Enzyme-linked immunosorbent assays for the detection of Toxoplasma gondii-specific antibodies and antigens in the aqueous humor of cats.

Serum and aqueous humor samples, collected from 14 clinically normal cats and 96 cats with clinical evidence of intraocular inflammation, were assayed with ELISA for Toxoplasma gondii-specific immunoglobulin M (IgM), T gondii-specific IgG, T gondii-specific antigens, total IgG, and total IgM. Additionally, serum was assayed with ELISA for feline leukemia virus p27 antigen and antibodies against the feline immunodeficiency virus as well as with an immunofluorescent antibody assay for antibodies against feline coronaviruses. Calculation of the Goldmann-Witmer coefficient (C-value) for the T gondii-specific antibodies detected in aqueous humor established the likelihood of local antibody production. Serologic evidence of present or prior infection by an infectious agent was found in 81.9% of the clinically affected cats from which serologic results were available (77/94 cats). Seropositive results for toxoplasmosis were found in 74.0% of the clinically affected cats. Anterior segment inflammation was found in 93.1% (81/87 cats from which information was available) of the clinically affected cats, most of which were older males. Toxoplasma gondii-specific antibodies were not detected in the aqueous humor of 6 seropositive, clinically normal cats. The C-values for aqueous T gondii antibodies were greater than 1 in 44.8% of the cats and greater than 8 in 24.0% of the cats. Response to treatment with clindamycin HCl was positive in 15/20 (75%) of the T gondii-seropositive, clinically affected cats treated with this drug. In 13/15 (86.7%) T gondii-seropositive, clinically affected cats having a C-value greater than 1, response to treatment with clindamycin HCl was positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-linked immunosorbent assay for the detection of bovine papillomavirus

An enzyme-linked immunosorbent assay has been developed to detect and quantitate bovine papillomavirus in partially purified and in purified viral preparations, using rabbit antiserum against group-specific papillomavirus structural antigens and alkaline phosphatase-labeled affinity purified goat antibody to rabbit immunoglobulin G. Viral detection correlated well with negative-stain electron microscopy of the various preparations and peroxidase-antiperoxidase staining of paraffin sections of the original fibropapillomas. The technique is rapidly done and will detect minute amounts of viral protein (1 ng/ml).     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies

The immune response to bluetongue virus in sheep and cattle was studied by applying a newly developed indirect enzyme-linked immunosorbent assay (ELISA). Purified virus obtained by sucrose gradient centrifugation was used at a concentration of 0.01 optical density units (formula: see text) to coat individual wells (200 microliter) of a microtitration plate. Dilution of antigen was performed in 0.05 M carbonate buffer, pH 9.6, and adsorption lasted for at least 16 hours at 4 C. Coated plates retained their activity for 10 weeks when stored at 4 C. Sera recovered from experimentally infected sheep and cattle were tested together with known negative sera. A good correlation between results was obtained with the modified complement-fixation test and the ELISA; however, the ELISA proved to be more sensitive. The group specificity of the ELISA was proven by testing various type-specific sheep and cattle immune sera. The ELISA has potential for the detection of group-specific antibodies to bluetongue virus infection.     

Diagnosis of recent Toxoplasma gondii infection in cats by use of an enzyme-linked immunosorbent assay for immunoglobulin M

Subclinical Toxoplasma gondii infection was induced in young and adult cats by oral administration of tissue cysts. An antibody-capture ELISA to detect anti-Toxoplasma IgM-class antibodies in the serum of cats was developed. The serologic response to experimental infection was followed in the 2 groups of cats by use of anti-Toxoplasma IgM and IgG detection. This study shows that anti-Toxoplasma IgM-class antibody titers develop early in the course of experimental infection in cats and that the combination of IgM- and IgG-class antibody titer measurement can aid in the detection of recent subclinical toxoplasmosis.     

