A monoclonal antibody blocking ELISA detects antibodies specific for epizootic haemorrhagic disease virus.

The isolation of a monoclonal antibody (1G9/C9) with specificity for the epizootic haemorrhagic disease (EHD) serogroup has enabled the development of a highly sensitive and specific blocking ELISA (B-ELISA) for the detection of serum antibodies to EHD viruses. The assay was sensitive to blocking antibodies present in hyperimmune reference antisera to all six EHD serotypes tested but was unaffected by reference antisera to 19 South African and eight Australian serotypes of the related orbivirus bluetongue virus (BTV). The sensitivity of the EHD B-ELISA exceeded that of an indirect ELISA (I-ELISA) for EHD-specific antibody detection. Serum antibody titres to BTV and EHD in experimental and field sera, including a sentinel herd from which virus isolations were made, were examined in both the BTV and EHD B-ELISA tests. These results showed the B-ELISA was only sensitive to antibodies specific for the homologous serogroup in each case, even where sequential and mixed infections with each virus type occurred.     

Evaluation of the specificity and sensitivity of two commercial enzyme-linked immunosorbent assay kits, the serum plate agglutination test, and the hemagglutination-inhibition test for antibodies formed in response to Mycoplasma gallisepticum

Two commercial enzyme-linked immunosorbent assay (ELISA) kits, seven serum plate agglutination (SPA) antigens, and the hemagglutination-inhibition (HI) test for antibodies to Mycoplasma gallisepticum (MG) were compared for sensitivity and specificity using known MG-positive and MG-negative sera from leghorn chickens. All SPA antigens proved to be highly sensitive when testing MG-positive sera. Laboratory-prepared SPA antigens yielded fewer positive reactions when testing MG-negative sera than commercial SPA antigens. Both MG ELISA kits showed high rates of positive reactions when testing sera from birds given commercial M. synoviae bacterin, fowl coryza (Haemophilus paragallinarum) bacterin, inactivated infectious bursal disease virus vaccine, and to a lesser extent fowl cholera (Pasteurella multocida) bacterin. Immunization with Frey's medium with 12% swine serum-in-oil or Staphylococcus aureus-in-oil resulted in sera which yielded numerous positive ELISA reactions. During the first 1 to 3 weeks, antibodies induced by experimental infection with MG were better detected by the SPA test than by the ELISAs and the HI test, thus confirming the SPA test's importance in Mycoplasma diagnostic serology. The HI test can serve to confirm positive SPA results.     

An enzyme-linked immunosorbent assay for the detection of antibody to avian reovirus by using protein sigma B as the coating antigen

An enzyme-linked immunosorbent assay using the expressed protein sigma B as the coating antigen (sigma B-ELISA) for detecting antibody to avian reovirus (ARV) in chickens was developed and compared with a conventional ELISA. Both ELISA s and a serum neutralisation (SN) test were used to test the sera from experimentally vaccinated and farm chickens. The sigma B-ELISA could clearly distinguish the SN-positive and -negative sera in 38-week-old chickens. The correlation rate between SN and a sigma B-ELISA was 100 per cent (65/65), and that between SN and conventional ELISA was 84 per cent (55/65). With the sigma B-ELISA, all SN-negative sera had low absorbance values (below 0.06), and the absorbance values correlated closely with the SN titres. However, the sera which were antibody-negative by SN had various absorbance values, ranging from 0.07 to 0.39 in the conventional ELISA. Hence, the sigma B-ELISA had lower non-specific binding reactions than the conventional ELISA against sera from ARV -negative birds. Antibody against ARV could be detected by sigma B-ELISA after vaccination. Absorbance values peaked 4 weeks after vaccination at 2 weeks of age and were maintained until the birds were 27 weeks old. The results suggest that the presence of antibody against viral protein sigma B in birds may be used as a good indicator by the sigma B - ELISA for detecting immune status of a chicken flock or to detect chickens infected with ARV.     

