Evaluation of ELISA for the detection of Toxoplasma antibodies in swine sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from greater than 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving less than 10 EIU were negative in the other tests, and all those with greater than 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10-70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

OBJECTIVE: To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. DESIGN: Comparison of results from known positive and negative sheep populations. PROCEDURE: Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. RESULTS: ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. CONCLUSION: Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.     

The development and evaluation of an ELISA for the detection of antibodies to caprine arthritis-encephalitis virus in goat sera

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to caprine arthritis encephalitis virus (CAEV) in goat sera. The system was evaluated using some 1500 sera from flocks of known clinical history. From this data the interpretation limits of the system were determined. The ELISA system was compared with a gel precipitin test using 5800 sera. Of the positive sera, ELISA detected 97.3% and AGPT 61%. Further evaluation was made using 60 sera of known CAEV reactivity from the USA, and results agreed 100%. Indications are that antibody to the envelope glycoprotein gp135 is being detected. The ELISA system is more sensitive than the precipitin test and is presently being used in a CAEV flock accreditation scheme.     

Evaluation of a blocking ELISA for the detection of antibodies against Lawsonia intracellularis in pig sera

Background

Lawsonia intracellularis is a common cause of chronic diarrhoea and poor performance in young growing pigs. Diagnosis of this obligate intracellular bacterium is based on the demonstration of the microbe or microbial DNA in tissue specimens or faecal samples, or the demonstration of L. intracellularis-specific antibodies in sera. The aim of the present study was to evaluate a blocking ELISA in the detection of serum antibodies to L. intracellularis, by comparison to the previously widely used immunofluorescent antibody test (IFAT).

Methods

Sera were collected from 176 pigs aged 8-12 weeks originating from 24 herds with or without problems with diarrhoea and poor performance in young growing pigs. Sera were analyzed by the blocking ELISA and by IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test and the results of the blocking ELISA was modelled against the IFAT test with a "2 dependent tests, 2 populations, no gold standard" model.

Results

At the finally selected cut-off value of percent inhibition (PI) 35, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%. The positive predictive value was 0.82 and the negative predictive value was 0.89, at the observed prevalence of 33.5%.

Conclusion

The sensitivity and specificity as evaluated by Bayesian statistic techniques differed from that previously reported. Properties of diagnostic tests may well vary between countries, laboratories and among populations of animals. In the absence of a true gold standard, the importance of validating new methods by appropriate statistical methods and with respect to the target population must be emphasized.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of anti-chlamydial antibodies in pig sera

The evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against chlamydiae in pig sera is described. The most widely used serological test is the complement fixation test (CFT). The CFT has a lack of sensitivity and specificity because of low antibody titers and unspecific reactions. Eight conventionally raised pigs were exposed to a pathogenic strain of Chlamydia suis, four controls were mock infected. The immune responses was monitored by CFT and indirect ELISA. There was no agreement between CFT and ELISA data. These results were confirmed by a study with 191 sera from nine pig farms. As shown by ELISA and PCR chlamydiae are widespread in swine.     

改进试管凝集试验法检测布鲁氏菌病抗体

为证实改进的试管凝集试验（SAT）的使用价值,分别用微量试管凝集试验（MSAT）和SAT以国际标准血清为参比检测6份牛布鲁氏菌病阳性血清的抗体效价。结果表明：MSAT和SAT试验结果一致,而且MSAT具有稀释方法更简便、精确,省时省力,可节约血清、抗原等试验原材料,试验结果容易判定,重现性良好等优越性。     

Possibilities for standardization of ELISA for detection of Salmonella antibodies in sera and meat juices of pigs

Programmes for controlling salmonella infections in German piggeries are based on the meat-juice-ELISA conducted in various investigation centres by using different test-kits. A usual procedure for harmonization (standardisation) of results is the calculation of the percentage of antibody-concentration from field samples in relation to the extinctions of a set of control-sera with known antibody concentrations. Whether this system is still acceptable in case of using different test-kits seems to be questionable. In principle, difficulties arise by calculating field results from the regression curve of control-sera because the calculated percentages of antibodies do not represent the antibody concentration but, instead, the percentages of the extinctions measured, and secondly, because control-sera presently in use are directed against different salmonella serovars. In regard to the number of laboratories involved and because of a variety of test-kits used it seems to be more adequate to include only one anti-Salmonella Typhimurium standard-serum at a given antibody concentration which is to be tested repeatedly on every test-plate. Simultaneously, further controls should include another anti-Salmonella Typhimurium and one anti-Salmonella Choleraesuis serum which should provide results similar to the Danish system which is regarded as a standard. As well, a negative serum must be included in the test and a minimum difference in extinctions between this negative serum and the standard positive control-serum should be reached to prove the validity of results from the test plate.     

Development of an indirect ELISA based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in human serum

BackgroundBrucellosis is the most common zoonotic diseases worldwide. The aim of this study was to develop and evaluate the diagnostic performance of an indirect-ELISA (I-ELISA) method based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in a human serum.Materials and methodsThe study was conducted in two species-rich endemic areas of Iran (Tehran and Lorestan provinces). Serum samples of 102 patients and 150 healthy individuals were tested by the new kit and the commercial Vircell kit for the presence of anti-Brucella IgM antibodies. The disease status was confirmed by Wright agglutination test. The difference in the mean optical densities (OD) recorded by the new and the Vircell kits for patients and healthy individuals were tested using Two-tailed Student t-test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the new kit were informed using Receiver operating curve analysis. The results were used to guide the choice of cutoff. Agreements in ODs recorded by the new and the Vircell kit was visually inspected using Bland-Altman plot.ResultsThe new I-ELISA showed excellent diagnostic performance (sensitivity and PPV = 95.7%, specificity and NPV = 97.8%) for the diagnosis of brucellosis. The cut-off area for the antibody index (AI) was determined to be 8–10, where AIs less than 8 and greater than 10 were considered Brucella-negative and -positive, respectively. AIs of 8–10 show equivocal results, requiring re-testing. The Vircell kit showed low (36.8%) sensitivity and perfect (100%) specificity on the same samples. The Bland-Altman plot showed low agreement between both tests in recording the OD values for the same individuals.ConclusionThe new I-ELISA based on whole cell Brucella abortus S99 showed a good performance for the detection of Brucella spp. Lack of agreement between the new and the Vircell kit suggest that the performance of ELISA kits might be dependent on the geographical area under study. Hence, validation of the new and the Vircell kits is recommended prior to their implementation in other geographical areas.     

Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA.

A blocking ELISA was developed for the detection of antibodies to foot-and-mouth disease virus SAT1, SAT2 and SAT3 and for the quantification of antibodies on a single dilution of serum. The avidin-biotin system was used. The test was compared with the liquid-phase ELISA executed at the World Reference Laboratory for foot-and-mouth disease. It was found to have favourable logistics and combined high specificity with high sensitivity. The quantitative test using a single dilution of serum was resource saving and proved to be a reliable and precise method for the assessment of antibody levels.     

Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.     

A comparative study of ELISA and other methods for the detection of Brucella antibodies in bovine sera

Enzyme-linked immunosorbent assay (ELISA), using β-galactosidase and a fluorigenic substrate, was used for the detection of antibodies to Brucella abortus in bovine sera.Among 677 animals from 9 brucellosis-free herds, none reacted in the ELISA. Among 785 animals from 23 brucellosis-infected herds, 336 were positive in ELISA, 229 in the slow agglutination test (SAT), 185 in the complement fixation test (CFT), and 165 in the Rose-Bengal test (RBT).Experimental infections were conducted with two B. abortus strains. At slaughter on day 101, after intraconjunctival infection of heifers with B. abortus strain 19 organisms, 3 animals were positive in the SAT, 3 in the CFT, 4 in the RBT and 11 in the ELISA, and Brucella organisms could be cultivated from 10 animals; among these, 2 scored positive in the SAT, 3 in the CFT, 3 in the RBT and 8 in the ELISA test. Seventeen heifers were infected with organisms of B. abortus strain 2308. On day 101, 11 heifers were found to be carriers, all of which yielded positive results in the CFT, RBT and ELISA tests, but not in the SAT.     

Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera

Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.     

Detection of Trichinella spiralis nativa antibodies in porcine sera by ELISA using T. spiralis spiralis excretory-secretory antigen

Enzyme-linked immunosorbent assay examination of sera from pigs vaccinated with T. spiralis nativa infective larvae and/or challenged with T. spiralis spiralis larvae using a T. spiralis spiralis excretory-secretory antigen showed a significant cross-reaction between the two species of Trichinella. Eight of 12 pigs vaccinated with a high dose of T. spiralis nativa reacted positively 28 days postvaccination while the remaining four pigs had high but negative ELISA optical density readings. Five of six pigs challenged with the homologous species reacted positively 28 days postchallenge but the sixth pig remained negative despite having a muscle infection of 5.6 larvae/g of musculature.     

Specificity of the enzyme-linked immunosorbent assay (ELISA) for antibodies in the sera of specific pathogen-free lambs vaccinated with Pasteurella haemolytica antigens

Pooled serum from specific pathogen-free (SPF) lambs vaccinated with sodium salicylate extracted (SSE) antigens of Pasteurella haemolytica serotype A1 was shown to contain antibody to other A serotype SSE antigens when tested by the enzyme-linked immunosorbent assay (ELISA). Specific antibody to serotype A1 SSE antigens was demonstrated by absorption of the serum pool with heterologous serotype SSE antigens.The type-specific antigens of serotypes A1 and A9 were prepared by phenol—water extraction (PWE) of their respective SSE antigens. The PWE antigens were examined in a sandwich ELISA where rabbit IgG anti-P. haemolytica A1 cells or A9 cells was used as a coating layer to bind PWE antigens. The specificity of these antigens was demonstrated by marked reduction of reactivity between serum from SPF lambs vaccinated with SSE of serotypes A1 or A9.