Enzyme-linked immunosorbent assay methods for detection of feline leukemia virus and feline immunodeficiency virus.

Enzyme-linked immunosorbent assays have been widely used for diagnosis of FeLV and feline immunodeficiency virus (FIV) infections. Various ELISA kits for FeLV are available from several manufacturers. Although these tests are configured in a variety of formats, they are all direct antigen-detection systems for the viral core protein p27. On the other hand, ELISA for FIV exposure detects specific feline antibody to FIV. Basic immunoassay principles and the application of ELISA technology used in FeLV and FIV ELISA kits are described.     

An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.     

Ten-year study comparing enzyme-linked immunosorbent assay with the immunofluorescent antibody test for detection of feline leukemia virus infection in cats.

Immunodetection tests for feline retroviruses are powerful tools used in modern veterinary practice. Veterinarians must fully understand the characteristics--strengths and weaknesses--of the FeLV tests so that the information gained from them can be used properly. Any FeLV ELISA or immunofluorescent antibody (IFA) test is a method for detection of FeLV infection (the virus) and is not a diagnostic test for leukemia or other feline disease. From previous studies, it was determined that the most accurate test for detection of persistent FeLV infection is the IFA test, which detects FeLV antigens in cytoplasm of leukocytes in the blood of infected cats. In the study reported here, 1,142,600 FeLV IFA tests were performed between June 1972 and December 1990. During this period 19.8% of the IFA test results were positive and 78% were negative. Evaluation was not possible for the remaining 2.2% of the tests because of lack of enough leukocytes in the smears to evaluate, or nonspecific staining reactions. In 1979, 7 years after introduction of the IFA test, in-hospital FeLV ELISA were introduced, which enabled veterinarians to test for FeLV in their hospitals. Ever since that time, continual discrepancies have been reported between results of FeLV ELISA and IFA tests, particularly between positive ELISA results and their IFA test confirmation. A 10-year comparison was made between practitioner-performed in-hospital FeLV ELISA (n = 20, 240 tests) results and FeLV IFA test performed by a commercial laboratory. All samples tested by ELISA were submitted (for confirmation of results) by veterinarians from the United States, Canada, Europe, Japan, and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the detection of antibody to duck hepatitis virus by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to duck hepatitis virus (DHV) is described. The results of ELISA were compared with those of an agar gel diffusion precipitin (AGDP) test and a serum-neutralization (SN) test. The specificity of the ELISA was in accordance with the specificity of the AGDP and SN tests, but there was a difference in sensitivity. The positive detection rates of ELISA, SN test, and AGDP test for 93 clinical samples were 68.8%, 68.8%, and 18.8%, respectively. A positive/negative (P/N) value larger than or equal to 2.1 plus an absorbance value larger than or equal to 0.4 was used as a comprehensive positive standard for the ELISA. This eliminated false-positive reactions. The results showed that the ELISA was a rapid, sensitive, and accurate method for detecting antibody to DHV.     

An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the hemagglutination-inhibition (HI) test under conditions of both controlled and field exposure. During the course of temporal experimental infection (0-76 days) of specific-pathogen-free (SPF) chickens with AI subtype Hav9N2, the AI-ELISA was able to detect specific AI antibody as early as 8 days postinoculation (PI), and it measured rising levels of antibody through 35 days PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were negative through 35 days PI, and HI tests began to detect low levels of AI antibody only at 21 days PI. Following a secondary infection at 35 days PI with the same AI subtype, all tests measured rising levels of AI-specific antibody (35-76 days PI). However, the AGP test was positive at only the 7- and 14-day samplings postsecondary immunization. Under field conditions, the AI-ELISA was able to detect serum AI antibody in flocks from which highly pathogenic AI was isolated, but the AGP tests of these sera were negative.     

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.     

A double-antibody enzyme-linked immunosorbent assay for the detection of turkey hemorrhagic enteritis virus antibody and antigen

A highly sensitive and specific double-antibody enzyme-linked immunosorbent assay (ELISA) is described for the detection of antigen and antibody of turkey hemorrhagic enteritis virus (HEV). The assay utilizes a virus-neutralizing monoclonal antibody (MAb) to capture the antigen and turkey antiserum against HEV as the second antibody. Microtiter plates were first coated with a dilution of 1:3000 of the MAb (300 ng immunoglobulin/well) and are used for detection of both antigen and antibody. For antibody detection, MAb-coated plates were treated with an appropriate dilution of a cell-culture-propagated HEV antigen and then reacted with the test turkey serum. For detection of HEV antigen, MAb-coated plates were treated with appropriate dilutions of test antigens and then reacted with purified anti-HEV turkey immunoglobulins. The assay for HEV antibody detection was more sensitive and specific than previously described single-antibody ELISAs. Using the double-antibody ELISA, it was found that the spleen of HEV-infected turkeys harbors very high levels of antigen. Traces of HEV antigen are present in some other organs. Infectivity assay for HEV is found to be about two orders of magnitude more sensitive than the ELISA for detection of virus.     

