Evaluation of Competitive ELISA for Detection of Antibodies to Brucella Infection in Domestic Animals

Aim

To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis.

Methods

cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested.

Results

Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzyme-linked immunosorbent assay, and CFT were greater than 90%.

Conclusion

cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis.In accordance with EC Directive 91/68/EEC, flocks of sheep and herds of goats in the United Kingdom (UK)are monitored serologically to prove that they are free from Brucella melitensis. In 2006, competitive enzyme-linked immunosorbent assay (cELISA) was introduced to screen these animals as part of a surveillance program in Great Britain (GB), the territory including all of the UK except for Northern Ireland. It replaced the complement fixation test (CFT) because of its much higher specificity and ease of automation. Currently, in excess of 35 000 animals are tested annually.In 2001, a revision to the pig semen directive was introduced by EC Directive 99/608 so that CFT was replaced with the Rose Bengal test (RBT) as the test used for brucellosis on all pigs whose semen is used for artificial insemination. RBT and CFT were run in parallel in addition to cELISA prior to this date in order to assess the effects of changing the testing regime and, at the same time, to validate the use of cELISA for pigs. During 2001, all routine samples that were tested for artificial insemination purposes and were positive by RBT were also tested by cELISA and the results analyzed using different diagnostic thresholds. The aim was to set an appropriate threshold that would provide optimal specificity and sensitivity for cELISA.CFT, RBT, and indirect enzyme linked immunosorbent assay (iELISA) are the conventionally used tests for diagnosis of bovine brucellosis. These tests are described in the Manual for Diagnostic Tests and Vaccines for Terrestrial Animals produced by the World Organisation for Animal Health, previously the Office International des Epizooties (OIE) (1), and this manual gives details of all the diagnostic methods. It also describes the strain of Brucella required for antigen preparation and the procedure for standardization for each test.The cELISA for the detection of antibodies against Brucella spp. was adapted at the Veterinary Laboratories Agency (VLA) from the method described by MacMillan et al in 1990 (2). It was initially developed for the diagnosis of brucellosis in small ruminants and was tested extensively on British sheep and on sheep and goats from France. It has also since been tested on large numbers of cattle and pigs.The aim of this study was to bring together and compare all brucellosis testing results carried out using cELISA, RBT, and iELISA at the VLA since 1991. The samples had been collected and analyzed within the framework of various surveillance screening programs and experimental studies. The present study demonstrates the effectiveness of cELISA compared with other assays currently used as diagnostic tests of brucellosis in domestic animals.     

Assessment of Two Immunoassays for Detection of IgM Antibodies to Scrub Typhus Using a Serum Panel

Laboratory tests are necessary for diagnosis of scrub typhus (ST) especially in the absence of the distinctive eschar. Performance of an ELISA and ICT (immunochromatography) to detect IgM antibodies to scrub typhus was assessed using a panel of 346 sera chosen from healthy individuals, those with scrub typhus and scrub-typhus like illness. A sensitivity of 98.7% for ST IgM ICT and 97.4% for ST IgM ELISA was observed while specificity was 96.3% for ICT and 95.9% for ELISA. As excellent concordance (98.8%) was noted between the two assays, IgM ICT can be used for rapid diagnosis of scrub typhus.     

Analysis of Complement Fixation and Commercial Enzyme Immunoassays for Detection of Antibodies to Mycoplasma pneumoniae in Human Serum

The Meridian ImmunoCard (IC), GenBio ImmunoWELL-IgM, and Remel EIA commercial antibody tests are qualitative enzyme immunoassays that detect antibodies to Mycoplasma pneumoniae in serum. These tests were compared to an M. pneumoniae complement fixation (CF) assay, which uses a commercially available antigen component. The Meridian IC and the ImmunoWELL-IgM detect immunoglobulin M (IgM) only; the Remel EIA and the CF test detect both IgM and IgG antibodies. Detection of specific IgM antibody, which appears early in infection, can be, but is not always, indicative of a recent or current infection. Paired serum samples from 64 adult patients with probable M. pneumoniae infection were examined with each of the four tests. Thirty (47%) of the 64 acute-phase sera were IgM positive by Meridian IC, 26 (41%) were positive by Remel EIA, 24 (38%) were positive by CF, and 15 (23%) were positive by ImmunoWELL-IgM. When both the acute- and convalescent-phase serum samples from each patient were examined, 61 (95%) of the 64 patients were positive by CF, 60 patients (94%) were positive by Remel EIA, 52 patients (81%) were IgM positive by the Meridian IC, and 29 patients (45%) were IgM positive by the ImmunoWELL-IgM assay. The Meridian IC was more sensitive than the other tests for early detection of IgM antibodies. However, after examining paired serum samples, we concluded that the detection of IgM alone may not be useful for all cases of mycoplasma infection, especially in an adult population.     

