Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Evaluation of the critical parameters of a sensitive ELISA test using purified infectious bovine rhinotracheitis virus antigens

An enzyme-linked immunosorbent assay (ELISA) for the survey or titration of bovine sera for the presence of IgG antibodies against infectious bovine rhinotracheitis (IBR) virus was developed. The optimal conditions of serum dilution, antigen concentration, conjugate dilution, substrate concentrations, and reaction time were established using the signal/ noise (S/N) ratio as the determining criterion. Equilibrium density gradient purified IBR virus was used as antigen at an optimal concentration of 0.60 μg/cuvette. The use of purified antigen allowed the testing of sera at a 1 : 10 dilution without nonspecific reaction.The conditions of conjugate dilution, substrate concentration and reaction time were shown to have significant effects on the ELISA test. Results from 35 sera showed this optimized ELISA procedure to be as much as 1000-fold more sensitive than the serum neutralization plaque reduction assay. Numerous sera showing no neutralizing titer to IBR virus were found to be positive when examined by this ELISA method.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.     

Demonstration of antibodies against bovine leukemia virus (BLV) by blocking ELISA using bovine polyclonal anti-BLV immunoglobulin.

A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.     

The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.

In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.     

Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.     

Performance characteristics of an enzyme-linked immunosorbent assay performed in milk for the detection of liver fluke (Fasciola hepatica) infection in cattle

AIM: To determine the performance characteristics of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in bovine milk. METHODS: Serum and milk from liver fluke infected and non-infected cattle was assayed in a commercially available enzyme-linked immunosorbent assay. Serum test results were used to determine the "gold standard" infection status of cattle and milk ELISA results assessed by ROC analysis. RESULTS: ROC analysis suggested changes to the ELISA protocol, arriving at milk dilutions assayed considerably higher than those suggested by the manufacturer. With those changes, the ELISA performed with high sensitivity and specificity, 95 and 98.2%, respectively, for individual bovine milks (relative to sera). For bovine tank milks, sensitivity was lower, with bulk milks only testing positive if 60% or more of cattle milking in the herd were infected. CONCLUSIONS: The analysis of the ELISA's performance when used on individual bovine milks demonstrated high sensitivity and specificity. ROC analyses optimised the assay conditions and cut-off point suggested by the manufacturer for this commercial diagnostic assay. This would help with the identification and control of fasciolosis, enabling simpler sample collection.     

Diagnostic sensitivity and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams.

An enzyme-linked immunosorbent assay (ELISA) for antibody to Brucella ovis was compared with a standard complement fixation test. Sera of 176 rams from uninfected flocks gave 175 negative and one suspect ELISA reaction (diagnostic specificity 99.4%) whereas the complement fixation test yielded 167 negative, seven suspect and two anticomplementary reactions (diagnostic specificity of 96.0%). Diagnostic sensitivity was evaluated on sera of 79 rams from which B. ovis had been isolated. The ELISA showed 75 positive and four suspect reactions, while complement fixation test revealed 64 positive, 13 suspect and two negative results. Considering both positive and suspect reactions, the diagnostic sensitivity was 100% for ELISA and 97.5% for complement fixation test. The ELISA method was considered more specific, more sensitive and technically more advantageous than complement fixation test as a serodiagnostic test for B. ovis infection in rams.     

Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar canicola in ELISA for serodiagnosis of bovine leptospirosis

The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis.     

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis.     

Comparison of diagnostic tests for the detection of equine infectious anemia antibody

Two diagnostic tests are approved for detecting antibody to equine infectious anemia virus: the agar-gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (ELISA). A total of 420 sera from National Veterinary Services Laboratories check sets were tested with the AGID and competitive ELISA. A 100% correlation was obtained. The AGID and competitive ELISA were further used to test difficult samples with low levels of equine infectious anemia antibody (weak positives). A third test (Western blot) was also used with these weak positive samples to resolve any discordant results.     

Identification of bovine herpesvirus-1 polypeptides involved in serum neutralization

Bovine herpesvirus (infectious bovine rhinotracheitis virus)-infected cell antigens were solubilized with Nonidet P-40. The crude antigen extract was separated by reaction with bovine hyperimmune serum in line immunoelectrophoresis; individual immunoprecipitates were used to immunize rabbits. Rabbit sera possessing serum neutralizing activity were analyzed by reaction with crude antigen extract in immunoprecipitation sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Four virus-specified glycopeptides, with molecular weights of 69-75K, 77-81K, 82-92K and 108-115K, appeared to be involved in inducing serum neutralizing antibody.     

A standardized enzyme-linked immunosorbent assay for infectious bronchitis virus: comparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody levels in chickens

An enzyme-linked immunosorbent assay (ELISA) was developed to measure antibodies to infectious bronchitis virus (IBV) in chickens. The results are reported in IBV standard ELISA values calculated by comparing antibody levels in test sera with antibody levels in a series of standard reference sera. The IBV standard ELISA values were good indicators of responses to vaccination and the immune status of experimentally challenged birds. Although the assay was not serotype-specific, the sensitivity makes it ideally suited for determining the immune status of poultry flocks. The assay results compared favorably with other laboratory results, including virus-neutralization titers, hemagglutination-inhibition levels in sera, virus isolation from vaccinated/challenged birds, and the tracheal ring test results.     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Studies on the efficiency of absorbed bovine PPD in tuberculin and serological tests

The passive haemagglutination, enzyme-linked immunosorbent assay and indirect fluorescent antibody tests were applied to study the non-specific reactions in experimentally infected guinea pigs and tuberculin positive bovines. These cross-reactions were greatly decreased after absorption of either sera with avian PPD or bovine PPD antigen with anti-avian PPD serum. The use of both absorbed sera and antigen raised the specificity of PHA and ELISA to 100%. The use of absorbed sera rendered the IFA specific in 95%. The absorption has reduced the sensitivity of ELISA, IFA and PHA by 14, 27 and 29%, respectively.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of a neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2).

A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA.