Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili

Infectious bovine keratoconjunctivitis (IBK), caused by Moraxella bovis, is a disease of major importance in cattle industry. M. bovis has several virulence factors among which pili are crucial antigen for the protective capacity of vaccines against this disease. The production of vaccines against IBK therefore requires a reliable technique for cellular piliation level assessment on cells to be included as vaccine components. In this study we describe a specific whole-bacterial cell enzyme-linked immunosorbent assay (bact-ELISA) capable of detecting pili antigen on M. bovis cell surface. A sequential competitive bact-ELISA was developed using highly piliated M. bovis cells as antigen. Samples to be analyzed were allowed to react with anti-pilus serum prior to incubation in wells coated with piliated cells of M. bovis. This assay proved useful for the rapid, sensitive and reproducible evaluation of piliation on M. bovis cells, and represents an important tool for cellular piliation monitoring daburing M. bovis cells production in stirred bioreactors.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

猪戊型肝炎病毒抗体间接ELISA诊断方法的建立

本实验建立了应用高效融合表达的重组抗原PET32a-p214检测猪戊型肝炎病毒血清抗体的间接ELISA诊断方法。确定了抗原最适包被浓度为6μg/mL;血清最适稀释度为1∶100,作用时间为60 min;酶标抗体最适稀释度为1∶4 000,作用时间为60 min;判定标准为OD值≥0.339为阳性,OD值<0.339为阴性。实验结果表明该法特异性、敏感性和重复性均较好,与万泰公司戊型肝炎病毒抗体诊断试剂盒检测猪血清的符合率为98.6%。该方法的建立为猪戊型肝炎病毒抗体检测和进行猪戊型肝炎流行病学调查提供了一种简便快速的血清学诊断方法。     

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。     

Establishment of an efficient enzyme-linked immunosorbent assay for the detection of Eperythrozoon sius antibody in swine

The Eperythrozoon suis (E. suis) antigen was purified using a Sephadex G-200 chromatograph, and thereby, a high-affinity, specific E. suis antigen was collected and confirmed with Western blotting. Using this antigen, an enzyme-linked immunosorbent assay (ELISA) system to detect the antibody against E. suis in swine was established. There was no cross-reaction with swine sera, which were affected with Mycoplasmal pneumonia, swine fever, swine colibacillosis, or toxoplasmosis. A comparison of this ELISA system with an indirect hemagglutination (IHA) test using 78 swine samples revealed that the ELISA system significantly improved the sensitivity, specificity, and stability for the serodiagnosis of swine E. suis.     

Monoclonal-antibody-mediated enzyme-linked immunosorbent assay for detection of reticuloendotheliosis viruses

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of reticuloendotheliosis viruses (REVs). The assay uses a mixture of monoclonal antibodies (MCAs) prepared against a 62-kilodalton REV envelope glycoprotein (gp62) to capture antigen, rabbit anti-REV serum as detection antibody, and peroxidase-conjugated anti-rabbit IgG as indicator antibody. The MCAs were reactive with REV strain T, chick syncytial virus, and duck infectious anemia virus but unreactive against Marek's disease and avian leukosis viruses. The ELISA was compared with complement fixation test and REV immunofluorescent assay of infected fibroblasts, plasmas, and egg albumen from infected chickens. The lower limit of gp62 detection was about 120 ng of REV protein. Limit dilution of infectious REV was detected after 7-8 days of cultivation of infected fibroblasts.     

Evaluation of an enzyme-linked immunosorbent assay for diagnosis of trichinellosis in swine.

Experimental and field trials were conducted to evaluate an ELISA for its ability to detect Trichinella-infected domestic swine and to compare ELISA results with muscle-digestion test results. The ELISA used was a commercial double-antibody kit, containing an excretory-secretory antigen, and was evaluated principally for epidemiologic use. Experimentally induced infection in swine (4 groups of 3 pigs each; inoculated with 0, 50, 500 or 5,000 larvae) was detected as early as postinoculation week 4, with seroconversion of all inoculated swine by postinoculation week 8. The rate of seroconversion appeared to be affected by initial larval dose, time after inoculation, and immunocompetence of the individual host. Determination of antibody kinetics generally revealed rapidly increasing antibody titer, followed by its steady decrease in most pigs. Once seropositive, however, all pigs remained seropositive for the duration of the 10-week study. Presence of muscle larvae was confirmed in all infected pigs at termination of the study. We recognize that the experimental conditions may not be truly representative of those under which natural infection develops in pigs; however, the ELISA detected an infected pig with muscle larval density of 0.87 larvae/g of tissue. Results of a field trial (n = 310) indicated no muscle digestion test-positive pigs (35 g of diaphragm muscle digested/pig), but 3 samples tested positive by ELISA for specificity of 99.0%.     

Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum

Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity.     

Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.     

Use of enzyme-linked immunosorbent assay for detection of antibodies to Brucella ovis in sheep: field trial

An enzyme-linked immunosorbent assay (ELISA), using an autoclave-extracted soluble antigen, for the detection of serum antibodies to Brucella ovis in sheep was developed. The test seemed to be both sensitive and specific, on the basis of the control groups studied. The antigen showed no deterioration in prepared plates stored at -70 C for up to a year. The ELISA was used in conjunction with palpation of rams for epididymal lesions as a means to detect and control B ovis infection in a naturally infected flock. All rams were evaluated by the ELISA. At the time that the first blood sample was obtained, all positive and suspicious reactors were removed. With subsequent blood sample collections, only the positive reactors were removed. Brucella ovis was not isolated from any rams during the following year, and none of the mature breeding rams developed epididymal lesions.     

Prevalence of trichinosis in Canadian swine determined serologically by enzyme-linked immunosorbent assay.

Fifteen thousand three hundred and eighteen porcine sera from all regions of Canada were examined for the presence of anti-Trichinella antibodies using the enzyme-linked immunosorbent assay with an excretory-secretory antigen. Four sera (0.026%) revealed the presence of anti-Trichinella antibodies, with titers (optical density readings) that fell in the low positive or high negative range on repeated examinations. One animal originated in British Columbia and three in Ontario. Serological examination of swine in the herds at time of traceback did not reveal further animals with anti-Trichinella antibodies.     

Comparison of pepsin-digestion and enzyme-linked immunosorbent assay for the diagnosis of trichinosis in swine.

Comparison of parasitological and serological diagnosis of trichinosis in swine was carried out on 36 pigs given 15,400 infective larvae each by gavage. Circulating eosinophil levels were determined and sera were examined by enzyme-linked immunosorbent assay for anti-Trichinella antibodies. Two pigs were killed per day from days 15 to 29 postinfection. Muscle was examined by pepsin-digestion and comparable tissue was fed to a rat. Eosinophil counts increased at about day 6 and reached peak levels about day 25 postinfection and returned to approximate preinfection levels about two months postinfection in those pigs still in the study. Infective larvae were recovered from all pigs killed at greater than or equal to 18 days postinfection. Using the criterion of 5 x mean optical density readings of negative sera as positive, seroconversion occurred between days 19 and 26 postinfection. Use of a lower criterion of 3 x mean optical density readings of negative sera resulted in only three of 30 pigs killed greater than or equal to 18 days postinfection seroconverting less than or equal to 18 days postinfection, when infective larvae were first recovered in the musculature. In pigs, even in those heavily infected, there is a lag between the period that trichinae in musculature become infective and development of antibodies as detected by enzyme-linked immunosorbent assay which results in false negative reactions in many animals. This study demonstrated that the enzyme-linked immunosorbent assay using an excretory-secretory antigen should not be used to certify pork or pork products free of infective Trichinella larvae or safe for human consumption.     

Detection of antibodies against porcine parvovirus in swine sera by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibody against porcine parvovirus in swine sera. The antigen used for the assay was partially-purified virus treated with fluorocarbon and shown to contain 7 proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Of these proteins 83-, 64- and 60-K proteins reacted in Western immunoblotting with swine serum after infection with porcine parvovirus. Antibody responses were demonstrated by ELISA in pigs subcutaneously-infected with porcine parvovirus as by hemagglutination-inhibition (HI) test and Western immunoblotting reaction with the 83-, 64- and 60-K viral proteins. The results of ELISA on random swine-serum samples were well-correlated with those of the HI test. These findings indicate the usefulness of the ELISA as a serological tool for porcine parvovirus infection.     

Use of an enzyme-linked immunosorbent assay for the detection of IgG antibodies to rinderpest virus in epidemiological surveys

A comparison was made between an indirect enzyme-linked immunosorbent assay (ELISA) and the virus neutralisation test (VNT) for the detection of antibodies to rinderpest virus in field sera. The results did not agree for 6 per cent of the sera tested where 3 per cent of the samples gave ELISA positive/VNT negative and 3 per cent gave ELISA negative/VNT positive. The latter sera all had high levels of IgM antibody, which may indicate animals being at an early stage of infection or detection of a non-specific reaction. The ELISA results give a representative picture of the immune status for field surveys and a greater number of sera can be assayed with relative ease, compared to the traditional serum neutralisation test.