Enzyme-linked Immunosorbent Assay For Diagnosis Research Articles

Synthetic p72 Peptide-Based Indirect- Enzyme-Linked Immunosorbent Assay for Diagnosis of African Swine Fever Virus Infection

Synthetic p72 Peptide-Based Indirect- Enzyme-Linked Immunosorbent Assay for Diagnosis of African Swine Fever Virus Infection

Development and Laboratory Evaluation of a Simple, Field-Applicable Coproantigen Enzyme-Linked Immunosorbent Assay for Diagnosis of Taeniasis in Northern Peru.

Coproantigen detection by enzyme-linked immunosorbent assay (coAg ELISA) is a vital tool for detecting and treating cases of Taenia solium taeniasis. However, the assay's procedures require costly materials and sophisticated equipment, which are typically inaccessible in rural settings where the disease is endemic. To overcome these barriers, we developed and evaluated a field-applicable coAg ELISA. The field coAg ELISA was developed and evaluated across four phases using known positive and negative stool samples collected from northern Peru. Phase I focused on field assay development, phase II on a small-scale performance evaluation, phase III on a large-scale evaluation, and phase IV on the use and reliability of a colorimetric scale card. All samples were processed using the field and standard assay procedures and compared using signal-to-noise ratios, correlation tests, performance characteristics, and agreement statistics where appropriate. The field coAg ELISA using reagents stored at -20°C and commercially available water and milk powder, and relying on spontaneous separation of the supernatant, had performance comparable to the standard assay. The field coAg ELISA was strongly correlated with the standard in both the small- and large-scale laboratory evaluation (r = 0.99 and r = 0.98, respectively). Finally, the field assay had an almost perfect agreement between independent readers (kappa = 0.975) and between each reader and the spectrophotometer. The field coAg ELISA demonstrated performance comparable to the standard, providing a low-cost alternative to the standard assay for identifying cases of intestinal taeniasis in a low-resource setting.

Evaluation of glutamic acid decarboxylase (GAD) 65 antibody detection methods for neurological and diabetic investigation in an Australian diagnostic laboratory

The role of anti-glutamic acid decarboxylase (GAD) 65 autoantibodies in autoimmune neurological conditions is evolving, but testing recommendations remain unchanged in Australia with GAD enzyme-linked immunosorbent assay (ELISA) and immunoblot as the only two Therapeutic Goods Administration approved testing methods available in Australia. Common practice is for use of ELISA in diagnosis of type 1 diabetes mellitus (T1DM) and use of immunoblot for diagnosis of GAD65-associated neurological disease. We observed a cohort of patients with negative immunoblot results and positive ELISA in the context of GAD-associated neurological disease without T1DM. In the absence of robust consensus guidelines on preferred testing modalities, we sought to determine if ELISA could have a superior role in the diagnosis of GAD-associated neurological disease when compared to immunoblot in this paper. We tested for anti-GAD65 autoantibodies on 55 patient samples, 40 samples requested for neurological disease and 15 type 1 diabetes samples with detectable anti-GAD65, using two testing platforms: Euroimmun anti-GAD enzyme-linked immunosorbent assay (ELISA) and. Euroimmun EuroLine immunoblot for paraneoplastic neurologic syndromes. These results were correlated against the clinical scenario. Positive ELISA results had a sensitivity of 100% and specificity of 91% for GAD65-related neurological disease. Immunoblot showed sensitivity of 43% and specificity of 76% for GAD65-related neurological disease. ELISA proved more sensitive and specific for GAD65-related neurological disease compared to immunoblot, raising questions about the role of this testing modality in neurological disease. We propose that ELISA should be used as a sole diagnostic method for all GAD65 antibody-related neurological disease over immunoblot. The presence of anti-GAD65 antibody on immunoblot is of doubtful clinical significance.

Development of CD163 receptor-based enzyme-linked immunosorbent assay for diagnosis of porcine reproductive and respiratory syndrome virus.

The online version contains supplementary material available at 10.1007/s13205-022-03376-z.

Comparison of Commercial Enzyme-linked Immunosorbent Assays for Diagnosis of Contagious Agalactia Caused By Mycoplasma Agalactiae.