酶联免疫法测定鸡肉中尼卡巴嗪残留

为了建立鸡肉中尼卡巴嗪残留标志物4,4’-二硝基均二苯脲（DNC）残留酶联免疫（ELISA）检测方法。将N-琥珀酰-L-丙氨酰-L-丙氨酰-L-丙氨酸4-硝基苯胺（SAN）与载体蛋白偶联作为抗原免疫动物,获得抗DNC抗体,在此基础上建立了鸡肉中尼卡巴嗪残留的检测方法。人工抗原中SAN与载体蛋白的结合比约为12：1,血清效价1：2000倍,鸡肉中检测限为9．2μg／kg,添加回收率在49．4％～118％之间,批内、批间变异系数均小于20％。该方法在灵敏度、精密度和准确度方面均能满足兽药残留筛选检测要求。     

Prevalence of Toxoplasma gondii infection in cats in Georgia using enzyme-linked immunosorbent assays for IgM, IgG, and antigens

Serologic prevalence of Toxoplasma gondii infection was determined using enzyme-linked immunosorbent assays detecting immunoglobulin M (IgM), immunoglobulin G (IgG), and circulating T. gondii antigens (Ag) in 81 healthy cats and 107 cats with clinical signs referable to toxoplasmosis. A higher prevalence of infection was detected using the three assays together in healthy cats, clinically ill cats, and combined healthy and clinically ill cats than when IgG class antibody detection alone was used. IgM titers greater than or equal to 1:256 and IgG titers greater than or equal to 1:512 were present more frequently in cats with clinical signs of disease. Prevalence of present or prior infection as defined by these three assays combined increased with advancing age in both groups of cats.     

Enzyme-linked immunosorbent assay for the detection of Salmonella enteritidis infection in chickens

An ELISA was developed and tested for its ability to detect antibodies against Salmonella enteritidis in chickens. Various features of the ELISA were evaluated and optimized. The outer membrane protein antigens selected by use of the protein immunoblotting method made the assay specific and sensitive. The assay was evaluated in chickens experimentally infected with S enteritidis. Blood samples collected at weekly intervals after experimental infection with S enteritidis were analyzed by ELISA. Results of the ELISA were compared with those of conventional serum plate and microagglutination tests. The ELISA was more sensitive and specific in the detection of S enteritidis infection than the other 2 conventional tests.     

Enzyme-linked immunosorbent assay for the detection of antibodies to infectious bronchitis virus

An enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis (IB) virus is described. The immune response of chickens following vaccination with IB virus was monitored using this test, and the titers were compared with those obtained by serum neutralization. The ELISA appears to be suitable for IB serology.     

Enzyme-linked immunosorbent assay for measurement of allergen-specific IgE antibodies in canine serum

A micro-ELISA, using horseradish peroxidase-conjugated anti-canine IgE and polystyrene microtitration wells for detection of allergen-specific IgE in canine serum, was developed. Specificity of anti-canine IgE was confirmed by reversed cutaneous anaphylaxis evaluations, gel-precipitation reactions, immunoelectrophoresis, immunoaffinity chromatography, and heat inactivation. Individual allergen blanks were used to account for variable nonspecific binding among various allergens, and results were normalized using 4 reference sera. Coefficients of variation for intra-assay and interassay variability ranged from 0.77 to 5.66% and 3.15 to 9.83%, respectively. Results observed with wells coated with mixtures of various allergen extracts yielded results approximately equal to results (average) of wells containing individual components. Agreement between ELISA and skin test results ranged from 43 to 64%, depending on allergen used.     

Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle

An adaptation of the enzyme-linked immunosorbent assay has been shown to detect antibodies in the circulation of cattle infected with Hypoderma bovis. Strong positive reactions were obtained from all infected cattle, even those which harboured only one larva. A strong cross reaction was observed in sera taken from cattle infected with H lineatum, but not in cattle infected with Fasciola hepatica or Ostertagia ostertagi.     

Enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in goats

Using a heat and sonicated Mycobacterium paratuberculosis Cordoba antigen (COA1) and the commercial protoplasmic-antigen (PPA-3) as antigens, an ELISA for detecting goat antibodies was standardized. When 2 reference populations, 1 positive (17 goats) and the other negative (63 goats) to disease, were used, this test showed 87.5% sensitivity and 93.6% specificity for COA1, and 88.2 and 95.2%, respectively for PPA-3. Absorption with M phlei was performed; no significant differences were found for COA1, but a lower sensitivity was found with PPA-3. This test was not especially affected by cross-reactivity with other mycobacterial disease because when 9 goats with M bovis infection were included in the M paratuberculosis control group, the specificity was only slightly different for absorbed (94.4%) and nonabsorbed sera (91.7%) for COA1, and (93.1 and 94.4%, respectively) for PPA-3. This test was used to study the percentage of seropositive goats for M paratuberculosis in 3 herds with different prevalences. Among 251 goats in southern Spain (Huelva), 40% were found positive for COA1 and 41% for PPA-3. Among 242 goats studied in southern Spain (Córdoba), 10.0% were positive for COA1 and 13.0% for PPA-3. In the Canary Island population of 176 goats, 3% were positive for COA1 and 0.5% for PPA-3. According to the accuracies of both positive and negative predictions, our test could be applied to populations with high prevalence to prevent additions to the herd and to cull infected animals (with 40% prevalence, the positive and negative predictive values are 90%), and to prevent adding infected animals to populations with moderate or low prevalence.     

Enzyme-linked immunosorbent assay for detection of transmissible gastroenteritis virus antibody in swine sera

An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantification of serum antibodies to transmissible gastroenteritis virus (TGEV) in swine. Sera from pigs inoculated with cell culture-origin TGEV or gut-origin TGEV were tested for anti-TGEV antibody by ELISA and by serum virus-neutralization test (NT). The ELISA detected antibody 3 days (av) sooner than did the NT when sera from pigs inoculated with cell culture-origin TGEV were tested and 1 day sooner than did the NT when sera from pigs inoculated with gut-origin TGEV were tested. The ELISA appeared to be more sensitive than the NT, since ELISA was more responsive to low-level antibody and ELISA titers exceeded NT titers.     

Enzyme-linked immunosorbent assay for detection of antibody to Pseudomonas aeruginosa and measurement of antibody titer in horse serum

Enzyme-linked immunosorbent assay (ELISA) was used for detection of immunoglobulin (Ig) M and IgG antibodies against a serologically common antigen (original endotoxin protein), protease, and elastase of Pseudomonas aeruginosa. The P aeruginosa antibody in horse sera was measured, using ELISA. Horseradish peroxidase-labeled rabbit anti-horse IgM and IgG antibodies were used for enzyme-labeled antibody conjugate. 5-Aminosalicylic acid and H2O2 were used for substrate. Sera collected from a vaccinated horse, a newborn foal, and 72 healthy racehorses were investigated for antibodies against P aeruginosa by ELISA and passive hemagglutination procedure. Changes in IgM and IgG antibody titers with vaccination were clear by ELISA. In the newborn foal, significant amounts of IgM and IgG antibodies from colostrum were present on the 1st day after birth. It was shown by ELISA that the level of antibodies in the newborn decreased initially and then increased. Some antibodies against original endotoxin protein, protease, and elastase of P aeruginosa were detected in almost all the healthy racehorses investigated.     

Enzyme-linked immunosorbent assay for detection of antibody to avian encephalomyelitis virus in chickens

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to avian encephalomyelitis virus (AEV) has been developed for determining whether existing AEV control programs adequately protect breeder hens. A partially purified AEV antigen was bound to microcuvettes for reaction with specific primary antibody. A second antibody, rabbit anti-chicken immunoglobulin G (IgG) conjugated with horseradish peroxidase, was employed to react with bound primary IgG. The relative amount of bound primary IgG was detected using ortho-phenylenediamine as a substrate for enzymatic production of a chromogen by horseradish peroxidase. Intensity of absorbance of the chromogen at 490 nm was related to the bound primary antibody by the titration method. Negative antisera were surveyed to establish an appropriate positive/negative cutoff level at twice the mean absorbance of negative sera at a 1:100 dilution. The test reagents for the ELISA were optimized by reagent titrations utilizing known positive and negative antisera for discrimination. The optimized ELISA had a coefficient of variation of from 1.2 to 3.3 for within-assay titer and of 2.4 for between-assay mean titer. Even though the ELISA detected only specific IgG, it was as accurate as the virus-neutralization test for evaluating the immune status of hens to AEV. Moreover, the ELISA was more economical in the use of reagents, time, and personnel and was free from dependence on susceptible embryos. Since ELISAs can be standardized and measured with manual or automated instruments, the derived ELISA can be easily and economically used to evaluate the immune status of breeder hens in commercial poultry operations.