A monoclonal antibody-blocking enzyme-linked immunosorbent assay for the detection of serovar-specific antibodies to Haemophilus paragallinarum

Two monoclonal antibody-blocking enzyme-linked immunosorbent assays (B-ELISAs) were developed to detect serovar-specific antibodies to Haemophilus paragallinarum. One assay detected antibodies against serovar A and the other antibodies against serovar C. The assays were evaluated with sera derived from disease-free chickens as well as chickens experimentally immunized and/or challenged with H. paragallinarum strains 0083 (serovar A), Modesto (serovar C), or HP31 (serovar C). When tested with 440 negative sera (170 from a specific-pathogen-free and 30 from each of nine commercial layer flocks), both tests gave only a single false-positive reaction. The use of the B-ELISAs with the experimentally produced sera showed the assays to be serovar specific. With the exception of one serum, the serovar A B-ELISA detected antibodies only in the chickens vaccinated with 0083. Similarly, with the exception of one serum, the serovar C B-ELISA detected antibodies only in those chickens vaccinated with Modesto or those chickens challenged with HP31. Overall, the serovar A B-ELISA had a specificity of 99.7% and a sensitivity of 78.7%, whereas the serovar C B-ELISA had a specificity of 99.8% and a sensitivity of 64.7%.     

gIII antibody responses in pigs following vaccination and challenge to Aujeszky's disease.

Serological responses to a genetically engineered Aujeszky's disease "marker" vaccine (dl gIII + dl tk) were monitored using a blocking-ELISA (B-ELISA), a serum neutralisation test (SNT) and an indirect ELISA (I-ELISA). The B-ELISA is capable of differentiating pigs vaccinated with the above vaccine from natural infection. The SNT and the I-ELISA indicated that the pigs responded to vaccination and challenge. All three tests showed that the controls and the in-contact pigs always reacted negative for antibodies. The B-ELISA was able to detect pigs challenged with a field isolate 24 days post-challenge. These pigs remained positive until 110 days post-challenge when last tested. These findings indicate that the B-ELISA could be used successfully with this vaccine in a control eradication programme. This trial also shows that the vaccine virus did not spread to the in-contact pigs and also the vaccinated and challenged pigs did not transmit the disease to other susceptible pigs when they were introduced 14 days after challenge.     

Comparison of the Q-fever complement fixation test and two commercial enzyme-linked immunosorbent assays for the detection of serum antibodies against Coxiella burnetii (Q-fever) in ruminants: Recommendations for use of serological tests on imported animals in New Zealand

Abstract

AIM: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods.

METHODS: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA.

RESULTS: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from noninfected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity.

CONCLUSIONS: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.     

Dot immunobinding assay in comparison with enzyme-linked immunosorbent assay for the detection of bluetongue virus antibodies in sheep

A total of 384 sheep serum samples collected from two organised sheep farms was tested by dot immunobinding assay (DIA) and indirect enzyme-linked immunosorbent assay (I-ELISA) for the presence of bluetongue virus (BTV) antibodies. The results of both these assays were compared to find a sensitive, specific, rapid, easily performed and economical test for the diagnosis of bluetongue disease. DIA detected BTV antibodies in 210 samples (54.94%) and I-ELISA detected 157 positive samples (40.88%). Competitive ELISA (C-ELISA) was performed to check the discrepancies in I-ELISA and DIA. On the basis of these tests the overall agreement, relative specificity and sensitivity between ELISA and DIA were 75%, 87.6% and 100%, respectively. DIA was found to be a rapid, sensitive, easily performed and economical test as compared to ELISA.     

Development of a Neutralizing Monoclonal Antibody-based Blocking ELISA for Detection of Equine Herpesvirus 1 Antibodies

A single-dilution, sensitive and specific monoclonal antibody-based blocking enzyme-linked immunosorbent assay (B-ELISA) was developed as an alternative to the cumbersome virus neutralization test (VNT) for detection of equine herpesvirus-1 (EHV-1) antibodies. Neutralizing monoclonal antibodies (1H6 and 9C6) raised against EHV-1 (Hisar-90-7 strain) and sera from 70 horses (30 known negative and 40 known positive for EHV-1 antibodies by VNT) were used for standardization of the B-ELISA. Using a single serum dilution of 1:250 in B-ELISA, 100% specificity was obtained with both monoclonal antibodies (Mabs) in comparison to VNT. Similarly, the sensitivity of the B-ELISA was 92.5% and 100% with 1H6 and 9C6 Mabs, respectively. A very high correlation coefficient (r = 0.85) was observed between B-ELISA and VNT that was significant at the p < 0.01 level. B-ELISA detected a more than 3-fold rise in antibody titres in paired serum samples collected from mares aborting owing to EHV-1 infection. Mab 9C6 was chosen for testing 231 field sera from apparently healthy vaccinated and non-vaccinated horses from organized breeding farms belonging to 11 Indian states, and from Bhutan, by B-ELISA and VNT. There was very good agreement between the results obtained by both VNT and B-ELISA (K = 0.9438). Of 231 field sera, 144 samples were negative for EHV- 1 antibodies by both VNT and B-ELISA and 81 were positive by both tests. Two samples negative by VNT were found positive in B-ELISA. On the other hand, four weakly positive samples in VNT (VN antibody titre 0.9 1.2 log10) were negative in B-ELISA. The Mab (9C6)-based B-ELISA was found to be a suitable alternative to VNT for screening large numbers of field sera and enabled confirmatory EHV-1 serodiagnosis.     

Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma gallisepticum in experimentally infected chickens

An indirect enzyme-linked immunosorbent assay (ELISA) was developed and tested for its ability to detect humoral response to Mycoplasma gallisepticum in chickens. Two antigens were used in the solid phase of the assay. Antigen 1 was a membrane-derived sodium dodecyl sulfate (SDS)-solubilized preparation; Antigen 2 was prepared in the same manner as Antigen 1 but was passed through an immunoadsorbent column containing rabbit anti-medium antibodies. Test conditions were optimized for incubation times and temperatures. Antigen, serum, and enzyme conjugate concentrations were standardized, and reproducibility was determined. A baseline value, representing a positive or negative result, was established independently for both antigens. The assay was then used to detect anti-M. gallisepticum antibodies in experimentally infected chickens. Serum samples collected at 0, 2, 5, 7, 10, 14, 21, 28, and 35 days postinfection (PI) were analyzed by serum plate agglutination (SPA), hemagglutination-inhibition (HI), and ELISA with both Antigens 1 and 2. ELISA was found to be less sensitive but more specific than SPA and more sensitive than HI. The ELISA was more sensitive with Antigen 1 than with Antigen 2. The former assay correctly identified 79% of the serum samples positive for M. gallisepticum by 7 days PI and 100% of the positive birds by 35 days PI. When the absorbance values for each group of birds were averaged, the ELISA successfully identified the M. gallisepticum-infected birds as uniformly positive 7 through 35 days PI and correctly identified all other groups negative for M. gallisepticum through 35 days PI.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of antibodies to caprine arthritis-encephalitis virus in goat serum.

An indirect enzyme-linked immunosorbent assay (ELISA), was evaluated for its ability to detect serum antibodies against caprine arthritis-encephalitis virus (CAEV). The ELISA was compared to three other serological immunoassays, agar gel immunodiffusion test (AGIDT), immunoblot assay (IBA), and a fixed-cell immunoperoxidase assay (FCIPA). A total of 511 samples, from 40 farms representing a variety of goat breeds and ages were tested. An estimate of the ELISA sensitivity and specificity was made, relative to combined test results of the three other CAEV serological assays. The degree of agreement of test results among these four assays was evaluated. The number of positives detected by the ELISA, AGIDT, IBA and IPA tests was 193, 154, 204 and 163, respectively. Of the 511 sera tested, 172 were positive to any two or all three of these tests, and were defined as reference positive. A total of 237 samples were negative to all three reference tests, and were defined as reference negative. Relative to these references, the ELISA had a point estimate of 98.3% sensitivity and 97.9% specificity. There was good agreement between the ELISA and the other three assays with a kappa statistic of agreement greater than 0.7 for all three comparisons. The ELISA is therefore considered a suitable assay, with high sensitivity and specificity, for detection of antibodies to CAEV in serum.     

Comparison of three serological tests in the diagnosis of Brucella infection in unvaccinated cattle in Eritrea

Three serological methods, the Rose-Bengal test (RBT), the complement-fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (I-ELISA) were compared for the detection of Brucella-infected animals in unvaccinated cattle herds in Eritrea. In this study, 71 herds first were classified as positive or negative for Brucella infection on the basis of at least one animal being seropositive by RBT and CFT. All the 159 RBT-positive samples from the 26 seropositive herds and 214 RBT-negative samples randomly selected from the seropositive herds and from the 45 negative herds were tested further by CFT and I-ELISA. Using the ELISA titer as main predictor, and incorporating the RBT results, a logistic model was built to predict the CFT-negative or -positive status of individual sera and to estimate sensitivity and specificity. Whilst the ELISA titers (< or =20) accurately predicted all the negative sera in herds that were also negative by the CFT, the number of seropositive animals was higher by ELISA in herds that had positive animals. Serum samples which give higher degrees of agglutination with the RBT need not be re-tested with CFT; consideration of the seropositive status of a herd should be taken into consideration on defining the cut-off optical density readings for ELISA.     