IntroductionContagious agalactia (CA) is a disease affecting small ruminants with worldwide distribution and caused by several mycoplasmas, especially M. agalactiae. The main option for systematic diagnosis under monitoring control programmes is the enzyme-linked immunosorbent assay (ELISA) test.Material and MethodsThis study was designed to appraise the performance of two commercial indirect ELISA tests using M. agalactiae p48 protein and one using total protein, for antibody detection in small ruminants after natural infection with different M. agalactiae strains. We carried out the test evaluation using sera of confirmed M. agalactiae-positive goats with clinical signs. In addition, test agreement was assessed by kappa between the three commercial ELISA tests.ResultsAll three ELISA tests showed high validity scores (Youden’s J: 72.9–84%). The sensitivity values for the P48 protein-based tests were 76.9% and 84.6%, and was 79% for the total protein-based test. The specificity of all tests was 100%. In addition, between the total protein-based ELISA test and the other two ELISA tests based on the P48 protein, the agreement was substantial (kappa: 0.762–0.763) and the agreement between the latter two tests was almost perfect (kappa: 0.93).ConclusionThe validity parameters for all tests allowed their application for diagnostic purposes in lactating goats excreting M. agalactiae in milk and presenting clinical signs. The agreements show that any of these ELISA tests could be equally well used for diagnosis in programmes against CA.

Anti-BP180 and anti-BP230 enzyme-linked immunosorbent assays for diagnosis and disease activity tracking of bullous pemphigoid: A prospective cohort study.

Autoantibodies against BP180 and BP230 play major roles in bullous pemphigoid (BP). We are the first to describe the values of serum anti-BP180 IgG and anti-BP230 IgG enzyme-linked immunosorbent assays (ELISA) for diagnosis and disease monitoring of BP among Thai patients. We aimed to determine the diagnostic performance of anti-BP180 IgG and anti-BP230 IgG in BP, to correlate disease activity with autoantibody levels through follow-ups, and to relate BP comorbidities with disease activity and autoantibody levels. Consecutive patients suspected of having BP were included. Skin biopsy, direct immunofluorescence, and serum anti-BP180 IgG and anti-BP230 IgG tests were performed. BP disease area index (BPDAI) was evaluated at diagnosis and throughout follow-ups. Of 131 patients, 68 were diagnosed with BP, and 63 were included as controls. Sensitivity and specificity of serum anti-BP180 IgG were 69.1% and 90.5%, respectively, while those of serum anti-BP230 IgG were 55.9% and 85.5%, respectively. Using anti-BP180 and anti-BP230 IgG antibodies resulted in a 7% increase in sensitivity compared with using anti-BP180 IgG antibody alone. Significant correlation with BPDAI was found for both autoantibodies at diagnosis but only for anti-BP180 IgG at follow-ups (p = 0.013). BP patients with positivity to anti-BP180 or anti-BP230 IgG had significantly higher BPDAI than did those without (p = 0.005). BP was associated with neurological diseases (p = 0.025), while patients with diabetes had higher disease activity (p = 0.010). While both serum autoantibodies are useful for diagnosing BP in patients with suspicious clinical features, only anti-BP180 IgG allowed prediction of disease activity over time.

Analysis of the effectiveness of the use of enzyme-linked immunosorbent assay for the diagnosis of leukemia in cattle during health-improving activities

The aim of the work is to analyze the efficiency of using enzyme-linked immunosorbent assay in the diagnosis of cattle leukemia when carrying out health-improving measures in livestock enterprises that are unfavorable for this disease. Indicators of infection of cattle with leukemia virus on 6 farms of agricultural enterprises of the Tomsk region are presented. For serological diagnostics, the immunodiffusion reaction and enzyme immunoassay were used. It has been established that while carrying out health-improving measures in enterprises unfavorable for leukemia in cattle, the use of enzyme-linked immunosorbent assay for serological diagnosis of the disease enabled to identify a greater number of seropositive animals in comparison with a low-sensitivity immunodiffusion reaction. With a decrease in the herd infection rate to single cases of detection of animals infected with the leukemia virus at the final stages of rehabilitation of the enterprise, the number of additional seropositive animals detected by enzyme immunoassay increases. In the final period of herd recovery, the number of animals with a low level of antibodies to the leukemia virus, inaccessible to detection by immunodiffusion, increases. The use of an expensive and labor-consuming delivery of an enzymelinked immunosorbent assay to detect antibodies to the cattle leukemia virus is advisable at the final stages of an enterprise’s recovery. This enables to identify animals with a low level of antibodies to the leukemia virus, to speed up the negative result of a serological test of the entire herd and to exclude repeated outbreaks of infection of animals with this virus in a rehabilitated enterprise.

Development of an antigen-capture enzyme-linked immunosorbent assay for diagnosis of Aleutian mink disease virus.

Aleutian mink disease (AMD), caused by Aleutian mink disease virus (AMDV), is a very important infectious disease of mink. Currently, elimination of antibody- or antigen-positive animals is the most successful strategy for eradicating AMD, but the claw-cutting method of blood sampling is difficult to perform and painful for the animal. In this study, we aimed to establish an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) method for the efficient detection of AMDV antigens using fecal samples. A purified mouse monoclonal antibody (mAb) was used as the capture antibody, and a rabbit polyclonal antibody (pAb) was used as the detection antibody. The assay was optimized by adjusting a series of parameters. Using a cutoff value of 0.205, the limit of detection of the AC-ELISA for strain AMDV-G antigen was 2 μg/mL, and there was no cross-reaction with other mink viruses. The intra- and inter-assay standard deviations were below 0.046, and the correlation of variance (CV) values were 1.24-7.12% when testing fecal samples. Compared with conventional PCR results, the specificity and sensitivity were 91.5% and 90.6%, respectively, and the concordance rate between the two methods was 91.1%.

Evaluation of two enzyme-linked immunosorbent assays for diagnosis of bluetongue virus in wild ruminants

Bluetongue (BT) is a reportable re-emerging vector-borne disease of animal health concern. Enzyme-linked immunosorbent assays (ELISA) are frequently used in BT surveillance programs in domestic ruminants, but their diagnostic accuracy has not been evaluated for wild ruminants, which can play an important role as natural reservoirs of bluetongue virus (BTV). The aim of this study was to assess two commercial ELISAs for BT diagnosis in wild ruminants using control sera of known BTV infection status and field samples. When control sera were tested, the double recognition ELISA (DR-ELISA) showed 100 % sensitivity (Se) and specificity (Sp), while the competitive ELISA (C-ELISA) had 86.4 % Se and 97.1 % Sp. Using field samples, the selected latent-class analysis model showed 95.7 % Se and 85.9 % Sp for DR-ELISA, 58.2 % Se and 95.8 % Sp for C-ELISA and 84.2 % Se for the serum neutralization test (SNT). Our results indicate that the DR-ELISA may be a useful diagnostic method to assess BTV circulation in endemic areas, while the C-ELISA should be selected when free-areas are surveyed. The discrepancy between control and field samples point out that the inclusion of field samples is required to assess the accuracy of commercial ELISAs for the serological diagnosis of BTV in wild ruminants.

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.

Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.

Evaluation of anti-gal enzyme-linked immunosorbent assay for the diagnosis of Indian post-kala-azar dermal leishmaniasis.

Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1-99.1) and 98.1% (95% confidence interval: 90.1-99.9), and 98.5% (95% CI: 92.4-99.9) and 98.1% (95% CI: 90.1-99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.

Recombinant Expression and Purification of Human Cystatin C Biomarker in Escherichia coli for Prediagnostic Renal Disorders

