblot assay for the detection of the IgG class antihistones antibodies in a population of systemic lupus erythematosus. The correlation between antihistones antibodies and the main clinical features of SLE or between antihistones antibodies and the presence of anti-double-stranded-DNA antibodies were analysed. MATERIALS AND METHODS: Serum samples from 126 systemic lupus erythematosus patients, classified according to the criteria of the American College of Rheumatology, were analysed for the presence of antihistones antibodies using a dot blot assay and an ELISA. Antihistones subfractions antibodies were assessed using the immunoblot technique on 88 out of the 126 sera. Serum samples from 50 blood-donors were analyzed as negative controls. RESULTS: The sensitivity of antihistones antibodies assessed by dot blot assay and ELISA was 69% and 54% respectively, and was lower than that of anti-double-stranded-DNA antibodies (83%). The sensitivity of the immunoblot assay for the detection of antihistones antibodies was 72%. Incidence of autoantibodies against histones H1, H2 A, H2B, H3 and H4 was 60%, 53%, 48%, 36% and 29.5% respectively. We found a correlation between the presence of antihistones antibodies, detected by the dot blot assay and ELISA, and the presence of anti-double-stranded-DNA antibodies. Antihistones antibodies detected by ELISA were correlated with renal disease in systemic lupus erythematosus; they showed a specificity, a positive and a negative predictive value for renal disease in systemic lupus erythematosus higher than those of anti-double-stranded-DNA antibodies. CONCLUSIONS: The sensitivity of the dot blot assay for the detection of antihistones antibodies is better than that of ELISA, but the latter technique could detect some cases negative by ELISA. Antihistones antibodies detected by ELISA have an important predictive value in the renal complications in systemic lupus erythematosus, better than that of AdsDNA. Antibodies to histone H1 were the most frequent antihistones autoandibodies in systemic lupus erythematosus and they were highly correlated with anti-double-stranded-DNA antibodies and renal disease.

Autoantibodies↗
PubMed2005

Factors associated with variation in bulk-tank-milk Salmonella Dublin ELISA ODC% in dairy herds.

Our objective was to determine factors that contribute to variation in bulk-tank-milk Salmonella Dublin enzyme-linked immunosorbent assays (ELISA) corrected optic-density measurements (ODC%) in dairy herds. We constructed hierarchical mixed models with repeated bulk-tank-milk ELISA ODC% in 31 Danish dairy herds. Four models included different combinations of explanatory factors, and we compared how well these models described the variation in the data. Herd was included as a random effect nested within Salmonella status and barn type. Detection of Salmonella Dublin or Salmonella Typhimurium by bacteriological culture of individual faecal samples or of slurry samples was associated with higher bulk-tank-milk ELISA ODC%, as was apparent Salmonella prevalence, the mean ELISA ODC% or mean-yield-corrected ELISA ODC% in milk samples collected from all individual cows. However, combinations of risk factors that included number or prevalence of cows with a very high ELISA ODC% provided better models, indicating that the effect of the cow-level explanatory variables on the bulk-tank-milk ELISA ODC% was related to the activity of the infection in the herd. Barn type (loose housing or tie stalls) was not associated with the variation in bulk-tank-milk ELISA ODC% in these models, which might be useful in planning of surveillance programs and intervention strategies.

Animals↗
PubMed2006

Bayesian estimation of sensitivity and specificity of serum ELISA and faecal culture for diagnosis of paratuberculosis in Greek dairy sheep and goats.

Latent class models were used to estimate the sensitivity (Se) and the specificity (Sp) of a serum ELISA and a faecal culture (FC) method for the diagnosis of paratuberculosis separately, in sheep and goats. The estimates were obtained by a Bayesian method. Possible dependence of diagnostic errors was investigated by comparing models where independence was assumed to models allowing for conditional dependence given the true disease status. ROC analysis for the serum ELISA was also performed and optimized cut-off values based on the misclassification cost term were determined. No evidence of conditional dependence was found. Assuming independence, posterior medians and 95% credible intervals for the Se(ELISA), Sp(ELISA), Se(FC) and Sp(FC), were 63% (42, 93%), 95% (90, 98%), 8% (2, 17%) and 98% (95, 100%) in goats and 37% (10, 80%), 97% (93, 99%), 16% (2, 48%) and 97% (95, 99%) in sheep. AUC was calculated 0.702 for sheep and 0.847 for goats. For the serum ELISA, there is need of species- and purpose-specific cut-off selection. For instance, with 20% prevalence situation and assuming equal and five-fold cost of a false negative to a false positive test result, the optimal cut-off is 0.3 and 0.05 in sheep, respectively, while it is 0.6 and 0.1 in goats, respectively. Serum ELISA performed better in goats than in sheep. Lowering the cut-off, in relation to the one recommended by the manufacturer, improved Se(ELISA) without seriously compromising Sp(ELISA), in either species.

Animals↗
PubMed2004

Development of a monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus.

Peste des petits ruminants (PPR) is an acute febrile, viral, disease of small ruminants with great economic importance. A competitive-ELISA (c-ELISA) test was developed for detection of antibodies to PPR virus in the sera samples of goats and sheep. The test uses monoclonal antibody to a neutralizing epitope of haemagglutinin protein of the virus. Based on the distribution of known negative sera samples (n=933) in respect of PPR virus antibodies in the test, a cut-off value was set as 38%. This value was the result of mean of negative population added with two times the standard deviations. A total of 1668 sera samples from goat and sheep and 32 sera from cattle were screened by c-ELISA and virus neutralization test (VNT). Efficacy of c-ELISA compared very well with VNT having high relative specificity (98.4%) and sensitivity (92.4%). The sensitivity of c-ELISA for PPR sero-surveillance could further be increased (95.4%), if the target population is non-vaccinated. c-ELISA test correlated well with VNT (r=0.845) for end-point titration of PPR virus antibody in 64 goat sera samples. It could clearly separate infected population from uninfected in field sera. Using c-ELISA test paired sera samples from 13 goats provided a clear diagnosis of PPR virus infection. Furthermore, antibodies to PPR virus could be successfully detected during 1 year after vaccination in four goats inoculated with an experimental PPR vaccine. Findings suggest that the c-ELISA test developed can easily replace VNT for sero-surveillance, sero-monitoring, diagnosis from paired sera samples and end-point titration of PPR virus antibodies.

Animals↗