Chemiluminescent immunoassay in comparison with the indirect ELISA as reference method for detecting Salmonella antibodies in swine meat juice

The efficiency of chemiluminescent immunoassay (CLIA) in detecting Salmonella antibodies in the meat juice of slaughter swine was compared with the indirect ELISA (BgVV method). Based on the screening test results of 987 meat juice samples obtained from different laboratories in Germany, a good level of agreement between the two systems was obtained with a kappa value of 0.824 at 20% cut-off and 0.798 at 40% cut-off. At 20% and 40% cut-off levels, a sensitivity of 96.2% and 97.3%, respectively, and a specificity of 94.6% and 95.1%, respectively, were demonstrated between CLIA and ELISA. The detecting LPS antigen was tested for specificity and a cross-reaction with two E. coli and Yersinia strains was found when tested with ELISA. This reaction was not observed in CLIA, possibly because of the broader measurement spectrum of this test which allows a more distinctive definition of immunologic reactions. The same explanation can be given for the increased number of meat juice samples which were positively detected only in ELISA but not in CLIA. The positively classified samples in screening were further tested for reciprocal titers in both test systems, and a higher correlation between screening and titration results was obtained for CLIA. Towards the end of the study, a preliminary comparison of CLIA with two available commercial ELISA test kits was conducted and the same tendency was observed, namely, wider detection range of CLIA compared to the other tests. Based on the results of this study, CLIA can be used as a reference method in detecting Salmonella antibodies in the meat juice of slaughter pigs.     

Control panels of meat juice samples for a Salmonella enzyme-linked immunosorbent assay.

In the Danish pig production system, an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies in meat juice is used for Salmonella surveillance. Quality control (QC) of this ELISA was previously based on repeated testing of control serum samples. The purpose of the study reported here was to collect, characterize, and implement a panel of meat juice pools for supplemental internal QC. Muscle samples for extraction of meat juice were collected from slaughter pigs of 5 herds infected with Salmonella spp. and from 4 herds without Salmonella infection. A QC panel with 39 pools of meat juice, yielding ELISA optical density (OD) values covering the full range of expected OD values, was prepared and tested repeatedly to determine mean and SD OD values. Each pool was tested twice on each microtitration plate, and the results were used to determine limits for validity of future tests. This QC panel was included as an internal QC to be tested every month. Besides the QC panel, 2 panels containing 100 samples of meat juice with OD above the positive cut-off value and 100 samples with OD below that value were prepared for quarterly control of the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of the ELISA. The inclusion of these panels in the QC system will provide information about drifts in DSe and DSp of the test. The procedures described here can be applied to other tests where meat juice samples are used for testing.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

Development and Bayesian evaluation of an ELISA to detect specific antibodies to Sarcoptes scabiei var suis in the meat juice of pigs

Samples of ear scrapings, serum and diaphragmatic muscle were collected from 271 fattening pigs at the slaughterhouse. The scrapings were examined for the presence of mites, and tests for specific antibodies to Sarcoptes scabiei var suis in the serum and meat juice were made with an experimental ELISA. The cut-off value for the meat-juice ELISA was estimated at an optical density of 0.5 by receiver operating characteristic curve analysis, on the basis of the cut-off value for the serum ELISA of 0.4. The results of the three tests were used in a Bayesian model to estimate the characteristics of each test. The specificity of the tests of the ear scrapings was considered to be 1 and their sensitivity was estimated by Bayesian analysis to be 0.86, with a 95 per cent confidence interval (CI) of 0.73 to 0.99. The sensitivity of the meat juice ELISA (0.71, 95 per cent CI 0.6 to 0.8) and its specificity (0.77, 95 per cent CI 0.66 to 0.89) were comparable with the sensitivity (0.73, 95 per cent CI 0.6 to 0.8) and specificity (0.81, 95 per cent CI 0.69 to 0.95) of the serum ELISA.     

