Detection of the K99 antigen of Escherichia coli in calf faeces by enzyme‐linked immunosorbent assay (ELISA)

Summary

An enzyme‐linked immunosorbent assay (ELISA) is presented for the detection of the K99 antigen of Escherichia coli in calf faeces. False‐positive reactions were not observed with K99‐negative strains and with several viral antigens. Only bovine coronavirus caused slight positive reactions which could be eliminated by a blocking test. As compared with the conventional procedure for the detection of the K99 antigen, ELISA seemed to be at least as sensitive and had the advantage that samples could be stored at —20° C before testing. In addition many samples could be handled at the same time and the results became available quickly. By carrying out the assay as a blocking test, specific antibody against K99 in serum or colostrum could be detected and titrated.     

Detection of flagellar antigen ofCampylobacter jejuni andCampylobacter coli in canine faeces with an enzyme-linked immunosorbent assay (ELISA) — New prospects for diagnosis

A new diagnostic procedure was developed to detect the flagellar antigen ofCampylobacter jejuni andCampylobacter coli in canine faecal specimens and was tested on faecal samples from random-source dogs obtained from the local dog pound. Extraction of acid-soluble proteins was performed on faecal specimens and the extracted material was evaluated using species-specific monoclonal antibodies in an enzyme-linked immunosorbent assay. The assay detected allC. jejuni orC. coli infected specimens compared with direct selective faecal culture. One of 18 faecal specimens culture-negative forC. jejuni was identified as positive by the assay, i.e. a false positive rate of 1 of 18 (5.6%) and a corresponding specificity of 94.4%. These results suggest that the screening procedure developed to detect flagellar antigens ofC. jejuni andC. coli in canine faecal samples should be further investigated as a diagnostic alternative to culture.Abbreviations ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - OD optical density     

Latest developments in the enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assays (ELISAs) for the serologic detection of both antigen and antibody in monitoring programs of commercial poultry flocks have begun to be recognized as an improvement over more conventional diagnostic procedures. The feasibility of employing double-antibody sandwich assays for the detection of virus without prior isolation of virus has been demonstrated and shows promise as the method of choice for the detection of lymphoid leukosis virus shedding. The versatility of indirect ELISA for the measurement of antibody induced by a wide variety of potential pathogens using a single basic overlapping ELISA system has also been demonstrated. It shows potential as a likely candidate to replace some of the more costly and time-consuming or less sensitive conventional serologic methods that do not overlap. Although some aspects of the two major types of immunoassays currently used in poultry health may need some modifications or improvements before delivery for routine use, it is likely that the use of computer-assisted ELISA will gain increased acceptance and use as the preferred way to efficiently and accurately monitor the health of poultry flocks on a broad scale.     

Diagnosis of swine trichinosis by enzyme-linked immunosorbent assay (ELISA) using an excretory- secretory antigen

An excretory-secretory (ES) antigen was used in a serodiagnostic enzyme-linke immunosorbent assay (ELISA) for swine trichinosis. ELISA procedures included a double- antibody test, using either an anti-swine IgG or a protein A enzyme conjugate, and a triple-antibody test using a pig IgG heavy-chain specific second antibody with a conjugated third antibody. The ES antigen was effective in eliminating all false-positive reactivity in sera from farm-raised hogs. The triple-antibody procedure was more sensitive and demonstrated a greater efficiency in detecting positive animals and early seroconversions. Naturally-infected pigs with worm burdens as low as 0.01 larvae per gram (LPG) of diaphragm were seropositive using these procedures. Seroconversion in experimentally-infected animals receiving low doses of muscle larvae (500) occurred considerably later than in animals receiving high doses (10000). This might account for false-negative reactions in naturally-infected animals with very low (less than 0.1 LPG) worm burdens.     

Rapid detection of viral-specific antibodies by enzyme-linked immunosorbent assay (ELISA)

The development of three separate rapid ELISAs for detecting antibodies in host serum to three different viruses is described. These include: 1. A direct antigen assay using enzyme labelled anti-canine Ig for detecting antibodies to canine parvovirus, 2. A competitive ELISA using a feline infectious peritonitis virus-specific monoclonal antibody labelled with enzyme, and 3. A competitive ELISA using an equine infectious anemia virus-specific monoclonal antibody and enzyme labelled antigen, p. 26. The utility and benefits of each of the three approaches is emphasized.     

An enzyme-linked immunosorbent assay (ELISA) for von Willebrand factor antigen (vWf-Ag) in canine plasma.

A quantitative enzyme-linked immunosorbent assay (ELISA) has been developed to measure canine von Willebrand factor antigen (vWf-Ag) in plasma of the dog. A vWf-Ag antiserum was raised in rabbits and purified by preabsorption with the low molecular weight vWf-Ag-deficient fraction of canine cryoprecipitate, followed by affinity chromatography on protein-A Sepharose. The rabbit anticanine vWf-Ag IgG was used to bind the vWf-Ag of the test plasmas to the solid phase and to prepare the enzyme-antibody conjugate in ELISA. Normal rat serum was used as blocking agent. The standard curve was linear (r2 greater than 0.98) and reproducible after logit-log transformation. The interassay coefficient of variation (CV) in test plasmas with various vWf-Ag concentrations was never greater than 7.7%. Assayed values in dilutions of pooled normal canine plasma added to canine vWf-Ag-deficient plasma were linear between 0 and 100% (r2 = 0.99) and indicated excellent analytical recovery of vWf-Ag. In 18 dogs with various internal diseases, including von Willebrand's disease and haemophilia A, the coefficient of correlation between the results of the ELISA and those of electroimmunodiffusion (EID) was 0.93.     

Detection of bovine antibodies to the outer membrane of ruminal Bacteroides succinogenes by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) was used to detect bovine serum antibodies directed to the outer membrane antigen of a ruminal bacteria, Bacteroides succinogenes. The outer membrane antigen of B. succinogenes was highly reactive against homologous antiserum, compared with rabbit sera raised against B. ruminicola subsp. ruminicola, B. ruminicola subsp. brevis and Selenomonas ruminantium. The titers of sera from colostrum-deprived calves were negligible level, while those of sera from colostrum-fed calves were relatively high. The mean titer of sera from 10 day-old calves was significantly (p less than 0.01) higher than that of 40 day-old calves, and was significantly (p less than 0.01) lower than that of adult cattle. The mean titer of sera from dairy cows which fed high-roughage diet was higher than that of feedlot cattle which fed high-concentrate diet. These results suggest that the antibodies against the outer membrane antigen of B. succinogenes transfer to calves via the colostrum, and that the titers of cows are affected by the way of feed management.     

Serodiagnosis of porcine cysticercosis by enzyme-linked immunosorbent assay (ELISA) using fractionated antigens

The sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Taenia solium cysticercosis was evaluated in experimentally and naturally infected pigs, using T. solium larval scoleces and its fractionated 1st and 2nd peaks on Sephadex G-200 as antigens. First peak antigen gave maximum sensitivity and highest antibody titres. The overall sensitivity of this test was found to be 91.5, 95.8 and 70.8% with scolex, 1st and 2nd peak antigens, respectively. False positive reactions occurred in 9.09% of uninfected pigs with scolex and 1st peak antigens and cross-reactions occurred in 25% of Taenia hydatigena-infected animals using scolex and 2nd peak antigens. No cross-reaction was observed using 1st peak antigen. The specificity of the test was 92.3, 96.2 and 92.3% with scolex, 1st and 2nd peak antigens, respectively.     

Antigenic relationships between the serotypes of Pasteurella haemolytica demonstrable by enzyme-linked immunosorbent assay (ELISA)

Mice and rabbits were immunised with sodium salicylate extracts (SSE) prepared from each of 12 serotypes of Pasteurella haemolytica, and the antisera to each were used in cross-indirect haemagglutination (IHA) tests and cross-enzyme-linked immunosorbent assays (ELISA) to study antigenic relationships between the serotypes. An indirect micro-ELISA demonstrated common antigenic relationships which were not apparent by IHA. Antisera from both species revealed considerable shared antigenicity between all the serotypes. Rabbit antisera presented clearer differences between the A biotypes on one hand and the T biotypes on the other, the T biotypes exhibiting much less cross-relatedness than that shown between the A serotypes.     

Enzyme-linked immunosorbent assay, using monoclonal antibody, to detect enterotoxic Escherichia coli K99 antigen in feces of dairy calves

A modified, double-antibody, enzyme-linked immunosorbent assay (ELISA) was developed to detect the K99 pilus antigen of enterotoxic Escherichia coli (ETEC) in feces of calves. Extremely high positive to negative ratios (greater than 200) were obtained by using monoclonal antisera as the primary antibody. Strong positive reactions were obtained with strains of E coli known to produce the K99 antigen; however, non-enteropathogenic E coli (strains not producing the K99 antigen), Salmonella, Proteus, Klebsiella, Pseudomonas, Staphylococcus, Streptococcus, and rotavirus produced negative results. Seventy-five fecal samples, 8 from healthy calves and 67 from calves with neonatal calf diarrhea were examined with the K99 ELISA for the presence of ETEC. Rotavirus test and fecal culture results were available on feces from calves with diarrhea and were used with the K99 ELISA results to determine the specific cause of the disease. Enterotoxic E coli was the predominant agent detected in the feces of 29 diarrheal calves less than 5 days of age. Mixed infections of rotavirus and ETEC were also common in these calves, but rotavirus infections alone were not detected. In 38 calves greater than or equal to 5 days, rotavirus was detected without ETEC. Of these calves, only 2 produced positive tests with the K99 ELISA. Salmonella sp and Proteus sp were detected from 5 of 67 calves with diarrhea.     

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of dourine

The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable for testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.     

An enzyme-linked immunosorbent assay (ELISA) for infectious bursal disease virus.

The application of the indirect enzyme-linked immunosorbent assay (ELISA) for detection of infectious bursal disease virus antibodies in chicken serum was investigated. The test procedure involved the coating of concentrated infectious bursal disease virus antigen onto polystyrene tubes, followed by the addition of chicken anti-infectious bursal disease virus serum and horseradish peroxidase labeled rabbit anti-chicken globulin. As an indicator substrate, 5-aminosalicylic acid, with the oxidant H2O2 was added. The reaction was stopped by 3M NaOH and the colour intensity of the reaction mixtures read in a spectrophotometer at 449 nm. The ELISA test was found to be a precise, sensitive and reproducible means of measuring infectious bursal disease virus antibodies in chicken and turkey sera.     

Identification by transfer blot of antigens reactive in the enzyme-linked immunosorbent assay (ELISA) in rabbits immunized and a calf infected with Cryptosporidium sp

Soluble and particulate fractions of Cryptosporidium sp. oocysts from cattle were obtained by homogenization and sonication. Electrophoresis of the soluble fraction in polyacrylamide gels with sodium dodecyl sulfate and silver staining revealed the presence of 41 bands. Enzyme-linked immunosorbent assay (ELISA) of sera from rabbits immunized with either fraction and from a calf 40 days after infection showed that the animals produced specific antibodies. Enzyme-linked immunoelectrotransfer blot tests revealed the presence of five antigens with the rabbit sera and nine with the calf serum. ELISA proved to be an appropriate test for diagnosis of cryptosporidiosis. Selection of reactive antigens may improve the quality of diagnosis and/or reveal the presence of protective materials in the parasite.