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。     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

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。     

HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis

The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.     

Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar canicola in ELISA for serodiagnosis of bovine leptospirosis

The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis.     

应用重组N蛋白—ELISA检测猪繁殖与呼吸综合征抗体的研究

利用昆虫杆状病毒表达系统表达的猪繁殖与呼吸综合征重组N蛋白作为抗原，包被聚苯乙烯微量反应板，建立了猪繁殖与呼吸综合征重组N蛋白-ELISA诊断方法。试验证明，该方法对其他8种猪疫病阳性血清均无交叉反应，对标准阳性样品的检出率为100%。具有敏感性高、特异性强的特点。     

Evaluation of an enzyme-linked immunosorbent assay in the diagnosis of bovine tuberculosis

The behavior of the immunoenzymatic system was tested in the serologic diagnosis of bovine tuberculosis in different populations from areas free of the disease, and in other areas with different prevalence. The specificity registered in the 4009 samples showed 94.4%, while the range of sensitivity increased proportionally to the number and distribution of lesions found in the anatomopathological test. Overall the sensitivity of the ELISA was 47%. When animals from groups with different prevalence of the disease were studied, a significant correlation (78%) between the ELISA system and the Intradermal Tuberculin Test (IT) was observed in the groups with low prevalence of the disease, animals reactive to both tests were found in all the units. The results of the work and the inherent advantages of the ELISA technique allow recommending this ELISA in programs for control and/or eradication of bovine tuberculosis.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of infectious bovine rhinotracheitis infection, with results of a preliminary survey

An indirect enzyme linked immunosorbent assay (ELISA) procedure was evaluated against the serum neutralisation test (SNT) for the detection of antibodies to infectious bovine rhinotracheitis virus (bovine herpesvirus type l), using 2028 sera from 166 dairy and 172 beef cattle herds. The results showed the ELISA to give high levels of agreement with the SNT in classifying positive and negative sera (98% and 97% respectively). Such disagreements as did occur involved weakly reactive sera with SNT titres of % or less. A number of sera (n=123) with trace neutralising activity of doubtful diagnostic significance were found to give marginal reactivity with ELISA. ELISA absorbance values were found to be highly correlated with SNT titres (r=0.909) on an overall basis, though agreements were lower with individual sera. The ELISA procedure was quicker, cheaper, and detected more reactors than the SNT. It also allowed results to be obtained with a number of sera which were unsuitable for testing by SNT because of their cytotoxic nature. Analysis of ELISA results showed reactors to be present in 57% of tested sera, representing 81% of cattle herds. Reactor rates for sera and herds in the South Island, (37% and 58%), were significantly lower than for those in the North Island (64% and 88%). Antibody prevalence was also found to be significantly lower in districts having a low annual rainfall (<850 mm), and to be lower in beef cattle than in dairy cattle. A surprising exception to the latter occurred in low rainfall districts, where dairy cattle showed significantly lower reactor rates than local beef animals.     

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 the serodiagnosis of pythiosis in dogs

Pythiosis (caused by the aquatic oomycete Pythium insidiosum) is a devastating and often fatal cause of either severe transmural gastroenteritis or locally invasive subcutaneous disease in dogs living in the southeastern United States. Although early diagnosis is essential for successful treatment, tools available for this task are limited. Therefore, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P insidiosum antibodies in canine serum. A soluble mycelial extract of P insidiosum was utilized as antigen in the ELISA, which was used to evaluate serum from 43 dogs with pythiosis, 8 dogs with lagenidiosis (another canine oomycosis), 16 dogs with nonoomycotic fungal or algal infections, 22 dogs with nonfungal gastrointestinal or skin disease, and 55 healthy dogs. Results were expressed as percent positivity (PP) relative to a strong positive control serum run on each plate. Medians and ranges for each of the 5 groups were as follows: pythiosis (81.7%, 50.6-98.5%), lagenidiosis (17.3%, 11.3-29.2%), other fungal or algal infections (8.2%, 4.7-15.4%), nonfungal gastrointestinal or skin disease (6.2%, 3.9-20.7%), and healthy dogs (6.7%, 3.0-15.2%). When using a cutoff value of 40% PP, the sensitivity and specificity of the ELISA both were 100%. In addition, ELISA values measured after successful surgical therapy in 2 dogs showed a decrease of anti-P insidiosum antibody concentrations into the normal range as early as 2 months after treatment. We conclude that the ELISA is a sensitive and specific test for the diagnosis of canine pythiosis, and may be a useful tool for monitoring response to medical or surgical therapy.     