Evaluation of ELISA for detection of Trichinella antibodies in muscle juice samples of naturally infected pigs

The performance characteristics of an ELISA test for trichinellosis in pigs applied to muscle juice was assessed using 314 samples collected from pigs located in endemic areas of Croatia. Peptic digestion was used as the reference method. The diagnostic accuracy of the two compared dilutions (1:10 and 1:100) was considered to be high because the area under the curve (AUC) index was 0.922 and 0.920 for each dilution, respectively. In this study the two graph-receiver operating characteristic (TG-ROC) analysis was used as a tool for selecting cut-off points. Sensitivity, specificity, likelihood ratios, efficiency and Youden's index were used as indices of test accuracy. The cut-off values that minimize overall misclassification cost under an assumption of 3% prevalence were calculated. Our results indicate that the ELISA applied to muscle juice is a highly accurate test and can be adapted to process a large number of samples.     

Comparing the results of the serological detection of Salmonella antibodies in blood serum and meat juice from different muscles from slaughter pigs

The German Salmonella Monitoring Programme started by the QS-System in 2002 (Blaha, 2004) is mandatory due to the so-called "Salmonella Regulation for Pigs" since 2007 (Anonym, 2007). The Regulation does not clearly prescribe the specific muscle which is to be taken as source of the meat juice. Thus, at different slaughter plants meat samples are also taken from different muscles and several scientific papers describe various muscles as source of the meat juice, too. The objective of this study was to compare the serological results of meat juices from three different locations (diaphragm pillar, neck, belly muscle) to each other and to those of the blood serum from exactly the same animals. All samples were simultaneously tested for Salmonella antibodies by two serological tests (Salmo-type Pig Screen, LDL, Germany; HerdChek Swine Salmonella, IDEXX, Germany). Comparisons were carried out between the various sample kinds per animal and between the two test systems. The analysis of all results of the meat juices revealed in both test systems a clear decline of the OD% values from the diaphragm pillar to the neck to the belly muscle. The average OD% values of all samples were higher when measured by the HerdChek ELISA (IDEXX, Germany) than by the Salmotype ELISA (LDL, Germany), especially in blood serum. Since the results of the meat juice samples gained from the diaphragm pillar were in both test systems by far the closest to the results of the corresponding serum blood samples, it is recommended to amend the "Salmonella Regulation for Pigs" by prescribing meat from the diaphragm pillar as the only muscle for gaining meat juice.     

Diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in individual milk and bulk milk samples of dairy herds

The objective of the study was to determine the diagnostic performance of the Pourquier ELISA for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Map) in individual milk samples and in bulk milk samples. For individual milk samples the specificity of the Pourquier ELISA was estimated by testing a panel of individual milk samples from certified Map-free herds. The relative sensitivity of the assay in individual milk samples and agreement of the results with those of serum samples was estimated by testing panels of paired serum-milk samples from seropositive cattle, whole-herd investigations, and moderate or heavy shedders. The specificity of the ELISA for individual milk samples was still 99.8% at a cut-off of 20% sample to positive (S/P) value, clearly lower than the cut-off defined by the manufacturer (30% S/P). The relative sensitivity for individual milk samples as compared with positive serum samples was 87% for a cut-off of 20% S/P, and 80% for a cut-off of 30% S/P. The sensitivity of this ELISA for detection of high shedders was >90% both for individual milk and serum samples, also agreement was very good (kappa=0.91 for all paired samples). The specificity of the Pourquier ELISA in bulk milk samples was investigated by testing bulk milk samples from certified Map-free herds. Feasibility of bulk milk testing was investigated by titrating ELISA positive individual milk samples in negative milk. In addition, 383 bulk milk samples from herds with a known within-herd seroprevalence were tested. The specificity of the ELISA for bulk milk samples was 100% at a cut-off of 12.5% S/P. At the cut-off recommended by the manufacturer (30% S/P) performance of the bulk milk ELISA related to herd status (> or =2 seropositive cows) was rather poor, corresponding with a sensitivity of 24% and a specificity of 99% relative to serology. However, at the revised cut-off for bulk milk of 12.5% S/P and a within-herd seroprevalence of > or =3%, sensitivity and specificity relative to serology were 85% and 96%, respectively. Given the current herd-level seroprevalence in The Netherlands, these test characteristics corresponded with positive and negative predictive values for bulk milk of 67% and 94%, respectively. In conclusion, the diagnostic performance of the Pourquier ELISA for individual milk samples creates opportunities for a cheaper and more feasible testing scheme, while the diagnostic performance for bulk milk samples warrants further consideration.     

