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。     

检测牛结核抗体新型ELISA方法的建立及应用

利用分子克隆和Splicing by overlapping extension(SOE)技术,表达了重组蛋白rM70-83-E6,Western blot分析rM70-83-E6具有很好的特异性。以蛋白rM70-83-E6作为诊断抗原建立了间接ELISA方法,ELISA诊断方法的判定标准,即S/P≥0.5为阳性,S/P<0.4为阴性,0.5>S/P≥0.4为可疑。ELISA方法的特异性为96.0%、敏感性为69.4%。对67份PPD皮试阳性和50份PPD皮试阴性(117份)血清样品进行了检测,并与PPD皮试比较。67份PPD皮试阳性血清样品中有46份为ELISA检测阳性,阳性符合率为68.7%,50份PPD皮试阴性牛血清都为阴性,阴性符合率为100%,本ELISA方法与PPD皮试诊断方法总复合率为82.1%。研究表明,ELISA方法具有很好的开发和应用前景。     

大豆抗原蛋白血清抗体间接ELISA检测方法的建立

旨在建立检测血清大豆抗原蛋白抗体的间接ELISA方法。经琼脂糖凝胶层析纯化大豆抗原蛋白,以不同剂量皮下注射免疫小鼠,采用方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白抗体的间接ELISA方法,利用该方法检测小鼠免疫后血清抗体水平。通过方阵滴定法确定11S蛋白最佳包被浓度为5.0μg/mL,血清稀释倍数为1∶800;7S蛋白抗原最佳包被浓度为2.5μg/mL,血清稀释倍数为1∶1 600;两者的批内、批间系数均小于10%,重复性较好,通过ELISA法确定11S和7S蛋白的最佳免疫次数为2次,免疫剂量为1 000μg/kg。结果表明本试验初步建立大豆抗原蛋白抗体检测间接ELISA方法,具有很强的特异性、敏感性和重复性,可用于大豆抗原蛋白过敏反应的临床检测。     

Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog

An enzyme-linked immunosorbent assay (ELISA) was compared to an indirect immunofluorescence assay (IFA) for detection of IgG antibodies to Borrelia burgdorferi in dog sera. The concordance of the two tests was 93.5% for sera from dogs from Maryland (n = 93), 98.0% for sera from dogs from North Carolina (n = 446), and 97.2% for the combined sample groups (n = 539). Twenty-five of the 27 samples with discordant or low positive results were tested, and showed immunoblot reactions to 1 to 10 different bands. Reaction patterns and intensity of the bands were quite variable, and did not explain a reason for the discordance.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle

Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398 bp) and SapA-C (1422 bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P < 0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P ? 0.45: positive; S/P < 0.4: negative; 0.45 > S/P ? 0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications.     

Development and initial evaluation of an indirect enzyme-linked immunosorbent assay for the detection of Leptospira interrogans serovar hardjo antibodies in bovine sera.

Outer sheath antigen from Leptospira interrogans serovar hardjo type hardjoprajitno and acetic acid extracted antigens from serovar hardjo types hardjoprajitno and hardjobovis were evaluated in an immunoassay for ability to detect hyperimmune rabbit serum to serovar hardjo. The degree of cross-reactivity with hyperimmune rabbit sera to L. interrogans serovars pomona, copenhageni, grippotyphosa, canicola and sejroe, and Leptospira biflexa serovar patoc was also measured for each antigen. All of the antigens reacted with the antiserum to L. interrogans serovar hardjo. The outer sheath antigen however, also showed wide cross-reactivity with the antisera to all of the serovars of L. interrogans tested and with the antiserum to L. biflexa serovar patoc. The acetic acid extracted antigen from either type hardjoprajitno, or type hardjobovis, showed a high degree of specificity for serovar hardjo antiserum. The hardjobovis acetic acid extracted antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting, and was incorporated into an indirect ELISA for detection of anti-serovar hardjo antibodies in bovine serum. This ELISA showed a relative specificity of 100% with 156 bovine sera which were negative at a dilution of 1:100 in the microscopic agglutination test (MAT) for L. interrogans serovars hardjo, pomona, sejroe, icterohaemorrhagiae, copenhageni, canicola, and grippotyphosa. The relative sensitivity of this assay with 192 bovine sera which had serovar hardjo MAT titres of > or = 100 was 95.3% (95% confidence limit = 2.99%). The degree of cross-reactivity with 289 bovine sera which had serovar pomona MAT titres of > or = 100 (with no detectable serovar hardjo MAT titres) was approximately 1.0%. This assay was: easily standardized, scored objectively, repeatable, semi-automated and used a non-hazardous antigen that can be routinely prepared in gram amounts.     

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 validation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against duck swollen head hemorrhagic disease virus

An indirect enzyme-linked immunosorbent assay (iELISA) was developed for detecting antibody to duck swollen head hemorrhagic disease virus (DSHDV) using purified whole virus by sucrose density gradient ultracentrifugation as a coating antigen. Antiserum against DSHDV strains HY-99 (hyperimmume serum) was prepared in 30-day-old ducks by vaccination with inactivated DSHDV and used as positive sera. The iELISA test was optimized with different reagents or dilutions. The validation results showed that this assay was only specific for antibodies against duck viral swollen head hemorrhagic disease. The OD450 value for positive serum diluted 1:800 was also determined to be greater than the positive threshold. The highest coefficient of variation value was 3.66% for the intra-assay and 5.79% for the interassay, which were all less than 10%. This assay has been successfully applied to the examination of the duck sera clinically. These results in this study indicate that the newly-developed iELISA offers a precise, specific, sensitive, and reproducible means of measuring DSHDV antibodies in duck sera.     