Comparison of Three Immunoassays for Detection of Antibodies to Strongyloides stercoralis

Due to the limited sensitivities of stool-based microscopy and/or culture techniques for Strongyloides stercoralis, the detection of antibodies to this intestinal nematode is relied upon as a surrogate for determining exposure status or making a diagnosis of S. stercoralis infection. Here, we evaluated three immunoassays, including the recently released InBios Strongy Detect IgG enzyme-linked immunosorbent assay (ELISA) (InBios International, Inc., Seattle, WA), the SciMedx Strongyloides serology microwell ELISA (SciMedx Corporation, Denville, NJ), and the luciferase immunoprecipitation system (LIPS) assay performed at the National Institutes of Health (NIH), for their detection of IgG antibodies to S. stercoralis. A total of 101 retrospective serum samples, previously submitted for routine S. stercoralis antibody detection using the SciMedx assay, were also evaluated by the InBios and LIPS assays. The qualitative results from each assay were compared using a Venn diagram analysis, to the consensus result among the three assays, and each ELISA was also evaluated using the LIPS assay as the reference standard. By Venn diagram analysis, 65% (66/101) of the samples demonstrated perfect agreement by all three assays. Also, the numbers of samples considered positive or negative by a single method were similar. Compared to the consensus result, the overall percent agreement of the InBios, SciMedx, and LIPS assays were comparable at 87.1%, 84.2%, and 89.1%, respectively. Finally, the two ELISAs performed analogously but demonstrated only moderate agreement (kappa coefficient for the two assays, 0.53) with the LIPS assay. Collectively, while the two commercially available ELISAs perform equivalently, neither should be used independently of clinical evaluation to diagnose strongyloidiasis.     

Diagnostic Accuracy of Immunoassays for the Detection of Antibodies to Citrullinated Proteins

Anticitrullinated protein/peptide antibodies (ACPA) are highly specific for rheumatoid arthritis (RA). They can be found early in the disease course and are associated with more severe joint destruction and disease activity. In the last 4 years, important progress has been made in the detection and identification of ACPA, improving antigenic composition and epitope recognition. Consequently, many ACPA-ELISA kits have been developed by several manufacturers and are now commercially available. However, albeit their widespread use in clinical laboratories, the use of some kits has not been accompanied by a clinical validation nor by a comparative evaluation of their diagnostic accuracy. In addition, full automation of ACPA assays featuring ease of use, rapid response, and high productivity is just beginning to appear on the market and also deserves clinical and analytical validation. This review will consider the most relevant characteristics of the ACPA-ELISA assays and will describe the results of a comparative study performed with all the currently available second- and third-generation commercial methods.     

Evaluation of Six Immunoassays for Detection of Dengue Virus-Specific Immunoglobulin M and G Antibodies

The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis.     

Evaluation of Three Immunoassays Used for Detection of Anti-Rubella Virus Immunoglobulin M Antibodies

Three automated assays (Abbott AxSYM, Bayer ADVIA Centaur, and bioMerieux VIDAS) used for the detection of rubella virus-specific immunoglobulin M were evaluated. A total of 57 samples from individuals with evidence of infection with rubella virus were used to estimate sensitivity, and 220 samples from blood donors and individuals attending an antenatal clinic who had no evidence of recent infection were used to estimate specificity. Seroconversion panels comprising an additional 31 samples from four individuals were used to determine clinical sensitivity. Samples containing potentially cross-reacting substances were also tested. The sensitivities of the three assays ranged from 84.2 to 96.5%, and the specificities ranged from 96.8 to 99.9%. The Abbott AxSYM assay detected more reactive samples than the other two assays when a panel of 57 positive samples was tested. Bayer ADVIA Centaur detected more reactive samples in the seroconversion panels than the other two assays. All three assays evaluated reported a reactive result in 1 or more of the 48 samples containing potentially cross-reacting analytes. The assays demonstrated comparable performance in testing of a well-characterized panel of samples.     