The suitability of ELISA for the detection of antibodies against Mycobacterium avium ssp. paratuberculosis in bulk milk samples from Rhineland-Palatinate

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached.The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels.The estimated within-herd seroprevalences ranged between 0 % and 37 %.There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146.To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing.Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.     

Necessity of double testing of meat juice samples from pigs using ELISA for the determination of Salmonella status in pig farms]

According to the test protocol of the "meat juice ELISA" for detection of salmonella antibodies in pigs, all meat juice samples and serum controls are to be tested in duplicate. Results from routine investigations of repeatedly double tested meat juice and serum samples have been used to analyze the effect of double testing versus single testing with regard to the reliability of the final result. In case of an individual animal, testing of samples in duplicate increases the reliability of the results significantly, especially, if samples are retested at different occasions. In contrast, such a difference between mono and double testing of samples is not of importance when a group of animals is tested in order to determine the mean antibody rate in a herd. Here, results from double testing practically do not contribute to a higher reliability of the final result. This observation provides the possibility to reduce the costs for investigation programmes drastically.     

Monitoring porcine reproductive and respiratory syndrome virus infection status in swine herds based on analysis of antibodies in meat juice samples

An indirect ELISA test was developed as a novel tool aimed at monitoring the herd infection status of swine herds. Meat juice samples from pig carcasses were analysed for the presence of antibodies against porcine reproductive and respiratory syndrome virus (PRRSV). A study of samples from herds with known PRRS status was undertaken. The PRRS status of the herds was evaluated based on the analysis of blood samples by another serological test (blocking ELISA) capable of differentiating between infection with PRRSV of the American type and European type. The specificity of the indirect ELISA test on meat juice samples was 0.98. The sensitivity of the test depended on the type of the PRRSV strain involved. The apparent prevalence in herds infected with the American type of PRRSV was 0.44. The apparent prevalence in herds infected with the European type of PRRSV was 0.64. Herd level sampling and herd level criteria for assessing the PRRS status of herds by the new test were developed. Herds were classified as PRRS negative or PRRS seropositive based on 10 meat juice samples collected randomly at slaughter throughout a 3-month-period. Herd PRRS status classification by the indirect ELISA was validated in 47 herds by collection of blood samples from the herds. Eighteen herds were classified as PRRS negative by both test systems. Twenty-nine herds were classified as PRRS seropositive by both test systems. Acceptable herd classification was achieved using this test.     

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.     

Dual-label time-resolved fluoroimmunoassay for simultaneous quantification of haptoglobin and C-reactive protein in meat juice from pigs

A new method was developed to simultaneously measure 2 acute-phase proteins (APPs) by time-resolved immunofluorometry. The assay, based on double-label quantification of haptoglobin (Hp) and C-reactive protein (CRP) in meat juice samples from pigs, was constructed by use of a combination of europium and samarium chelate lanthanides as labels. Meat juice samples from 154 pigs were used for analytic and clinical validation of the assay through determination of precision, accuracy, limit of detection, and quantification. The analytic performance of the assay was satisfactory, with good intra-assay and interassay precision and accuracy. The levels of Hp and CRP were increased in the meat juice samples of diseased animals compared with healthy ones. According to the results, higher sensitivity could be achieved if the cut-off values of both proteins were taken into account for clinical relevance rather than used individually. Since the dual assay saved both time and sample, it could be used as a rapid and sensitive screening test in porcine production.     

