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PubMed1994

Detection of autoantibodies against M2, LKM-1, and SLA in liver diseases by standardized uniform ELISA-techniques.

Antibodies directed against soluble liver antigen (SLA), liver kidney microsomal antigen (LKM-1-AG), and antimitochondrial antigen M2 (M2-AMA) are critical serological markers for the differential diagnosis of autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). The exact diagnosis of autoimmune hepatitis and PBC is of great clinical relevance, as it leads to different therapeutic strategies. In the present work, a simple and reliable ELISA test system is described, which applies the same test principle for the detection of three different species of autoantibodies important for the diagnosis of chronic liver disease. The ELISA assays are based on a competitive inhibition of binding of positive standard antibodies by patients sera containing antibodies of unknown specificity. The purified immunoglobulins of clinically and serologically clearly defined patients with SLA or LKM-1 positive AI-CAH and with M2 positive PBC were used as coating- and detection antibodies in the ELISAs. From homogenized rat liver the fractionated 100,000g supernatant was employed for the SLA ELISA, the microsomal preparation served as antigen for the LKM-1 ELISA and the mitochondrial preparation was used for the M2 ELISA. In 1,500 sera of patients with the differential diagnosis of a hepatobiliary disease, 17 gave a positive signal in the SLA-ELISA, 12 in the LKM-1-ELISA, and 72 in the M2-ELISA. The results of the ELISAs were compared with Western blotting and immunofluorescence staining pattern on cryostat sections and Hep2 cells. The antibody profiles of several patients are described in detail.

Adult↗
PubMed2000

Identification of Staphylococcus aureus enterotoxins A and B genes by PCR-ELISA.

We developed PCR-enzyme linked immunosorbent (ELISA) assays to detect Staphylococcus aureus enterotoxins A and B genes. The assays use internal biotin-labelled oligonucleotides as capture probes for immobilizing and subsequently detecting target sequences on microtiter plates. The detection limits of the PCR-ELISAs were approximately 250 gene copies, versus 2500 gene copies by agarose gel analysis. The sensitivity of the assays, as determined from a reference panel of 46 coded samples that included DNA purified from 31 different bacterial species and strains, SEA and SEB plasmid controls, and no-template controls was 100%. No cross-reactivity was observed with DNA from non-staphylococcal species. Using 27 clinical isolates of S. aureus, the SEA PCR-ELISA identified the enterotoxin A (sea) gene in 26 samples, and the SEB PCR-ELISA identified the enterotoxin B (seb) gene in all 27 samples. Compared with conventional antigen capture ELISAs for SEA and SEB toxins, the PCR-ELISAs showed overall superior detection limits. The sensitivity and specificity levels of the SEA PCR-ELISA and the SEA toxin ELISA were comparable within their respective detection thresholds, but the sensitivity and specificity of the SEB PCR-ELISA was much greater than that of SEB toxin ELISA.

Bacteria↗
PubMed1998

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus.

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.

Animals↗
PubMed1990

Two ELISA's for measurement of protein S, and their use in the laboratory diagnosis of protein S deficiency.

Protein S is a vitamin K-dependent coagulation factor involved in the regulation of the anticoagulant activity of activated protein C (APC). Heterozygosity for hereditary protein S deficiency is considered as a risk factor for the development of thrombotic disease. To measure the plasma concentration of protein S, two enzyme-linked immunosorbent assays (ELISA) have been developed: the ELISA-p (using polyclonal antibodies both as catching and tagging antibodies) and the ELISA-m (using polyclonal antibodies as catching antibodies and a monoclonal antibody as tagging antibody). The performance of these assays has been compared with that of an immunoradiometric assay (IRMA) for total protein S. Results obtained with both ELISA's in plasma's of healthy volunteers, patients with hereditary protein S deficiency type I and patients using oral anticoagulant treatment correlate very well with those of the IRMA: r = 0.974 (n = 57) for ELISA-p and r = 0.966 (n = 57) for ELISA-m. The intra- and inter-assay variation (n = 6) were calculated to be 5% and 11% for the ELISA-p and 4% and 10% for the ELISA-m. Both ELISA's can be used for the measurement of total protein S, but also for the measurement of free protein S and C4b-BP-PS complexes, after PEG precipitation. The ELISA-m was further used to measure free protein S in healthy volunteers (n = 40), patients with a protein S deficiency type I (n = 20) and patients with unexplained familial thrombosis (n = 76). In the first two groups measured and calculated free protein S levels were very similar. In the third group 7 patients were identified with total protein S in the normal range, but with measured free protein S and the ratio between the measured free protein S and calculated free protein S below the lower limits of the normal range.