Development of an indirect enzyme-linked immunosorbent assay for detecting equine serum antibodies to the lipopolysaccharide of Salmonella abortusequi

An indirect enzyme-linked immunosorbent assay (IELISA) was developed for the detection of equine serum antibodies to lipopolysaccharide of Salmonella enterica subsp. enterica serovar Abortusequi (LPS), a causative organism of Equine Paratyphoid. The data presented demonstrates that horses immunized with S. abortusequi LPS developed antibodies detectable by the IELISA. By comparison, the tube agglutination test (TAT) did not detect antibody to S. abortusequi LPS as consistently as the IELISA. The data suggests that the IELISA may be a more suitable test for the detection of serum antibodies to S. abortusequi than the TAT.     

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (mvv) in sheep is described, in which microtitre plates are with a partly purified preparation of mvv. The antibodies bound are detected by a horseradish peroxidase conjugate.The results obtained with ELISA on a total of 493 serum samples from several commercial flocks were compared to those of a routine agar gel precipitation test (AGPT) and a complement fixation test (CFT).All samples which scored positive in AGPT, CFT or both (20.8%) were also found positive by ELISA. In addition, with ELISA a further 11.5% of the samples were positive. Serum samples from maedi-free flocks, from sheep suffering from sheep pulmonary adenomatosis and from lambs immunized against other viruses were all negative by ELISA. The assay has been used routinely for some years and proved to be specific, sensitive and suited for screening of large numbers of serum samples.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

Background

In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.

Results

The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value = sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.

Conclusion

The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.     

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.     

An indirect enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serovar 7 in pig serum.

Lipopolysaccharide (LPS) antigen was purified from Actinobacillus pleuropneumoniae serovar 7 by phenol-water extraction and fractionated on a, S-100 Sephacryl column. High molecular weight fractions of LPS purified from the S-100 column were pooled and used as antigen in an indirect serovar 7 ELISA. The ELISA was evaluated with sera from pigs experimentally infected with 11 different A. pleuropneumoniae serovars of biotype 1. Estimation of sensitivity and specificity of the A. pleuropneumoniae serovar 7 ELISA was performed using pig sera from herds naturally infected with A. pleuropneumoniae serovar 7 as well as sera from herds free of infection with A. pleuropneumoniae serovar 7. When compared to the complement fixation test (CFT) as a reference test, the ELISA showed much higher sensitivity and statistically equivalent specificity.     

Development of a competitive enzyme-linked immunosorbent assay for detection of turkey coronavirus antibodies

A competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of turkey coronavirus (TCV) antibodies. The cELISA utilized a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus)-expressed TCV nucleocapsid (N) protein and biotin-labeled TCV N protein-specific monoclonal antibody. Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test (IFAT) with 1269 reference, experimentally derived, and field-origin sera. Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses. The cELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related coronavirus, but did not detect antibodies specific for other avian viruses. A high degree of concordance was observed between the cELISA and IFAT; sensitivity and specificity of the cELISA relative to IFAT were 92.9% and 96.2%, respectively. Western immunoblot analyses provided additional evidence of cELISA specificity. The findings indicate that the cELISA is a rapid, sensitive, and specific serologic test for detection of TCV antibodies in turkeys.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) using a detergent-solubilized antigen of purified virus was developed for detection of antibody against porcine transmissible gastroenteritis (TGE) virus in swine serum. The ELISA demonstrated antibody responses in pigs immunized intramuscularly with the attenuated TO-163 strain of TGE virus and in pigs orally infected with the virulent Shizuoka strain of the virus. The results of the ELISA were well correlated with those of the neutralization test. These results indicate the usefulness of the ELISA as a serological tool for TGE virus antibody.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for the detection of allergen-specific IgE antibodies in dogs

Twenty-eight atopic dogs, 22 pruritic, non-atopic dogs and 10 healthy dogs were ELISA tested. For calculations of diagnostic specificity and sensitivity, positive ELISA test results in non-atopic dogs were considered false positive results. The absence of any positive results in the atopic dogs was considered false negative results. The atopic dogs were tested both with ELISA and an intradermal test, utilising allergen extracts from the same manufacturer, to determine the frequency of positive allergen reactions in the ELISA test compared with the intradermal test. The Prausnitz-Küstner test was performed to evaluate the significance of a positive ELISA test result. Based on cross-tabulations with clinically defined atopic dermatitis, the ELISA test showed a sensitivity of 53.6% and a specificity of 84.4%. The correlation between the ELISA and the intradermal test was poor. Positive Prausnitz-Küstner tests were not obtained using sera from dogs that were intradermal test negative for the tested allergens, even though sera had high levels of IgE as measured by the ELISA. These findings question the significance of a positive ELISA test result and indicate that the test is not measuring functional allergen-specific IgE.     

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.     

鹿副结核病抗体间接ELISA检测方法的建立

本研究以副结核杆菌亲和层析抗原为检测抗原,检测以草分枝杆菌抗原吸收的待检鹿血清,建立检测鹿副结核病血清抗体的间接酶联免疫吸附试验,确定其抗原最佳包被浓度为40μg/mL,血清样品稀释度为1:80,兔抗鹿IgG辣根过氧化物酶标记抗体稀释度为1:8000。经特异性试验和重复性试验证明该方法特异性高、重复性好。对不同地区4个鹿场的760头份鹿血清进行副结核病抗体检测,其中阳性61头份,阳性率为8%,获得副结核病在我国鹿群中的血清流行病学资料,从而为防制鹿副结核病提供一定的依据。     

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 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.