血清C肽免疫检测方法的对比分析及临床意义探讨

采用四种常规免疫检测方法同时检测血清C肽,分析检测方法间的差异性并研究检测差异对临床判断胰岛β细胞功能的影响。随机选取病人51例152份血清样本,其中正常糖耐量13例,空腹血糖受损8例,糖耐量减低9例及2型糖尿病21例。免疫检测方法包括放射免疫法一种、化学发光法两种和电化学发光法一种。四种方法检测血清C肽的结果相互之间总体相关系数在0.74~0.99之间;检测数值间差异均显著（P〈0.0001）。在监测血清C肽的变化节律方面,四种免疫检测的变化趋向性表现一致,对临床判断无显著差异。     

Antibodies to Spermatozoa: XI. The Use of Immunobeads for the Detection of Sperm Antibodies in Serum

ABSTRACT: Two procedures were developed and evaluated that used either larger or smaller volumes for the detection of sperm antibodies in serum by means of an indirect immunobead test (IBT). The immunobeads, coated with rabbit antibody to each of the major human immunoglobulins (IgG, IgA, or IgM), were mixed with preparations of donor sperm, onto which antibody had been coated by passive transfer from various serum samples. The results of the IBT could be evaluated in various ways: (1) positive or negative; (2) if positive, whether binding is to the tail, the head, or the head and tail of the sperm cells; (3) if positive, whether binding is by IgG, IgA, or IgM. The diverse IBT results were obtained from a group of 50 serum samples; these sera were also tested by two sperm agglutination methods; the gelatin agglutination test (GAT) and the tube-slide agglutination test (TSAT). There was an excellent agreement between the IBT and the GAT; it was not as good between the IBT and the TSAT. However, considering both agglutination methods together, 90% of the IBT-positive sera were agglutination-positive. In terms of morphological sites, tail binding occurred in 27 of 31 sera, head binding in 12 of 31 sera, and head-tail binding in 15 of 31 sera. The number with tail binding was very close to the number that were GAT-positive (26). As for the immunoglobulins, the most frequent class was IgG. IgA was 83% as frequent and IgM was only 25% as frequent as IgG. In a larger group with only IgG and IgA, of 31 IBT-positive sera, 26 showed IgG and 23 showed IgA; 18 showed both. Hence, only eight showed IgG exclusively, and only five showed IgA exclusively. One final point is that several sera with GAT titers of only 4 were IBT-positive, adding strength to the concept that such a low GAT titer does have antibody significance.     

Application of Mouse Antibodies to Somatic Antigen for Detection of Salmonella enteritidis by Competitive ELISA

Competitive ELISA estimation based on application of polyclonal mouse antibodies to somatic antigen O:9, 12 was developed. The optimization of the protocol is reported. The optimal concentration of immobilized somatic antigen O:9, 12 was found to be 4.9 ×10⁴ cells ml^-1; optimal concentration of mouse IgG was 6.25 μg ml^-1; and the optimal concentration of peroxidase labelled antibody to mouse IgG was 8 μg ml^-1. The tested antibody exhibited neither cross reactions with chosen strains (serotypes) of salmonellas group 04 (B), 07 (C₁), 08 (C₂-C₃), nor with members of Enterobacteriaceae: Escherichia coli, Klebsiella pneumonia, Citrobacter freundii and non-fermenting bacterium Pseudomonas fluorescens. Application of chemiluminiscent substrates increased the sensitivity of S. enteritidis detection up to three times. Competitive ELISA tested on model samples produced results comparable with standard cultivation techniques for Salmonella spp.     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

Development of a Polyclonal Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ehrlichia ruminantium

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater.     