Blood Coagulation Disorders↗
PubMed1989

Comparison of different ELISAs for the detection of monoclonal antibodies to human interferon-alpha. Implications for antibody screening.

Three enzyme-linked immunosorbent assays (ELISA) were compared in the initial screening of some 400 hybridoma supernatants for antibodies to a recombinant human interferon-alpha subtype, 4a (IFN-alpha 4a). In these assays, (i) the antigen was coated directly to polystyrene microtitre plates (ELISA-PS), (ii) the antigen was coated directly to nitrocellulose (ELISA-NC), or (iii) the antigen and mouse antibody were reacted in solution and the resulting complex immobilized to a solid support precoated with polyclonal rabbit anti-IFN-alpha antibody (ELISA-SW). The ELISA-PS detected eight antibodies, the ELISA-NC 15 and the ELISA-SW 18. The interferon specificity of the MAbs detected by each of the ELISAs was confirmed by neutralization of IFN-alpha antiviral activity and Western immunoblotting analysis. The results suggest that in ELISAs, the presentation of an antigen and its recognition by antibodies is substantially influenced by the method used in the immobilization of antigen and the type of solid support used. The ELISA-SW proved optimal for screening hybridoma supernatants for antibodies to IFN-alpha 4a, and is recommended for screening for antibodies to other soluble antigens.

Animals↗
PubMed1995

Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus.

Two novel formats of ELISA for the detection of antibodies against swine vesicular disease (SVD) virus were developed. One of the tests described is a monoclonal antibody-based competitive ELISA (MAC-ELISA). In this test, specific antibodies in serum are detected due to their ability to compete with a neutralizing monoclonal antibody (MAb). The second is an indirect trapping ELISA which employs isotype-specific MAbs to detect swine IgG or IgM specific for SVD virus. The diagnostic sensitivity and specificity of the MAC-ELISA was studied on 5671 field sera of known origin, enabling the cut-off level to be defined. Using the MAC-ELISA, 100% of sera from infected pigs were found positive, whereas only 0.45% of negative sera gave a false-positive result. A positive correlation between MAC-ELISA and virus neutralizing titres was recorded for pig sera collected sequentially after experimental infections. The results from the isotype-specific ELISA revealed the dynamics of the antibody response to SVD virus in pigs. The first antibodies were detectable as early as 3 days after experimental infection. Up to the 10th day, demonstrable antibodies were exclusively of the IgM class. IgG developed later, between 11 and 14 days postinfection and remained at a plateaux level throughout the whole investigation period. The two tests satisfy different diagnostic requirements: the MAC-ELISA is useful as a screening test, the isotype-specific ELISA has potential application for the determination of stage of infection. Both tests benefit from the use of MAbs in terms of specificity and standardization and have advantages over the virus neutralization test.

Animals↗
PubMed2006

Serum laminin assayed by Slot-Blot-ELISA in patients with combined viral hepatitis C and schistosomiasis.