Evaluation of two enzyme-linked immunosorbent assays for serodiagnosis of Aleutian mink disease virus infection in mink

Background

Aleutian disease in mink is caused by infection with Aleutian mink disease virus (AMDV). In Sweden, the infection most commonly causes classical Aleutian disease in which the immune system fails to neutralize the virus and the infection becomes persistent. Diagnosis of AMDV infection is based on serological methods that detect virus-specific antibodies. Traditionally counterimmunoelectrophoresis (CIEP) has been the preferred method, but in order to enable automation interest has been paid to other antibody detecting systems. Recently, at least two different ELISA systems that detect antibodies to AMDV have been manufactured; one is based on an in vitro grown AMDV as antigen, and the other system is based on the AMDV capsid protein VP2 as antigen. The aim of this study was to evaluate the two ELISA systems for detection of antibodies to AMDV using CIEP as the gold standard.

Results

When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the ELISA with the VP2 antigen had a sensitivity of 99.7% and a specificity of 98.3% compared to CIEP (n = 364). Analysis of samples with the AMDV-G antigen based ELISA employing an assay cut-off value based on the negative control samples, as suggested by the manufacturer, resulted in a sensitivity of 54.3% and a specificity of 93.2% with reference to CIEP as the gold standard (n = 359). When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the AMDV-G ELISA had a sensitivity of 37.6% and a specificity of 98.3%.

Conclusions

The ELISA system based on VP2 antigen had high sensitivity and specificity, and was concluded to be an alternative to the CIEP as a diagnostic tool for AMDV antibodies. In contrast, the AMDV-G ELISA suffered from low sensitivity when compared to CIEP.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange

Abstract The purpose of this study was to evaluate a serodiagnostic test (enzyme-linked immunosorbent assay; ELISA) for sarcoptic mange in dogs and to characterize the assay antigen, based on the mite Sarcoptes scabiei var. vulpes. The ELISA, applied to sera from 359 dogs suspected of having sarcoptic mange, showed a sensitivity and specificity of 92 and 96%, respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the antigen employed in the ELISA revealed polypeptide bands with molecular weights ranging between 14 and 164 kDa. In Western blot analyses antigens of molecular weights between 62 and 64 kDa dominated. Particularly dominant were antigens of 164 and 147 kDa. These were found to have isoelectrical points in the range of 5.7–6.9. Sera from dogs infected with Cheyletiella sp., Demodex canis, Linognathus setosus and Otodectes cynotis, as well as from dogs allergic to fleas, were negative in the ELISA. Résumé— Le but de cette étude est d'évaluer un test sérologique ELISA pour le diagnostic de la gale sarcoptique chez le chien et de caractériser l'antigène révélateur, extrait de l'acarien Sarcoptes scabiei var. vulpes. Le test ELISA, lors d'une étude conduite avec les sérums de 359 chiens suspects de gale sarcoptique a démontré une sensibilité et une spécificité de 92 et 96%, respectivement. L'électrophorèse en gel polyacrilamide dodécyl sulfate de sodium de l'antigène utilisé dans l'ELISA a révélé des bandes polypeptidiques de poids moléculaire compris entre 14 et 164 kDa. Dans l'analyse en Western blot, les antigènes de poids moléculaire compris entre 62 et 164 kDa étaient les plus abondants, notamment ceux de 164 et 147 kDa. Ces derniers ont des points isoélectriques compris entre 5.7 et 6.9. Les sérums de chiens infectés par des Cheyletiella sp. Demodex canis, Linognathus setosus et Otodectes cynotis, ou par des chiens allergiques aux puces, se sont révélés négatifs en ELISA. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Evaluation d'un test ELISA pour le diagnostic sérologique de la gale sarcoptique canine). Veterinary Dermatology 1996; 7 : 21–28.] Resumen El objetivo de este estudio fue el de evaluar una pruba serodiagnóstica (prueba de inmunoadsorción ligada a enzima; ELISA) para la sarna sarcóptica en el perro y caracterizar el antigeno prueba, basado en el ácaro Sarcoptes scabei, var. vulpes. El ELISA, aplicado a sueros de 359 perros sospechosos de padecer sarna sarcóptica, mostró una sensibilidad y especificidad del 92 y 96%, respectivamente. La electroforesis en gel de poliacrilamida dodecil sulfato sódico (SDS-PAGE) del antigeno usado en el ELISA reveló bandas de polipétidos con peso molecular entre 14 y 164 kDa. En el análisis Western blot, predominaron los antigenos de pesos moleculares entre 62 y 164 kDa. Los antigenos entre 164 y 147 kDa fueron especialmente predominantes. Estos tuvieron puntos isoeléctricos entre 5.7 y 6.9. Los sueros de perros infectados por Cheyletiella sp., Demodex canis, Linognathus setosus y Otodectes cynotis, asi como el de perros alérgicos a las pulgas fueron negativos en el ELISA. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Evaluation de una prueba de immunoadsorcion ligada a enzima (ELISA) para el diagnostico serologico de la sarna sarcóptica canina). Veterinary Dermatology 1996; 7 : 21–28.] Zusammenfassung— Ziel dieser Studie war, einen Serodiagnostiktest (Enzyme-Linked-Immunosorbent-Assay, ELISA) für Sarkoptesräude des Hundes zu überprüfen und das Testantigen zu charakterisieren, das auf der Milbe Sarcoptes scabiei var. vulpes basiert. Der ELISA-Test, der bei den Sera von 359 Hunden mit Sarkoptesverdacht angewendet wurde, zeigte eine Sensitivität von 92% bzw. 96%. Die Natriumdodecylsul-fatpolyacrylamid-Gelelektrophorese (SDS-PAGE) des Antigen, das im ELISA verwendet wurde, zeigte Polypeptid-Banden mit Molekulargewichten zwischen 14 und 164 kDa. In der Wester-blot-Analyse dominierten Antigene mit einem Molekulargewicht zwischen 62 und 164 kDa. Besonders dominierend waren Antigene von 164 und 147 kDa. Bei diesen stellte man isoelektrische Punkte im Bereich von 5,7 bis 6,9 fest. Die Sera von Hunden, die mit Cheyletiella sp., Demodex canis, Linognathus setosus und Otodectes cynotis infiziert waren, fielen ebenso wie die Hunde mit Allergie auf Flöhe im ELISA negativ aus. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Die Auswertung eines Enzym-Linked-Immunosorbent-Assay (ELISA) für die serologische Diagnose der kaninen Sarkoptesräude). Veterinary Dermatology 1996; 7 : 21–28.]     