Introduction: Chronic kidney disease is a worldwide health problem and Glomerular filtration rate (GFR) is the most frequently used criteria in the assessment of rental function. Cystatin C as a member of type 2 cystatin superfamily and cysteine-protease inhibitor is found in high concentrations in all biological fluids. This study is aimed to study cystatin C as a potent biomarker for clinical measurement of renal disorders and other diseases. Materials and Methods: In this study, the human cystatin C construct was analyzed by bioinformatics software. It was cloned and expressed to produce an appropriate antigen for anti-cystatin C (anti-Cys C) obtained from mice Balb/C as a crucial point in the improvement of an enzyme-linked immunosorbent assay (ELISA) method. Serum samples were given from 32 hospitalized patients with renal failure and cardiovascular disease and non-hospitalized patients were tested by ELISA method using anti-Cys C obtained from mice Balb/C. Results: Our findings indicated 0.36-2.4 mg/L as the best conclusion for antigen cystatin C in patients’ sera and 1/50 dilution for anti-Cys C obtained from mice Balb/C and showed a relationship between patients with high creatinine and high concentration of cystatin C. In case of five cardiovascular disease patients with normal upper limit of Creatinine we obtained cystatin C lower than kidney failure and raising of cystatin C in 6 patients with increased TSH were seen. Conclusions: Polyclonal anti-Cys C antibodies were obtained through the immunization of Balb/C mice can be employed as an anti-Cys C in ELISA for diagnosis of some renal dysfunction.

Development of indirect enzyme-linked immunosorbent assay for diagnosis of canine leptospirosis.

Aim:This study was taken up to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) for screening antibodies against Leptospira spp. in canines.Materials and Methods:An i-ELISA was developed using outer membrane protein extracted from Leptospira interrogans serovar canicola used for coating the well with concentration of 0.5 µg/µl. A total of 250 serum samples from clinically affected and apparently healthy dogs were collected along with relevant epidemiological data at Teaching Veterinary Clinical Complex, Veterinary College and Research Institute, Namakkal, and subjected to i-ELISA.Results:Out of 250 sera samples, 140 (56.00%) were found to be positive by i-ELISA. All the sera samples were subjected to microagglutination test (MAT) with panel of 12 different serovars. A total of 71 (28.40%) sera samples were positivity to MAT excluding the sera samples positive to L. interrogans serovars canicola and icterohaemorrhagiae in vaccinated dogs. Sensitivity and specificity of i-ELISA were higher in compared with MAT was 91.54% and 58.10%, respectively.Conclusion:An indirect ELISA developed for the detection of canine antileptospiral antibodies proved to be highly sensitive, rapid and easy to perform and overcome the drawbacks of MAT.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans.

BACKGROUNDAmphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing.OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera.METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections.PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana.MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

The value of mucin 7 in the detection of bladder cancer

Objective To investigate the diagnosis value of mucin 7 in plasma and urine in the detection of bladder cancer.Methods Expression of mucin 7 was detected quantitatively in plasma and urine from 23 patients with bladder cancer and 23 patients as control group by enzyme-linked immunosorbent assay(ELISA) from November 2012 to September 2013.There were 15 were male and 8 were female in the 23 bladder cancer patients,aged from 41 to 95 years,with an average of 64 years.Of the 23 bladder cancer pathology,12 with high grade,11 with low grade,11 with invasive disease and 12 with noninvasive disease.There were 19 males and 4 females in the 23 patients in control group,aged from 28 to 85 years,with an average of 61 years.Results The amount of mucin 7 in plasma was significantly higher in bladder cancer group than that in control group [(7.43±4.54) ng/ml versus (4.55±.1.98) ng/ml,P=0.017].While there was no significant difference in urine between the two groups [(12.44±7.1 1) ng/ml versus (11.96±8.41)ng/ml,P=0.840].There was no significant differences in the amount of mucin 7 had in different grades and stages of bladder cancer (P＞0.05).Conclusions Expression of mucin 7 in plasma of patients with bladder cancer is significantly higher than that in control group.Mucin 7 expression has no significant correlation with the grading and staging of bladder cancer.Detecting mucin 7 expression quantitatively with ELISA for diagnosis of bladder cancer is a method with certain value. Key words: Mucin 7; Bladder cancer; Quantitative detection; Enzyme-linked immunosorbent assay

Usefulness of desmoglein 1 and 3 in serodiagnosis of pemphigus vulgaris and its correlation with disease activity - ELISA study

