Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis.

Sera from Canadian pigs (brucellosis free, n = 14037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.     

Comparison of dot immunobinding assay, enzyme-linked immunosorbent assay and immunodiffusion for serodiagnosis of paratuberculosis.

A rapid, simple and inexpensive dot immunobinding assay (DIA) was evaluated for the serodiagnosis of paratuberculosis in cattle. The assay was performed on nitrocellulose strips which were dotted with purified protoplasmic antigen of Mycobacterium paratuberculosis. After incubation with test serum samples, the bound antibodies were detected using an enzyme-amplified immunostaining procedure. The efficacy of DIA as a screening test for paratuberculosis was compared to that of an enzyme-linked immunosorbent assay (ELISA), a modified agar gel immunodiffusion (mAGID) test, and an AGID test using 329 serum samples from cattle which were examined for M. paratuberculosis infection by a sensitive fecal culture technique. The DIA and ELISA had comparable results and both of the enzyme immunoassays had higher sensitivity than tests based on AGID. The sensitivity of all four tests was influenced by the intensity of fecal bacterial shedding. Preabsorption of sera with Mycobacterium phlei increased the sensitivity of both enzyme immunoassays. the specificity but reduced the sensitivity of both enzyme immunoassays.     

Persistence of reactivity against the 45 k Da glycoprotein in late trichinellosis patients

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.     

Studies on bovine leukosis. VII. Further experience with an ELISA for the detection of antibodies to bovine leukosis virus

An enzyme linked immunosorbent assay (ELISA) and the agar gel immunodiffusion test with bovine leukosis virus glycoprotein as antigen (AGIDT-BLV gp) were further used to test 633 bovine sera for antibodies to BLV. Both tests detected the same number of sera positive (149) or negative (464) for antibodies. Nine sera were negative in the ELISA but found to be weakly positive (2 sera) or bending the control line (7) in the AGDT-BLV gp. On the other hand 11 sera were scored negative in the AGIDT-BLV gp but were weakly positive (9 sera), positive (1), and strongly positive (1) in the ELISA. Both tests are used routinely in this Institute as they complement each other, specially if sera with low antibody titers are under investigation. It is concluded that ELISA can fully replace radioimmunoassays in the serodiagnosis of enzootic bovine leukosis.     

Detection of antibody to Bordetella avium using a particle concentration fluorescence immunoassay (PCFIA).

A fluorescent assay for detection of antibody to Bordetella avium was developed and evaluated against current serological techniques. Bordetella antigen was coated on submicron polystyrene beads and used in an antibody sandwich technique with commercially available goat anti-turkey fluorescein-labeled IgG. Positive control sera were taken from turkeys from which B. avium was isolated. Negative control sera were from turkeys from which no B. avium was isolated and which tested negative by agglutination and enzyme immunoassay. A commercial fluorescence concentration analyzer was used to quantitate the epifluorescence in a special 96-well plate designed to minimize background fluorescence. The particle concentration fluorescence immunoassay was found to be as sensitive as the enzyme immunoassay, with good reproducibility. It required less time and smaller quantities of sample and reagents.     

Studies on Bovine Leukosis

Summary

An enzyme linked immunosorbent assay (ELISA) and the agar gel immunodiffusion test with bovine leukosis virus glycoprotein as antigen (AGIDT‐BLV gp) were further used to test 633 bovine sera for antibodies to BL V. Both tests detected the same number of sera positive (149) or negative (464) for antibodies. Nine sera were negative in the ELISA but found to be weakly positive (2 sera) or bending the control line (7) in the AGIDT‐BLV gp. On the other hand 11 sera were scored negative in the AGIDT‐BLV gp but were weakly positive (9 sera), positive (1), and strongly positive (I) in the ELISA. Both tests are used routinely in this Institute as they complement each other, specially if sera with low antibody titers are under investigation. It is concluded that ELISA can fully replace radioimmunoassays in the serodiagnosis of enzootic bovine leukosis.     

Evaluation of three different antigens in an indirect enzyme-linked immunoassay for the detection of antibodies against Brucella abortus SRB51 in vaccinated heifers

The live attenuated Brucella abortus SRB51 (SRB51) is a partial O-chain-deprived mutant. The relative lack of the polysaccharide prevents it from inducing antibodies detectable by most of the serological tests used for the diagnosis of bovine brucellosis. The performance of three antigens used in an indirect enzyme-linked immunoassay test for detecting SRB51 antibodies were evaluated. A homogeneous group of twenty-five 10-month-old Hereford heifers was used. The animals were bled on day 0 and then subcutaneously vaccinated with 2 ml of a commercially available SRB51 vaccine (Schering-Plough) containing 1x10(7) to 3.4x10(7) viable cells. Blood samples without anticoagulant for sera obtaining were then collected at days 30, 90, 210 and 360 post-vaccination. To detect the SRB51 antibodies, Brucella ovis hot saline extract, B. ovis RLPS (RLPS), and SRB51-RLPS were used. The buffered antigen plate agglutination test and an indirect enzyme-linked immunoassay (I-ELISA) using the smooth LPS (SLPS) antigen from B. abortus were used as control tests. All the sera samples were negative in the BPA test and in the standard I-ELISA using the SLPS. The SRB51-RLPS and the B. ovis RLPS antigens performed better than the B. ovis hot saline extract antigen.     