Rapid Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Peste des Petits Ruminants Virus

Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be <50 on the basis of the mean PI plus 3 standard deviations for sera from PPRV-negative ruminants. The relative specificity and sensitivity of the rapid c-ELISA were 98.5% (722 of 733 serum samples) and 93.4% (234 of 249 serum samples), respectively. The rapid c-ELISA sensitively detected PPRV antibodies in hyperimmune sera (virus neutralization test [VNT] titer, >512), even at dilutions ≥512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was ≤128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field.     

Identification of Brucella abortus Antibodies in Cattle Serum by Single Radial Diffusion

Single radial diffusion combined with the Rose Bengal test permitted rapid identification of all of the Brucella abortus-infected cattle in a study group of 689 animals.     

六种TSH检测方法的对比研究

采用六种免疫检测方法检测血清促甲状腺激素（TSH）,分析检测方法间的差异性和相关性及对临床诊断的影响。纳入研究的样品150份,甲亢、甲减和表面健康人各50份。免疫检测方法包括一种放射免疫分析法（RIA）,一种免疫放射分析法（MRIA）,四种自动化检测方法（三种化学发光免疫分析法和一种电化学发光免疫分析法）。结果表明,六种免疫方法在检测血清TSH时相互之间存在不同程度差异（P〈0．05）,其中RIA法在甲亢组和表面健康组的检测值与其他方法的检测值间均不具有相关性（P〉0．05）;IRMA法在甲亢组的检测值与四种自动化检测法的检测值相关系数较低（r为0．38—0．41）;四种自动化检测法两两之间的检测值问均高度相关（r为0．92—0．99）。在临床诊断符合率方面,四种自动化检测法相互间无显著差异;IRMA法在甲亢组同四种自动化检测法有显著差异（P〈0．01）,而RIA法不能将甲亢病人同表面健康组区分,不宜用于甲亢诊断。     

胶体金法和TPPA法检测血清梅毒抗体的比较与分析

目的 通过梅毒螺旋体明胶凝集试验（TPPA）和胶体金（Colloidal Gold,TPAb）检测血清梅毒抗体的比较,探讨胶体金法和TPPA法检测血清梅毒螺旋体抗体结果之间的关系.方法 通过对本院首次用TPPA法检测阳性后的血清标本,再用TPAb法复查,共616例,以及急诊术前和腔镜检查前用TPAb法筛查出阳性的血清标本,再用TPPA法复查,共88例,并对7份两种方法共同阳性梅毒抗体血清用阴性血清进行倍比稀释,对稀释后的同一份血清分别用TPPA法和TPAb法进行定性检测,以比较两种方法灵敏度.结果 TPPA法检出616例阳性,用TPAb法复查612例阳,符合度99.35％;TPAb法共检测出88例阳性,用TPPA法复查77例阳性,符合度87.5％.结论 稀释实验表明TPAb法灵敏度比TPPA法更高,能检测出更低的下限.     

两种检测方法测定抗甲状腺球蛋白抗体和抗甲状腺过氧化物酶抗体结果的比较

目的 应用化学发光免疫分析法（CLIA）和放射免疫分析法（RIA）分别测定血清抗甲状腺球蛋白抗体（抗TgAb）和抗甲状腺过氧化物酶抗体（抗TPOAb）.方法 采用CLIA法和RIA法测定304例不同类型甲状腺疾病患者和38名健康对照者血清抗TgAb和抗TPOAb水平.结果 CLIA法测定血清抗TgAb和抗TPOAb水平与RIA法测定的结果具有良好的一致性.结论 CLIA法测定血清抗TgAb和抗TPOAb可以用来替代RIA法.     

Parallel Detection and Quantification Using Nine Immunoassays in a Protein Microarray for Drug from Serum Samples

A protein microarray system for detection and quantification of nine prohibited drugs in serum is described. Chemically modified slides were chosen as the microarray substrates because of their suitable for drug-BSA printing. The developed protein microarray was able to preserve the biological function of the haptens, when immobilized on the microarray surface and demonstrated binding with their corresponding antibodies. The microarray could also be used for quantitative analysis when mouse IgG was chosen as an internal control for data processing. There was no qualitative difference between the results obtained using the protein microarray and ELISA. The protein microarray technology should be applicable to performing, simultaneously, large scale screening tests for many different analytes in serum.