OBJECTIVES: Hepatic schistosomiasis and chronic hepatitis C virus (HCV) are the most prevalent agents causing hepatic fibrosis in humans. Laminin (LA) has been related to liver fibrosis and subsequent development of portal hypertension in chronic liver disease. There are no available data describing the pattern of laminin in combined HCV and schistosoma-infected patients, thus the rationale of this study was to assess the serum LA as an index of liver fibrosis in patients with schistosomiasis and/or chronic viral hepatitis C and to evaluate a developed Slot-Blot Enzyme-Linked Immunosorbant Assay (Slot-Blot-ELISA) as a method of estimation. DESIGNS AND METHODS: This study included four groups: group I included 34 patients with schistosomiasis, group II included 58 patients infected with HCV, group III included 68 patients with combined chronic viral hepatitis C and schistosomiasis and group IV included 50 healthy individuals who served as a control group. Serum LA was measured in the different groups quantitatively by ELISA and semi-quantitatively by Slot-Blot-ELISA. RESULTS: Significantly higher serum LA concentrations measured by ELISA were found in patients with combined chronic viral hepatitis C and schistosomiasis than in patients with either chronic HCV (P = 0.005) or schistosomiasis (P < 0.001) alone. Serum LA was significantly higher in the patient groups than the control group (P < 0.001). Serum LA concentration was positively correlated with fibrosis grading scores. Semi-quantitative results of serum LA using the developed SB-ELISA were found to have approximately the same power of ELISA results in different groups. The overall sensitivity, specificity, positive predictive value, negative predictive value and efficiency of ELISA for estimation of serum LA were 85.6%, 84.0%, 94.5%, 64.6% and 90%, respectively and for SB-ELISA were 87.5%, 82.0%, 94%, 67.2% and 88%, respectively. CONCLUSIONS: Serum LA was significantly increased in patients coinfected with HCV and Schistosoma mansoni. The newly developed Slot-Blot-ELISA is a simple, rapid and highly sensitive assay for detection of LA in hepatic fibrosis. Moreover, all steps were performed at room temperature without the need to use expensive equipment, and this may enhance the application of this assay in screening programs. Further study is warranted for confirmation of SB-ELISA reproducibility in a large population.

Adult↗
PubMed2006

Toxoplasma gondii: comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs.

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7x10(7) viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10=2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as "gold standard" and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISAxIFAT (kappa = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 +/- 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs.

Agglutination Tests↗
PubMed2006

Agreement between three ELISAs for Mycobacterium avium subsp. paratuberculosis in dairy cattle.

During a 10-month period in 1999, 994 serum and tissue samples were collected from dairy cows at slaughter in eastern Canada. The sources of these cattle were from all four Atlantic Canadian provinces along with some cows from the state of Maine. The sera were used to assess the agreement of three commercially available ELISAs for Mycobacterium avium subsp. paratuberculosis. Two ELISAs were indirect absorbed ELISAs licensed for use in North America, the third was an indirect non-absorbed ELISA licensed for use in Europe. Overall, there was poor agreement between the three ELISAs. The highest and lowest kappa values were 0.33 and 0.18, which is fair and poor agreement, respectively. However, when only tissue culture-positive cattle were compared, the ELISAs had better agreement (kappa=0.37-0.51). The proportions of positive tests, however, were significantly different among the three ELISAs. The poor agreement among the three ELISAs is as concerning as the fact that these tests have low sensitivity. The implications are greatest when the tests are used at the cow level to make individual animal decisions, which is not the recommended method on the product labels. At the cow level, if the result obtained from one ELISA is positive, using a different ELISA in a pre-clinical animal has a high likelihood of giving a different result due to low predictive values of positive test results.

Animals↗
PubMed2000

Comparative sensitivity of dot-ELISA, PCR and dissection method for the detection of trypanosome infections in tsetse flies (Diptera: glossinidae).

A visually read dot-enzyme linked immunosorbent assay (dot-ELISA) developed for the detection of trypanosomes in tsetse flies (Glossina spp.) was evaluated in the laboratory and under field conditions. In the evaluation, the fly dissection method was used as a standard technique and compared to the polymerase chain reaction (PCR). In laboratory studies, 133 and 126 tsetse flies were experimentally infected with different stocks of Trypanosoma brucei and T. congolense, respectively. Twenty-five days after infection, the flies were dissected and tested for the presence of trypanosomes using dot-ELISA and PCR. Dot-ELISA detected 98.4% of T. brucei and 94% of T. congolense infections in tsetse midguts, while PCR detected 97.6% of T. brucei and 96% of T. congolense tsetse midgut samples. For field evaluation of dot-ELISA, 700 tsetse flies were caught and screened for trypanosome infections by dissection. Seven of these (1%) had trypomastigotes in the midgut, 23 (3.3%) in the proboscis and none had trypanosomes in the salivary glands. All the flies with midgut infections also had trypanosomes in their proboscides. Five of the seven flies (71.4%) with midgut infections revealed by dissection, were also positive for T. congolense by the dot-ELISA and PCR techniques. Dot-ELISA detected T. congolense infections in an additional 86 (12.4%) of the 700 flies dissected. Of the 23 infections in the proboscis, 16 were T. vivax. Dot-ELISA detected 13 of the 16 (81%) while PCR detected 15 of 16 (94%) T. vivax infections. No T. brucei infection was detected by any of the methods in all the 700 tsetse flies examined. The results obtained from both the laboratory and field studies indicate that the dot-ELISA and PCR techniques are sensitive and species-specific in revealing trypanosome infections in tsetse flies. While dot-ELISA required a single test to detect T. congolense, several primer pairs were needed for PCR. The potential use of dot-ELISA as a tool for studying the epidemiology of trypanosomosis, while considering its field applicability and relatively lower cost is discussed.

Animals↗
PubMed2002

Development of an ELISA for detection of antibodies to avian reovirus in chickens.

An enzyme-linked immunosorbent assay (ELISA) using the expressed sigmaC and sigmaB proteins which induce neutralizing antibodies as the coating antigen (sigmaC-sigmaB-ELISA) for the detection of antibodies to avian reovirus in chickens was developed and compared with serum neutralization and conventional ELISA tests. These assays were used to examine the sera from chickens vaccinated experimentally and farm chickens. The correlation rate between serum neutralization and a sigmaC-sigmaB-ELISA was 100% (156/156), and that between serum neutralization and conventional ELISA was 89.1% (139/156). The results revealed that preparation of an ELISA by using sigmaC and sigmaB of ARV as the coating antigen in detecting the field chicken sera in comparison with the conventional ELISA gave a titer more correlated to the serum neutralization test. The sigmaC-sigmaB-ELISA showed a higher correlation with the serum neutralization-positive and -negative sera than that obtained with conventional ELISA. This combination antigen may thus be the best suited for preparing an ELISA for improving the determination of the immune status of chicken flocks or for detection of chicken infections with avian reovirus.

Animals↗
PubMed1997

Evaluation of a quantitative double ELISA strategy for confirmation and differentiation of HIV infection.

The current HIV pandemic is complicated by the spread of distinct types and subtypes of HIV. The currently used conventional diagnostic tests have shown limitations in the detection of antibodies against all HIV-1 subtypes, as demonstrated by recent identification of HIV-1 subtype O. To evaluate quantitatively the diagnostic potential of a double ELISA strategy for the detection and partial differentiation of HIV-1, HIV-1 subtype O and HIV-2 infections blood samples were examined at five different test centers: Blantyre, Malawi; Abidjan and Daloa, Ivory Coast; Yaoundé, Cameroon; Munich, Germany. All tests results, including ELISA extinction values and Western blot profiles, were forwarded to Munich for final interpretation. An indirect anti-HIV-1/2 ELISA and a competitive anti-HIV-1 ELISA were used in combination for the initial screening of blood specimens. All anti-HIV positive and anti-HIV negative samples were subjected to immunoblot analysis. Independent of the diversity of the extinction profiles, and of the test manufacturer, the quantitative evaluation of the ELISA extinction values could define two extinction areas with a 100% predictive value for HIV-1 seropositivity and HIV seronegativity; extinction values > 2 by the indirect ELISA and < 0.2 by the competitive ELISA for an anti-HIV-1 subtype A to I positive result; extinction values < 0.2 by the indirect ELISA and > 1.0 by the competitive ELISA for an anti-HIV negative result. Additionally, the quantitative evaluation of the extinction profile provides partial information on the HIV-1 subtype as far as the distinction in group M and group O is concerned. In conclusion, the quantitative evaluation of this double ELISA strategy can reduce the number of blood specimens that require additional confirmatory testing in developing countries and can be superior to the immunoblot method during early seroconversion.

Antigens, Viral↗
PubMed1998

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.

Animals↗
PubMed1997

Evaluation of an ELISA for the routine diagnosis of Dictyocaulus viviparus infections in cattle.