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.     

Comparison of serologic techniques for the detection of antibodies against feline coronaviruses.

The seroprevalence of feline coronavirus (FCoV) antibodies was studied in cats in southern Italy. One hundred twenty sera collected from cats belonging to catteries or community shelters and to households were tested for FCoV type I and II antibodies. The virus neutralization (VN) was performed and compared with indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Ninety-six sera tested positive for FCoV antibodies by VN and ELISA. Interestingly, ELISA revealed 2 more positive sera than did the VN test and 3 more positive sera than did the IFAT. All results were confirmed by Western blotting. ELISA proved to be more sensitive and detected a seroprevalence of about 82%. Considering the cross-reactivity of FCoV type I and type II, ELISA was able to detect antibodies against both serotypes, allowing the use of the assay as a reference test for sera screening. The high prevalence of antibodies observed indicates that FCoVs are common in southern Italian cat populations.     

Use of an avidin-biotin enhanced dot-immunobinding assay to detect antibodies for avian mycoplasma in sera from Iowa market turkeys

A dot-immunobinding assay, amplified with avidin and biotin (DAB assay), was used to detect serum antibodies to Mycoplasma iowae in immunized turkeys. The DAB assay was used to test serum samples from 122 commercial market turkey flocks obtained from four Iowa processing plants. The samples were pooled and tested for the presence of antibodies to four species of Mycoplasma spp. considered to be important pathogens for turkeys: M. gallisepticum (MG), M. iowae (MI), M. meleagridis (MM), and M. synoviae (MS). The occurrence of antibodies against these mycoplasmas, as determined by the DAB assay, were 5.7% for MG, 18.0% for MI, 77.9% for MM, and 9.8% for MS.     

Development of a monoclonal blocking ELISA for the detection of antibody to Mycoplasma bovis in dairy cattle and comparison to detection by PCR

A monoclonal antibody blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma bovis in cattle sera. The assay was highly specific and sensitive and there was no cross-reaction detected. This method revealed a high prevalence of antibodies (60%) to M. bovis in dairy cattle in North Queensland. The diagnostic potential of this B-ELISA for the detection of antibody to M. bovis was compared with its detection by PCR. There was a strong positive correlation between PCR and B-ELISA titers. Thus, the B-ELISA appears to be a valuable and reproducible tool in the serodiagnosis of M. bovis infection in cattle.     

Enzyme-linked immunosorbent assay for detecting antibodies to Aujeszky's disease virus in pigs

An indirect micro enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Aujeszky's disease virus in pigs is described. A control antigen prepared from infected cells was included for each serum tested. Of 243 sera from serologically positive farms, 175 (72 per cent) and 147 (60 per cent) were positive by the ELISA test and microtitre serum neutralisation test, respectively. Failure to include a control antigen for each serum would have resulted in 14 sera (6 per cent) being differently recorded. Results for sera from experimental and field infections indicated that seroconversion was more quickly detected by the ELISA test than the microtitre serum neutralisation test. In addition to greater sensitivity the ELISA test has other advantages over the serum neutralisation test. ELISA is a rapid, cheap test which is not dependent on a continuous supply of cell cultures and which can be readily automated.     

Novel purification method for recombinant 3AB1 nonstructural protein of foot-and-mouth disease virus for use in differentiation between infected and vaccinated animals.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for differentiation of animals infected with foot-and-mouth disease virus (FMDV) from vaccinated animals. The test was based on a highly pure and concentrated preparation of recombinant 3AB1 protein obtained by expression in a prokaryotic system, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro elution. Experimental- and field-serum samples from naive, vaccinated, and infected cattle were tested for anti3AB1 antibody using the ELISA. A cutoff level was set at 35% of the maximum absorbance obtained with a positive control serum (FMDV-infected animal, 21 days postinfection [dpi]). This assay could detect antibodies from sera of animals experimentally infected by contact (n = 118) with a sensitivity of 97.5%. The specificity was 100%, based on negative test results obtained on 109 sera from naive animals. Remarkably, all sera from animals vaccinated either once (n = 102) or twice (n = 30) were negative. In addition, this 3AB1-ELISA could detect seroconversion at 7 dpi in animals inoculated intradermolingually. This assay constitutes an important tool for the rapid detection of FMDV outbreaks in a vaccinated population. In addition, it presents a reliable, economical, and simple method for testing large numbers of serum samples.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

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.