Evaluation of enzyme-linked immunosorbent assays with recombinant antigens for the serodiagnosis of equine Babesia infections

Two enzyme-linked immunosorbent assays (ELISA) with recombinant protein as antigens were evaluated by comparison with the indirect fluorescent antibody tests (IFAT) for the detection of specific antibodies to Babesia caballi and Babesia equi, respectively in 380 sera from experimentally infected, uninfected, and field horses. The high concordances of 92.4% (351/380) and 98.2% (373/380) between ELISA and IFAT for B. caballi and B. equi, respectively suggest that ELISA, especially for B. equi infection, could be alternative to the corresponding IFAT for serodiagnoses of equine piroplasmosis, although some improvements are required in ELISA for B. caballi.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs

Canine scabies is a challenging disease to diagnose because sarcoptic mites are hard to find on skin scrapings. The purpose of this study was to evaluate a serologic enzyme-linked immunosorbent assay (ELISA) as an aid in the diagnosis of canine scabies. In addition, serum samples were obtained post treatment to determine the duration and persistence of circulating scabies antibodies after resolution of natural infection. Nineteen dogs diagnosed with sarcoptic mange and 38 control dogs were tested. Sixteen scabies-infested dogs showed positive pretreatment ELISA results (84.2% sensitivity). Thirty-four control dogs showed negative ELISA results (89.5% specificity). In the 11 scabies dogs from which multiple post treatment serum samples were obtained, detectable antibodies were not present 1 month after treatment in four cases, but were present for 1-4.5 months post treatment in seven dogs. Our results suggest that this scabies ELISA test is useful in the diagnosis of canine scabies.     

About the presence of a thermolabile (TL) antigenic fraction in Leptospira interrogans

Antisera prepared with living serovars of Leptospira interrogans have a fraction anti-TL able to neutralize TL antigen of Leptospira strains. After neutralization, any leptospiral serovar is able to adsorb heterologous antisera prepared with living strains.     