An enzyme-linked immunosorbent assay (ELISA) that detects antibodies against Dictyocaulus viviparus in experimentally and naturally infected cattle was evaluated for its sensitivity, specificity, the moment of seroconversion and persistence of the anti-D. viviparus response and precision. The first three parameters were compared with those of an indirect haemagglutination assay (IHA). Specificity and sensitivity of both assays were assessed in sera collected from calves experimentally infected with pure isolates of D. viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Ascaris suum or Fasciola hepatica, and from parasite-naive calves. The specificity of both the ELISA and IHA was very high, 99.2% and 99.6%, respectively. The sensitivity of the ELISA (100%) was significantly higher than that of the IHA (78.1%). In experimentally infected cattle, D. viviparus-specific antibodies were first detected with the ELISA between days 28 and 42 post-infection (p.i.), whereas the IHA only became positive between days 42 and 70. With the ELISA, antibody levels persisted until day 168 p.i. The IHA remained positive until the end of the experiment (day 196). None of the vaccinated animals were seropositive with the ELISA, whereas 25% of the calves were seropositive with the IHA. The seroprevalence of D. viviparus infections was determined in a field study with 467 sera from cattle of 64 herds; 227 (48.6%) of the animals were seropositive with the ELISA whereas only 38 (8.1%) scored positive with the IHA. To determine the precision of the ELISA, a total of five laboratories participated in trials, in which panels of strong positive, positive, and weak positive candidate sera were tested blind according to an international (International Standard ISO 5725, 1986) standard procedure. The repeatability and reproducibility of the ELISA were 0-16% and 14-26%, respectively. After these promising results it was decided to introduce this ELISA in 1995 as a routine test in all Animal Health Services in the Netherlands, replacing the IHA and faecal examinations for lungworm.

Animals↗
PubMed1997

The time course of the specific antibody response by various ELISAs in pigs experimentally infected with Toxoplasma gondii.

With the aim of developing routine serological tests for monitoring the Toxoplasma infection status of Danish swine herds, four ELISAs based on tachyzoite antigen were set up: (1) an indirect ELISA for IgG-antibody; (2) a blocking ELISA for antibody to the membrane antigen, P30; (3) an indirect ELISA for IgM; (4) a reverse, antibody-catching IgM-ELISA. Groups of pigs (number between 6 and 10) were inoculated with tachyzoites of the RH-strain, tissue cysts of two complete strains, or oocysts in two doses (10(3) and 10(4). All inoculations were tolerated well. Irrespective of strain and stage used for inoculation, specific IgG and anti-P30 blocking activity appeared after 1-2 weeks, with OD-values stabilizing after 3-6 weeks and persisting throughout the study period (3-4 months). Specific IgM appeared quickly, but was short-lived (approximately 2 weeks). A cut-off OD-value of 0.36 for positive seroreaction in the indirect IgG-ELISA was determined on the basis of 69 sera from four herds, investigated in the dye-test (serum dilution 1:10) and ELISA. The chosen cut-off gave optimal combined sensitivity and specificity of 0.94 and 0.92, respectively, using the dye-test as a standard. Corresponding figures for the blocking ELISA were 37% inhibition as cut-off, with sensitivity and specificity of 0.94 and 0.94, respectively. Sera from a total of 87 pigs, experimentally infected with bacteria of the genera Salmonella, Yersinia or Actinobacillus and with the parasites Isospora suis, Trichinella spiralis or Ascaris suum, in no case produced cross-reactions in the IgG-ELISA. However, 3/9 pigs inoculated with 50 000 sporocysts of Sarcocystis miescheriana gave maximal OD-readings of 0.40-0.45 during the 13-15 weeks observation period. None of the sera from heterologously infected animals produced inhibitions in the anti-P30 blocking ELISA exceeding 36%.

Animals↗
PubMed1998

Comparison of three ELISA tests for seroepidemiology of bovine fascioliosis.

