Newcastle disease: an enzyme-linked immunosorbent assay for the control of antibodies in chicks

An enzyme-linked immunosorbent assay using a vaccine strain of Newcastle disease as antigen has been developed. It allows the control of the drop in the maternal antibody level of 1 and 28 days old chicks. Thus it can be used as alternative to the inhibition of hemagglutination method. A comparative study has shown a good correlation between the enzyme-linked-immunosorbent-assay and the inhibition of hemagglutination.     

Development and evaluation of an enzyme-linked immunosorbent assay for the serodiagnosis of pythiosis in dogs

Pythiosis (caused by the aquatic oomycete Pythium insidiosum) is a devastating and often fatal cause of either severe transmural gastroenteritis or locally invasive subcutaneous disease in dogs living in the southeastern United States. Although early diagnosis is essential for successful treatment, tools available for this task are limited. Therefore, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P insidiosum antibodies in canine serum. A soluble mycelial extract of P insidiosum was utilized as antigen in the ELISA, which was used to evaluate serum from 43 dogs with pythiosis, 8 dogs with lagenidiosis (another canine oomycosis), 16 dogs with nonoomycotic fungal or algal infections, 22 dogs with nonfungal gastrointestinal or skin disease, and 55 healthy dogs. Results were expressed as percent positivity (PP) relative to a strong positive control serum run on each plate. Medians and ranges for each of the 5 groups were as follows: pythiosis (81.7%, 50.6-98.5%), lagenidiosis (17.3%, 11.3-29.2%), other fungal or algal infections (8.2%, 4.7-15.4%), nonfungal gastrointestinal or skin disease (6.2%, 3.9-20.7%), and healthy dogs (6.7%, 3.0-15.2%). When using a cutoff value of 40% PP, the sensitivity and specificity of the ELISA both were 100%. In addition, ELISA values measured after successful surgical therapy in 2 dogs showed a decrease of anti-P insidiosum antibody concentrations into the normal range as early as 2 months after treatment. We conclude that the ELISA is a sensitive and specific test for the diagnosis of canine pythiosis, and may be a useful tool for monitoring response to medical or surgical therapy.     

Diagnosis of naturally occurring Fasciola hepatica infections in cattle with an enzyme-linked immunosorbent assay

Sera from 100 herds of cattle located in the state of Washington were examined with an enzyme-linked immunosorbent assay for antibody to Fasciola hepatica in a screening procedure that included 5 to 10 samples/herd. Twenty-eight herds contained infected cattle and F hepatica was most prevalent in 3 distinct geographic areas. Subsequent retesting of all sera available from 14 herds (mean of 109 samples/herd) revealed that the screening procedure correctly detected 7 of 7 operations in which greater than 40% of samples were positive or suspect and 3 of 3 operations in which 12% to 13% of the samples were positive or suspect. One of 3 herds considered negative after screening was found to contain a few (7%) positive samples and 1 herd considered possibly infected was negative on retest. These results were compared with those obtained by fecal examination for F hepatica eggs in 9 of the 14 herds. A good correlation (5 of 5) was found in which a high percentage (48% to 85%) of sera were positive or suspect. Fasciola eggs were not found in samples from 2 herds with few (7% to 12%) positive or suspect sera or in 2 herds that were negative by an enzyme-linked immunosorbent assay.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody to Pasteurella haemolytica: constructing an enzyme-linked immunosorbent assay titer

A 2-stage strategy was developed and evaluated for estimating serum antibody titer by use of ELISA and a series of dilutions. In stage 1, the linear response region and least-square estimate of the assay line slope were established from 9-point dilution assays. Provided that the reading was within the linear response region, this information was used in the stage-2 estimation of titer from a single absorbance reading. Operationally, 2 fixed dilutions were selected, one suitably low and one suitably high, to provide at least one reading within the linear region. The procedure should save considerable time when a large number of assays are to be performed. Stage 1 required approximately twenty 9-point assays, but all subsequent assays required only 2 fixed dilutions.     

Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.     

The enzyme-linked immunosorbent assay for detecting antibodies against Dirofilaria immitis in dogs

Antisera prepared in mice by injection of antigens from Dirofilaria immitis, Toxocara canis, Dipylidium canium and Fasciola hepatica and sera from Dirofilaria-infected and non-infected dogs were tested at different dilutions using an enzyme-linked immunosorbent assay (ELISA). For this ELISA, adult D. immitis antigen was fractionated by gel filtration methods and then absorbed with immunoadsorbent-fixed IgG fractions from mouse sera immunized with various parasites. The results indicated satisfactory discrimination between antisera to D. immitis and those to other parasites. A significant ELISA O.D. value was considered to be greater than or equal to 0.30 with a 1 : 250 dilution of the dog sera. However, the lack of significant differences between the O.D. value of microfilaraemic and amicrofilaraemic infections was observed. These facts suggest that the use of immunoadsorbent chromatography for canine dirofilariasis is especially useful for purifying antigens and eliminating cross-reactions against other parasitic infections, when immunological methods are used for serodiagnosis.     