Evaluation of ELISA for the detection of Toxoplasma antibodies in swine sera

Enzyme-linked immunosorbent assay (ELISA) is compared with the indirect fluorescent antibody test (IFAT), the indirect haemagglutination test (IHAT) and the latex agglutination (LA) test for the detection of toxoplasma antibodies in swine sera. The 100 swine sera examined represent ELISA values from greater than 0 to 154 EIU. The agreement was highest (0.67) between ELISA and IFAT with an ELISA cut-off value of 30 EIU, and between ELISA and the LA test with an ELISA cut-off value of 50 EIU (0.74). All sera giving less than 10 EIU were negative in the other tests, and all those with greater than 70 EIU were positive in 1, 2 or all of the reference tests. In order to avoid false positive results with ELISA, all sera giving 10-70 EIU should be confirmed with a test which has a good specificity, e.g. IFAT. ELISA is a sensitive test and is highly suitable for the screening of large amounts of samples, but it may be too complicated for screening toxoplasma antibodies in the laboratories of abattoirs.     

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/T. evansi), an indirect card agglutination test (LATEX/T. evansi) and a newly developed antibody detection ELISA (ELISA/T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals (n=8) were positive in all tests. Diagnostic specificity was highest in CATT/T. evansi (98%) followed by the ELISA/T. evansi (95%) and the LATEX/T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1-8%) false positive results were actually due to a specific reactions, and that LATEX/T. evansi and ELISA/T. evansi detected more immune trypanolysis positives than the CATT/T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/T. evansi with a 30% positivity cut-off and LATEX/T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/T. evansi with a 50% positivity cut-off and the CATT/T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.     

Detection of antibodies to S. enteritidis in broilers by means of indirect ELISA and chemiluminescent immunoassay (CLIA)

This study was conducted to develop a serological detection system for the monitoring of broiler flocks for Salmonella enteritidis infections. A specific S. enteritidis antigen (FG-Antigen) was used to compare the sensitivity and the specificity of the chemiluminescent immunoassay (CLIA) with those of the indirect ELISA. This comparison was performed using a total of 578 sera, which, depending on the microbiological and vaccination history, were categorized into groups. Most of the serum samples which were classified as positive showed higher titers in CLIA than in ELISA. Using the prevalence of positive reactors, significant differences between Groups were additionally demonstrated. The absorbance values of the passively immunized group showed the highest and those of the Salmonella-negative group the lowest correlation-coefficient. Using the mean net absorbance of the prevalence group, the ELISA system exhibited a sensitivity of 100% and a specificity of 96.2%, while CLIA had a sensitivity and a specificity of 85.7% and 96.2%, respectively. ELISA and CLIA can be used in the examination of non vaccinated flocks for S. enteritidis-infections as alternative to the bacteriological culture method. CLIA is distinguished for its fast and convenient procedure as well as for its wider measurement spectrum, while the indirect ELISA is almost as efficient as CLIA and requires less investment.     

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.     

Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA

Surveillance of porcine reproductive and respiratory syndrome (PRRS) in negative sow farms is usually performed by testing for the presence of antibodies against PRRS virus in serum with a commercial ELISA test. The objective of this study was to evaluate the feasibility of pooling serum samples for detection of PRRS virus antibodies by ELISA. The effect of pool size on the sensitivity and specificity of the ELISA test was evaluated by testing true positive samples and false positive samples, respectively, diluted in negative sera. The effect of three different cut-off values for the interpretation of the diagnostic test (0.4, 0.3 and 0.2) was evaluated as well. Furthermore, the obtained sensitivity and specificity estimates were used to calculate the herd sensitivity and herd specificity of surveillance protocols in different scenarios. The results showed that pooling serum samples to detect PRRSV antibodies resulted in a decrease in sensitivity and an increase in specificity, compared to testing individual samples, while the reduction of the s/p cut-off value recommended by the manufacturer (0.4) had the opposite effect. We describe an approach that can increase the herd sensitivity of a surveillance protocol for breeding herds, while maintaining high herd specificity and low testing costs. This can be achieved by sampling a larger number of animals and running the samples in pools. Therefore, the conventional monitoring protocols based on ELISA on individual samples can be improved by using pooled-sample testing.     