The aim of the present study was to compare the sensitivity, specificity and usefulness of the DIG-ELISA, DOT-ELISA and Indirect ELISA tests for determining the seroprevalence of fasciolosis in cattle under tropical conditions in Mexico. To standardize the tests, positive and negative sera to F. hepatica from 88 Holstein Freisian adult cows located in an enzootic area of fascioliosis and 88 crossbred adult cattle from a fluke-free area were used. For the epidemiological study, 85 crossbred cattle between 1 to 7 years of age were used. Animals were bled every two months, from March 1995 to September 1996 and the sera obtained were stored at -70 degrees C, until used. Indirect ELISA showed a sensitivity of 96.5% and a specificity of 98.8%, DIG-ELISA 97.5% and 80.0% and DOT-ELISA 93.1% and 95.4%, respectively. During 1995, Indirect ELISA yielded the highest levels of IgG anti-F. hepatica antibodies. However, in 1996, after animal treatment with triclabendazole, DIG-ELISA tended to show higher percentages of antibody-positive animals, but it was not significantly different (p>0.05) from the other tests. Comparisons made in parallel to the faecal sedimentation test demonstrated that all serological tests detected higher percentages of positive animals. Only one serum out of ten (10%) of Paramphistomum spp. cross-reacted with the DOT-ELISA test, but no cross-reaction was observed with sera from animals with other parasites. All ELISA tests were highly sensitive and specific; they may be recommended for use in seroepidemiological surveys for F. hepatica.

Animals↗
PubMed2001

Development and validation of an ELISA for the detection of leptospire-specific antibodies in rodents.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies in rodents was developed and validated with the microscopic agglutination test (MAT) and leptospiral cultures. Sonicated antigen from cultures of serovars tarassovi and pyrogenes was used. As conjugate, a combination of anti-rat and anti-hamster IgG labeled with peroxidase was used. The optimal cut-off point was determined by plotting the sensitivity and specificity for various cut-off point values by means of receiver operating characteristic (ROC) curve. Concordance between ELISA and each of the MAT titers was measured by kappa (kappa). Proportions of positive results were compared by means of McNemar's test. Total 214 rodents were trapped, but only 117 could be processed by the three techniques (culture, ELISA, MAT; 1:20, 1:40, 1:50) and used for statistical analysis. Although, MAT titers in rodents infected with the serogroup Ballum tended to be lower than those infected with the serogroup Icterohaemorrhagiae, all (20/20) were ELISA-positive and almost all (19/20) were MAT-positive.The percentage of positive results obtained by ELISA, 47.0% exceeded significantly the 40.2% obtained by MAT (1:50). Difference between ELISA and MAT (1:40) was not significant and no differences were observed between ELISA and MAT (1:20). Agreement, specificity, sensitivity and the consequent area under the ROC curve between ELISA and MAT were higher as MAT cut-off points were lowered, being optimal at 1:20. The fact that differences between ELISA and MAT were significant at 1:50, but not at 1:40 or 1:20, supports the suggestion that lower MAT titers should be considered positive in rodents. The ELISA developed to detect leptospire-specific antibodies had optimal sensitivity and specificity in relation to MAT and it is concluded that it may constitute a very useful indicator for epidemiological purposes of past or present leptospiral infection in rodents.

Agglutination Tests↗
PubMed1999

Binding properties of antibodies to prothrombin and beta2-glycoprotein I (beta2-GPI) assayed by ELISA and dot blot.

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards beta2-GPI (anti-beta2-GPI) or prothrombin (anti-II). They are mainly screened by ELISA using polyoxygenated plates. However, some authors have claimed that immunoblotting can also be used. Exposure of cryptic epitopes or increase of antigen density on its binding to either phospholipids or suitable plastic surfaces are the two hypotheses proposed for the interaction of beta2-GPI or prothrombin with their antibodies. Forty-five patients with aPL were studied: 25 with lupus anti-coagulant (LA) and anti-cardiolipin antibodies (aCL), 10 with LA alone and 10 with aCL but negative LA. All patients with LA and aCL were positive for anti-beta2-GPI by ELISA and dot blot, while 15/25 had anti-IIELISA and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti-beta2-GPI by ELISA or dot blot, whereas 6/10 had anti-IIELISA (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti-beta2-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to beta2-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with beta2-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 microg/ml) of beta2-GPI or prothrombin demonstrated that antibody binding reduction was more evident on dot blot than on ELISA. It was almost completely abolished on dot blot, while on ELISA a moderate inhibition was achieved even at the highest protein concentration. However, antibody binding on ELISA was virtually abolished when diluted sera were incubated with high protein concentrations applied to nitrocellulose membranes. We could infer that ELISA and dot blot detect antibodies with some differences in avidity but directed against native epitopes on beta2-GPI and prothrombin.

Adolescent↗