Development of an enzyme-linked immunosorbent assay for Bordetella avium

A Bordetella avium enzyme-linked immunosorbent assay (ELISA) was developed to detect serum antibodies in 1-day-old poults, experimentally infected turkeys, and naturally infected turkeys. The optimized procedure included use of a suspension of whole bacteria coated onto plastic microtiter plates, a 1:200 serum dilution, a 1:3200 dilution of commercially available goat anti-turkey IgG (heavy and light chain) conjugated with horseradish peroxidase, and 0.04% orthophenylenediamine as substrate. A sample/negative (S/N) ratio method of analysis was used to estimate antibody titer from absorbance values. The regression equation used to estimate antibody titers was derived from the testing of naturally infected turkey sera. The equation was derived by plotting the log10 titer of the sera against the S/N ratio at a 1:200 serum dilution. The ELISA was an effective method for detecting antibody to B. avium, and the procedure should prove useful for laboratories equipped for high-volume ELISA testing.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs

Canine scabies is a challenging disease to diagnose because sarcoptic mites are hard to find on skin scrapings. The purpose of this study was to evaluate a serologic enzyme-linked immunosorbent assay (ELISA) as an aid in the diagnosis of canine scabies. In addition, serum samples were obtained post treatment to determine the duration and persistence of circulating scabies antibodies after resolution of natural infection. Nineteen dogs diagnosed with sarcoptic mange and 38 control dogs were tested. Sixteen scabies-infested dogs showed positive pretreatment ELISA results (84.2% sensitivity). Thirty-four control dogs showed negative ELISA results (89.5% specificity). In the 11 scabies dogs from which multiple post treatment serum samples were obtained, detectable antibodies were not present 1 month after treatment in four cases, but were present for 1-4.5 months post treatment in seven dogs. Our results suggest that this scabies ELISA test is useful in the diagnosis of canine scabies.     

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.     

Quality control criteria for quantitative enzyme-linked immunosorbent assay of porcine immunoglobulins A and M

Using radioimmunoassay methods, quality control criteria were applied to monoclonal antibodies produced to measure porcine immunoglobulins by quantitative ELISA. Porcine IgM and IgA were purified to homogeneity and were used to produce murine hybridomas that secreted antibodies against IgM, IgA, and immunoglobulin light chains. A competitive ELISA was developed to measure IgM, and a sandwich ELISA was used to quantify IgA in serum and colostrum. Both ELISA were tested for specificity, accuracy, sensitivity, and precision. Monoclonal antibodies were specific for porcine IgM or IgA in serum and colostrum, and competitive and sandwich ELISA fulfilled all validation criteria.     

The relationship between the hemagglutination-inhibition test and the enzyme-linked immunosorbent assay for the detection of antibody to Newcastle disease

An assay of 364 chicken serum samples for Newcastle disease virus antibodies determined that a commercial NDV enzyme-linked immunosorbent assay (ELISA) had a 98.2% sensitivity and a 91.7% specificity relative to the NDV HI test. The ELISA values regressed significantly (F = 930, df = 1/362, P less than 0.001) on the hemagglutination-inhibition (HI) titers. The correlational coefficient was 0.85. For individuals, two tests can have the same result based upon chance alone. Kappa is a measure of agreement between two tests that corrects for this chance agreement. The kappa between the ELISA and HI test was calculated to be 0.84 (Z = 7.74, P = 0.00001), which indicates a highly significant agreement between the two tests.     

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of Borrelia burgdorferi exposure in dogs

OBJECTIVE: To evaluate the effectiveness of a commercially available ELISA kit for detecting antibodies against Borrelia burgdorferi in dogs. SAMPLE POPULATION: Banked sera from 440 military working dogs were used for serologic analyses. PROCEDURE: Serum samples were analyzed for antibodies against B burgdorferi by use of a commercially available ELISA and subsequently by western blot analysis as a confirmatory test. RESULTS: Results from the ELISA indicated that 89 (20%) samples were positive for exposure to B burgdorferi or canine Lyme disease vaccine, and 351 (80%) were negative. Follow-up testing by western blot analysis indicated that results for 109 (25%) samples were positive and 331 (75%) were negative for exposure. All samples that had positive results on the ELISA also had positive results on western blot analysis (true positives). Of the 351 samples that had negative results on the ELISA, only 331 had negative results on western blot analysis (true negatives). The remaining 20 samples had positive results on western blot analysis. By use of a standard 2 x 2 table, it was determined that the ELISA had a sensitivity of 82%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 94%. CONCLUSIONS AND CLINICAL RELEVANCE: The commercial ELISA kit evaluated in this study appeared to lack adequate sensitivity for detecting all potential cases of borreliosis in dogs. The ELISA was also unable to discriminate natural exposure from exposure attributable to vaccination, which could complicate interpretation of positive results and treatment of dogs with clinical signs.     