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.     

A comparison of five serological tests for bovine brucellosis.

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.     

Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection of bluetongue virus antibodies in cattle.

A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (BTV) is described for the detection of BTV antibodies to BTV in cattle sera. Dots of BTV antigens were adsorbed to nitrocellulose (NC) strips and/or NC mounted in the windows of dipsticks. After blocking the remaining sites of the NC paper with milk powder solution and immersion in the test sample, the NC strips and dipsticks were exposed to mAb. Bound mAb was detected with peroxidase conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in the test sample, BTV antigen sites were reactive with mAb as indicated by a brown colored dot in the presence of the enzyme substrate, hydrogen peroxide and diaminobenzidine. In the presence of sufficient anti-BTV antibodies no color reaction was observed. The performance of these assays in detecting anti-BTV antibody in field blood eluate samples, prepared from whole blood dried on filter paper, from 395 bluetongue-free cattle in Canada and 635 sentinel cattle in Florida, USA, was evaluated and compared with the standard competitive (C) ELISA. The specificity of the dipstick B-dot ELISA was identical to that of the C-ELISA in testing of BT-free Canadian cattle but not in the testing of samples from the sentinel cattle in Florida, resulting in values of 100% diagnostic and 88.9% relative specificity, respectively. Based on the C-ELISA, the specificity of the NC strip B-dot ELISA was low and in the same order as that of the dipstick assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotavirus antibody assays on monkey sera: a comparison of enzyme immunoassay with neutralization and complement-fixation tests.

An enzyme immunoassay (EIA) for the detection of rotaviral antibodies was developed, using a purified, cell culture-grown SA 11 viral antigen and alkaline phosphatase as an enzyme label. This technique was evaluated by comparative testing with tube neutralization and complement-fixation assays on a collection of simian sera. There was close correlation between positive and negative results obtained by EIA and by neutralization. The EIA was as easy to perform as complement fixation testing, but showed greater sensitivity and fewer nonspecific reactions. Thus, EIA was shown to be a very suitable test for routine detection of rotaviral antibodies in serum. Results of neutralization tests suggested that the monkeys (mostly rhesus macaques) in the present study were infected with viruses varying in their antigenic relatedness to SA 11 virus and to a British isolate of calf rotavirus.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Enzyme immunoassay for the diagnosis of brucellosis: chimeric Protein A-Protein G as a common enzyme labeled detection reagent for sera for different animal species

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody-enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.     

Comparison of serological techniques to measure antibody to Pasteurella haemolytica A1.

Analysis of 45 sera was performed employing five techniques which are currently in use in three laboratories to measure anti-Pasteurella haemolytica antibodies. The enzyme linked immunosorbent assay, passive hemagglutination, complement fixation and direct and indirect bacterial agglutination assays were employed and a relationship between tests in the measurement of anti-P. haemolytica antibodies was demonstrated. Regression analysis together with prediction and confidence intervals were tabulated also. The conclusion drawn from statistical analysis was that all five tests are similar in their ability to detect immune responses (antibody and antigen(s) interactions) to Pasteurella haemolytica.     

Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.     

用蓝舌病优质琼扩抗原进行ELISA的试验研究

经用125份牛羊血清进行定性定量精确比较和对原用解聚抗原检测过的3020份血清的平行重复试验证明,优质琼扩抗原与解聚抗原在ELISA中检测效果一致,具有等效性。     

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle.

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti-Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.     

An immunoenzyme test used for the detection of antibodies to rinderpest: the advantage of using a purified virus cultivated in a cell line

Two types of in vitro assays (enzyme immunoassay and sero-neutralization test) were compared for their ability to detect antibodies to rinderpest virus in field sera from West African bovines. Purified virus grown on Vero cells (Ag/Vero) or bovine kidney cells (Ag/BK), were tested as antigen in enzyme-linked immunoassays (EIA). The results of the comparative evaluation of the two antigens by EIA, prove that Ag/Vero did enhance the sensitivity and the specificity of the test in comparison to Ag/BK and offers a 94% correspondence with seroneutralization.