Adaptation of a surface antigen-based ELISA for the detection of antibodies against Neospora caninum in bovine milk

The aim of the present study was to determine whether an ELISA based on a 38-kDa surface antigen (NcSRS2) of Neospora caninum tachyzoites could be used to examine bovine milk for antibodies against N. caninum. A total of 797 undiluted milk samples from N. caninum-infected herds were examined in the p38-ELISA. Milk results of individual animals were compared with those obtained by the same ELISA for the corresponding serum samples. The linear correlation between milk and serum antibody results of individual animals was characterized by an adjusted R(2) of 0.644. The examination of the two-graph receiver-operating characteristics revealed an optimal cut-off of 0.150 to obtain similar results in the examination of milk and serum. With this cut-off, the test had a specificity and a sensitivity relative to the serum results of 93%. Using this cut-off, excellent agreement was observed between milk and serum results (Kappa 0.85; 95% CI, 0.781-0.920). A cut-off of 0.450 resulted in a specificity of 99% relative to the serum results. At this cut-off, the sensitivity of the test was 80% relative to the serum-ELISA and agreement was slightly lower (Kappa = 0.82; 95%CI, 0.749-0.887). An optimized linear regression model suggests that, in addition to the result in the p38-serum-ELISA, the variables abortion risk (ABRISK) (abortion between 100 and 269 days pregnant) and the age of the animal (AGE) affected the result of the p38-milk-ELISA. The optimized linear regression model had an adjusted R(2) of 0.8501.     

非洲猪瘟病毒p30基因的原核表达及间接ELISA抗体检测方法的建立

为建立检测血清中非洲猪瘟病毒（African swine fever virus,ASFV）抗体的间接ELISA方法,本试验将ASFV p30基因进行原核表达,采用SDS-PAGE和Western blotting方法对重组蛋白进行表达鉴定和免疫原性分析,随后以纯化的重组蛋白为包被抗原,经条件优化、特异性试验、敏感性试验和重复性试验,建立一种血清中ASFV抗体的检测方法。结果显示,ASFV p30基因成功克隆到原核表达载体pET-32a （+）中,获得pET-32a-p30重组质粒;转化大肠杆菌BL21（DE3）感受态细胞进行诱导表达,得到P30重组蛋白,重组蛋白大小约为42 ku,主要以包涵体形式存在;Western blotting结果显示,纯化后的蛋白具有良好的免疫原性;以纯化的P30重组蛋白为包被抗原,建立了检测ASFV抗体的间接ELISA方法,通过方阵试验对间接ELISA方法进行优化,最终确定了抗原最佳包被浓度为1.2 μg/mL,待检血清最佳稀释倍数为1：100,最佳封闭液为1% BSA,酶标抗体最佳稀释度为1：4 000,以此建立的ASFV间接ELISA方法临界值为0.322。本方法仅与ASFV阳性血清发生特异性反应,与猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒、伪狂犬病病毒、猪圆环病毒2型及猪流行性腹泻病毒阳性血清均无交叉反应,具有较强的特异性。该方法检测ASFV阳性血清灵敏度可达到1：1 600;批内重复性和批间重复性变异系数均<10%。本试验建立的间接ELISA方法具有良好的特异性、灵敏度和重复性,可初步应用于ASFV抗体的检测。     

Relationship of the enzyme-linked immunosorbent assay to indirect immunofluorescent antibody test for the detection of so-called chicken anemia agent antibodies in serum from broiler breeders.

Serum samples collected from breeder chickens ranging in age from 1 day to 55 weeks were tested for CAA antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA) test. The relationship of ELISA to IFA test was determined. The sensitivity of the ELISA relative to the IFA test was 82.64%, and the specificity of the ELISA relative to the IFA test was 56.25%. Agreement between the ELISA and the IFA test was highly significant (Kappa = 0.74, Z = 5.78). We concluded that the ELISA is as good as the IFA test for detecting CAA antibody in sera from chickens.