Comparison of the enzyme-linked immunosorbent assay (ELISA) and serum neutralisation test for the serodiagnosis of Aujeszky's disease

The serum neutralisation test (SNT) and the indirect enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease were compared, utilising 3202 sera from Aujeszky's disease free pig herds, and 304 SNT reactor and 245 non-reactor sera from Aujeszky's disease infected piggeries. ELISA was found to give good discrimination between positive and negative sera, results showing 96.9% and 99.7% agreement with the SNT in classifying positive and negative reactor pigs respectively. The ELISA appeared to detect a slightly higher proportion of reactors than did the SNT. Absorbance values obtained with ELISA showed a high degree of overall correlation with SW titres (r = 0.916), though correlations were lower when applied to individual sera. The ELISA was considered to be a rapid and convenient procedure, offering many advantages over the SNT for routine use.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Marek's disease virus

A reproducible enzyme-linked immunosorbent assay (ELISA) using Marek's disease virus (MDV)-infected cells for the detection of antibodies to MDV is described. The optimum number of MDV-infected chicken embryo fibroblasts (CEF) was 5 X 10(4)/well, and test sera were positive at 1:400 dilutions. Compared with a purified virus preparation, MDV-infected CEF produced high specific and low nonspecific reactivities. Wells coated with whole cells could be stored at 4 C or -20 C for at least 3 months without loss of reactivity. With antibody-negative sera, the cutoff absorbency was 0.20 units. The ELISA was 20-to-40-fold more sensitive than indirect immunofluorescence. Homologous combinations of antisera in wells coated with CEF infected with different MDV serotypes were more reactive at higher dilutions than were heterologous combinations. The procedure described is specific and suitable for large-scale screening of both chicken and monoclonal antibodies against MDV.     

Development and evaluation of an enzyme-linked immunosorbent assay for endotoxin in milk

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of endotoxin in milk samples. Bovine and rabbit antisera raised in response to vaccination with the J5 mutant of Escherichia coli 0111:B4 were used. Antiserum to this mutant has been shown to be cross-reactive with endotoxin from other gram-negative organisms. Known quantities of endotoxin were added to milk samples to generate a standard curve. Acid treatment of whole milk enhanced the detection of endotoxin as compared to untreated whole milk, skim milk and chloroform-treated milk. Milk samples from experimentally induced mastitic cows were then assayed for endotoxin content. Recovery of endotoxin, as measured by ELISA, positively correlated with the amount of endotoxin infused and the time post-infusion of sampling. However, when endotoxin from these samples was quantitated using the Limulus Amebocyte Lysate (LAL) assay, readings tended to increase, suggesting false-positive reactions with the LAL assay. Milk samples from cases of clinical mastitis were assayed by ELISA with 64% of these showing measurable levels of endotoxin. While further studies of this assay are needed, refinements may produce an assay important for clinical applications.     

Development of an enzyme-linked immunosorbent assay for the detection of phenothiazine tranquillisers in horses

An acepromazine (ACP) hapten was synthesised, coupled to bovine serum albumin and injected into a horse to produce antibodies to the drug. A competitive ELISA was developed whereby ACP attached to the solid phase via lysozyme competed with free ACP present in phosphate buffered saline, horse serum or horse urine for limiting amounts of antibody. The assay could detect the presence of ACP and, or, some of its metabolites in horse urine for at least 25 hours after intravenous injection of 0.1 mg kg-1 ACP maleate, but because of non-specific interference, horse serum could not be used. As little as 0.24 micrograms ml-1 ACP or its metabolites could be detected. The level of detection and the ease of performance of the assay make it an attractive alternative to the more complex methods currently available for the screening of horse urine samples at horse races, shows and sales.     

Comparison of enzyme-linked immunosorbent assay, indirect fluorescent antibody test, and direct agglutination test for detecting Toxoplasma gondii antibodies in naturally aborted ovine fetuses

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to greater than 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.     

Evaluation of an in-house enzyme-linked immunosorbent assay for quantitative measurement of serum total thyroxine concentration in dogs and cats

OBJECTIVE: To compare serum total thyroxine (T4) concentrations obtained with an in-house ELISA and a validated radioimmunoassay (RIA). DESIGN: Laboratory trial. SAMPLE POPULATION: 50 canine and 50 feline serum samples submitted for measurement of total T4 concentration with the RIA; samples were selected to represent a wide range of concentrations (< 6 to 167 nmol/L). PROCEDURE: Results of the ELISA and RIA were compared by calculating correlation coefficients, examining linearity, determining bias and precision, and evaluating clinical interpretations. RESULTS: Correlation coefficients for results of the 2 methods were 0.84 for the canine samples and 0.59 for the feline samples. Examination of bias plots revealed large variations in ELISA results, compared with RIA results. For the feline samples, the ELISA consistently overestimated total T4 concentration obtained with the RIA. When results of the 2 methods were categorized (low, borderline low, normal, borderline high, or high), results were discordant for 24 (48%) and 29 (58%) of the canine samples and for 18 (36%) and 28 (56%) of the feline samples (depending on whether borderline high ELISA results were considered normal or high). Reliance on results of the ELISA would have led to inappropriate clinical decisions for 31 (62%) canine samples and 25 (50%) feline samples. The ELISA coefficients of variation for the pooled canine and feline samples were 18 and 28%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Substantial discrepancies between ELISA and RIA results for T4 concentrations were detected. Thus, we concluded that the in-house ELISA kit was not accurate for determining serum total T4 concentrations in dogs and cats.