Course of feline leukemia virus infection and its detection by enzyme-linked immunosorbent assay and monoclonal antibodies

Monoclonal antibodies specific for 3 distinct epitopes of the species-specific determinants of feline leukemia virus (FeLV) p27 were used in an enzyme-linked immunosorbent assay (ELISA) for measurement of serum p27 in cats infected with FeLV. Group-specific antigen (GSA) of FeLV in peripheral blood leukocytes was also determined by an immunofluorescence assay. Antibodies to FeLV and the feline oncornavirus-associated cell membrane antigen (FOCMA) were also measured. Thirty-six cats were surveyed and assigned to 4 categories. Five developed persistent viremia (category 1), characterized by continuous expression of p27, GSA, and low antibody titers to FeLV and FOCMA. Eleven cats with transient viremia (category 2) and 13 cats that were never detectably viremic (category 3), as judged by absence of GSA and p27, developed increased antibody titers to FeLV and FOCMA. Seven cats were never viremic, as judged by the GSA in the peripheral blood leukocytes, but still had detectable serum p27 (category 4). Most category 4 cats developed high antibody titers against FOCMA and/or FeLV. Of 307 field cats examined, 7% of the healthy cats and 10% of the sick cats could be assigned to category 4. However, this difference was not significant (P greater than or equal to 0.05). Of 26 cats with neoplasms 2 (1 of 12 with lymphosarcoma) could be classified as category 4. Because virus could be isolated from 2 category 4 cats, they were considered immune carriers.     

Labeled avidin-biotin enzyme-linked immunosorbent assay for detecting antibody to infectious laryngotracheitis virus in chickens

A labeled avidin-biotin enzyme-linked immunosorbent assay (LAB-ELISA) for detecting antibody to infectious laryngotracheitis (ILT) virus in chicken sera was developed and compared with ordinary ELISA. Purified ILT virus, biotin-labeled anti-chicken IgG rabbit IgG conjugate, and horseradish-peroxidase-labeled avidin were used in the LAB-ELISA. When sera from farm chickens were tested by serum neutralization (SN) and two kinds of ELISA, the correlation rate between SN and LAB-ELISA was 50/50 (100%), and that between SN and ordinary ELISA was 39/50 (78%). In LAB-ELISA, all of the sera that were antibody-negative by SN had low absorbance (A) values (below 0.05), and the A values were closely correlated with the SN indexes. In ordinary ELISA, however, the sera antibody-negative by SN had various A values ranging from 0.06 to 0.32. LAB-ELISA had much lower nonspecific reactions than ordinary ELISA against sera from ILT-negative chickens, even when chickens were 30 weeks old. ILT antibody production after ILT vaccination could be detected by LAB-ELISA. A values peaked 5 weeks postinoculation and were maintained for 17 weeks.     

Monoclonal antibody based sandwich enzyme-linked immunosorbent assay for chicken growth hormone.

1. Monoclonal antibodies which bind to different epitopes of chicken growth hormone (cGH) were used to develop a homologous sandwich enzyme-linked immunosorbent assay (ELISA). 2. The first antibody, which is species specific, was immobilised on microtitre plates and concentrations of cGH in biological fluids were estimated by revealing bound hormone using a second, biotinylated monoclonal antibody. 3. The sensitivity was 0.024 ng/ml, which is at least ten-fold greater than current radioimmunoassays (RIA) and there was no cross-reactivity to other chicken pituitary hormones or to growth hormone from other species. 4. The accuracy and precision of the assay were similar to RIA, and the growth hormone concentrations measured in plasma samples by both RIA and this new ELISA showed a high degree of correlation. 5. The assay takes only 4 h using pre-coated plates which can be stored at 4 degrees C in sucrose. The advantages of being rapid and non-isotopic make this method attractive to both research and industrial laboratories.     

Development and validation of an enzyme-linked immunosorbent assay for feline trypsin-like immunoreactivity

OBJECTIVE: To develop and validate an ELISA for quantitative analysis of feline trypsin-like immunore-activity (fTLI). SAMPLE POPULATION: Purified feline cationic trypsin (fCT) and rabbit anti-fCT antiserum; blood samples from 63 healthy cats. PROCEDURES: A sandwich capture ELISA was developed, using anti-fCT antiserum purified by affinity chromatography that underwent biotinylation. Purified fCT was used for standards. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. A reference range was established by assaying serum samples from the 63 healthy cats. RESULTS: Sensitivity was 1.23 microg/L; working range was 2 to 567 microg/L. Ratios of observed versus expected results for 4 samples tested at various dilutions ranged from 90.0 to 120.7%. Ratios of observed versus expected results for 5 samples spiked with various concentrations of fCT ranged from 82.0 to 101.8%. Intra- and inter-assay coefficients of variability ranged from 9.9 to 11.1% and from 10.2 to 21.7%, respectively. The reference range for serum fTLI measured with this ELISA was 12 to 82 microg/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that an ELISA can be used to measure serum fTLI in cats. The ELISA was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

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.     

Detection of antibodies to mouse thymic virus by enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay was used to detect serum antibodies to mouse thymic virus, a herpesvirus that causes thymic lesions and immunosuppression. Antibodies were detected in mice that had received single or multiple injections of the virus and were also found in mice housed in contact with the experimentally infected animals. By contrast, mice not exposed to mouse thymic virus or those inoculated with an uninfected thymus preparation remained seronegative. A serological survey of eight mouse colonies revealed one positive colony, confirmed by virus isolation. These results show that the test is sufficiently sensitive and specific to be used for routine screening of mice.     

An enzyme-linked immunosorbent assay for the detection of Clostridium perfringens enterotoxin antibody.

An enzyme-linked immunosorbent assay (ELISA) was adapted to test serum antibody to enterotoxin of Clostridium perfringens type A. The test was evaluated using sheep, calf and guinea pig sera and compared with passive hemagglutination and immunodiffusion tests. The ELISA was found to be more sensitive than the other two tests and was completely free from nonspecific reactions. The method was considered to be technically advantageous and suitable for semiautomated procedures.     

H5 antibody detection by blocking enzyme-linked immunosorbent assay using a monoclonal antibody

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.