Use of an enzyme-linked immunosorbent assay in a bovine brucellosis eradication program

An enzyme-linked immunosorbent assay (ELISA) was developed and was compared with the complement fixation test (CFT) in a bovine brucellosis eradication program. The ELISA detected significantly more reactors than the CFT in both strain 19 vaccinated infected herds (1.79% versus 1.14%) and non-vaccinated infected herds (4.2% versus 3.59%) but not in either vaccinated or non-vaccinated brucella-free herds. The specificity for both tests in brucella-free herds was greater than 0.998. The specificity and sensitivity of the ELISA were compared with those of 3 other tests (the Rose Bengal test; the indirect haemolysis test [IHLT] and the CFT) on serum from 151 animals cultured at slaughter. The calculated specificity of the ELISA in this infected group was lower than for both the CFT and the IHLT (0.58 versus 0.67 versus 0.75). The sensitivity however was much greater (1.0 versus 0.73 versus 0.71). The value of the ELISA when used in an eradication program is discussed.     

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R² = 0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively.In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n = 253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.     

Evaluation of a recombinant enzyme-linked immunosorbent assay for detecting Chlamydophila psittaci antibodies in turkey sera

Chlamydophila psittaci (formerly Chlamydia psittaci) is one of the major pathogens associated with turkey respiratory disease. Devastating outbreaks with high mortality rates, similar to those of 1950 to 1970 in the USA occasionally occur, but respiratory signs without or with low mortality mostly characterize outbreaks now a day. Accurate diagnostic methods should be made available. The present study examined the sensitivity and specificity of a recombinant ELISA (rMOMP ELISA) for detecting Cp. psittaci major outer membrane specific antibodies in turkey sera. Test results were compared to those of immunoblotting and of a competitive ELISA (Chlamydia-psittaci-AK-EIA, R?hm Pharma, Germany) and an indirect ELISA (LPS/LGP) detecting antibodies to the lipopolysaccharide/lipoglycoprotein complex. The rMOMP ELISA was most sensitive as determined on serial dilutions of positive control sera originating from experimentally infected SPF turkeys. The competitive ELISA gave false positives since three negative controls reacted positive. For conventional sera, the sensitivities of the competitive ELISA, immunoblotting and the indirect ELISA were found to be 99.4, 93.1 and 82.2%, respectively, as compared to the rMOMP ELISA (100%). The specificities of the rMOMP ELISA, immunoblotting and the indirect ELISA were found to be 100% while the specificity of the competitive ELISA was only 2.7%. The rMOMP ELISA was chosen to compare the prevalence of chlamydiosis in 2002 with the one from 1992. In 2002, 188 on 200 (94%) turkey sera reacted positive compared to 175 on 200 (87.5%) in 1992 and like 10 years ago all examined farms were seropositive at slaughter. Interestingly, Belgian as well as French farms were seropositive.     

恩诺沙星在鸡肉组织中残留的ELISA检测方法研究

分别用N-羟基琥珀酰亚胺法和氯甲酸异丁酯法把恩诺沙星(ENR)与载体蛋白牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联制备免疫抗原和包被抗原,免疫新西兰大白兔得到ENR的多克隆抗体,建立了ENR间接竞争ELISA方法。结果表明:抗ENR血清效价达达1∶212以上,得到标准曲线的线性回归方程为Y=-0.2341X+0.1193(R2=0.9878),中值(IC50)为36 ng/mL,最低检测限(LOD)为10 ng/mL,标准曲线的线性范围为10~1000 ng/mL。批内变异系数为3.18%~7.64%,批间变异系数为9.69%~11.94%,鸡组织中的ENR的回收率为76.5%~89.42%。本试验建立的ELISA方法能够满足恩诺沙星兽药残留检测要求。     

孔雀石绿间接竞争ELISA检测方法的建立

采用无色孔雀石绿(Leucomalachite green,LMG)单克隆抗体建立了水产品中孔雀石绿(Malachite green,MG)残留的间接竞争ELISA(ciELISA)检测方法。结果表明,LMG-McAb最佳稀释倍数为1∶80 000,包被抗原最佳质量浓度为0.80μg/mL;竞争反应时的LMG理想稀释液为40%乙腈水溶液;标准曲线呈线性相关,相关系数R2=0.9823,最适检测范围1 ng/mL~256 ng/mL,最低检测限为1.29 ng/mL,批内和批间变异系数分别为4.307%和4.566%;鳗鱼肉样的平均添加回收率为90%~110%;该检测方法与隐性结晶紫、孔雀石绿、结晶紫的交叉反应(CR%)分别为40.67%、13.50%和5.89%,与其他抗生素无交叉反应。