Background: Pemphigus is an autoimmune blistering disease with three subtypes, which includes pemphigus vulgaris (PV), pemphigus foliaceus (PF) and paraneoplastic pemphigus (PP), which are characterized by circulating autoantibodies to the desmosomal glycoproteins desmoglein 3 (Dsg 3) and Dsg 1. Detection and quantification of these antibodies can be useful in diagnosis of PV. Aims: The purpose of this study was to evaluate the practical application of ELISAs for diagnosis of PV based on immunological reactivity of Dsg 1 and Dsg3. Materials and Methods: Based on clinical presentation and histopathologic confirmation, 10 patients previously diagnosed of PV were included in the study, out of which, five were with active lesions and five were under medication and hence were at remission stages. Sera of the patients were tested for Dsg 1 and Dsg 3 titres by Desmoglien enzyme-linked immunosorbent assay (ELISA) test. Results: Desmoglien ELISA was positive in all the patients, with higher values of Dsg antibody titers in patients with active disease. Dsg 3 titers exceeded the cut off value in patients with oral lesions and titers of both Dsg 1 and Dsg 3 were greater than cut value in patients with mucocutaneous involvement. Conclusion: Thus, the Dsg 1 and Dsg 3 provided objective and quantitative data which allowed differentiation of PV, and in view of these advantages; they are likely to become a routine technique in diagnostic laboratories.

An enzyme-linked immunosorbent assay for diagnosis of Fasciola gigantica infection in cattle and buffaloes.

The enzyme-linked immunosorbent assay (ELISA) was evaluated for the diagnosis of Fasciola gigantica infection in cattle and buffaloes. The excretory-secretory (E-S Ag) antigen of F. gigantica adult flukes obtained after invitro incubation was used as an antigen. The test was conducted with 276 sera collected from cattle and buffaloes which included 22 sera each from naturally infected cattle and buffaloes (known positive serum) and with similar number of samples with healthy cattle and buffaloes (known negative serum). The positive results were observed in 18 and 19 of the sera from naturally infected cattle and buffaloes with sensitivity of 81.8 and 86.3% respectively. Out of 188 serum samples which were found negative on faecal examination 32 (34%) sera of cattle and 40 (42.5%) sera of buffaloes were found positive by ELISA respectively. The sensitivity of the test was found to be 91.6 and 95.6% in cattle and buffaloes respectively.

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

Comparative study of direct and indirect immunofluorescence and of bullous pemphigoid 180 and 230 enzyme-linked immunosorbent assays for diagnosis of bullous pemphigoid

Direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA) are used for the laboratory diagnosis of bullous pemphigoid (BP). The diagnostic value of DIF and IIF on rabbit and monkey esophagus or human salt-split skin and commercial ELISAs was assessed. This was a single-center retrospective study where 313 patients with BP were compared with 488 control subjects. DIF was the most sensitive test (90.8%) whereas sensitivities for IIF on rabbit esophagus, IIF on monkey esophagus, IIF on salt-split skin, BP180 ELISA, and BP230 ELISA were 76.0%, 73.2%, 73.3%, 72.0%, and 59.0%, respectively. The sensitivity of the serologic tests was 88.8% altogether. The specificities for DIF, IIF on rabbit esophagus, IIF on monkey esophagus, IIF on salt-split skin, BP180 ELISA, and BP230 ELISA were 98%, 96.5%, 97.1%, 100%, 94.1%, and 99.2%, respectively. The retrospective nature of study was a limitation. Correlation of diagnostic data with clinical manifestations or disease course was not possible. In suspected BP, both serologic tests and DIF have to be performed because of a sensitivity issue. Although the ELISAs had a relatively low sensitivity, the serologic tests altogether almost reached the level of sensitivity of DIF. The specificities of all assays were excellent.

Limitations of the BP26 Protein-Based Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis

Brucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies against Brucella lipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity with Escherichia coli O157:H7 and Yersinia enterocolitica O:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectious Brucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis of Brucella-infected animals and humans, but a few results showed that BP26 couldn't react with all Brucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with various Brucella species, we infected sheep, goats, and beef cattle with common virulent reference Brucella species. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that all Brucella-infected individuals could produce high levels of antibodies against LPS, but only B. melitensis 16M- and B. melitensis M28-infected sheep and B. melitensis 16M- and B. abortus 2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed both Brucella species and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis.