Sensitivity Of ELISA Research Articles

Performance of p16INK4a ELISA as a primary cervical cancer screening test among a large cohort of HIV-infected women in western Kenya: a 2-year cross-sectional study

ObjectiveA biomarker with increased specificity for cervical dysplasia compared with human papillomavirus (HPV) testing would be an attractive option for cervical cancer screening among HIV-infected women in resource-limited settings. p16INK4a...

Performance of microscopy and ELISA for diagnosing Giardia duodenalis infection in different pediatric groups

Techniques for Giardia diagnosis based on microscopy are usually applied as routine laboratory testing; however, they typically exhibit low sensitivity. This study aimed to evaluate Giardia duodenalis and other intestinal parasitic infections in different pediatric groups, with an emphasis on the comparison of Giardia diagnostic techniques. Feces from 824 children from different groups (diarrheic, malnourished, with cancer and from day care) were examined by microscopy and ELISA for Giardia, Cryptosporidium sp. and Entamoeba histolytica coproantigen detection. Giardia-positive samples from day-care children, identified by either microscopy or ELISA, were further tested by PCR targeting of the β-giardin and Gdh genes. Statistically significant differences (P<0.05) were observed when comparing the frequency of each protozoan among the groups. Giardia duodenalis was more frequent in day-care children and Cryptosporidium sp. in diarrheic and malnourished groups; infections by Entamoeba histolytica were found only in children with diarrhea. Considering positivity for Giardia by at least one method, ELISA was found to be more sensitive than microscopy (97% versus 55%). To examine discrepancies among the diagnostic methods, 71 Giardia-positive stool samples from day-care children were tested by PCR; of these, DNA was amplified from 51 samples (77.4%). Concordance of positivity between microscopy and ELISA was found for 48 samples, with 43 confirmed by PCR. Parasite DNA was amplified from eleven of the 20 Giardia samples (55%) identified only by ELISA. This study shows the higher sensitivity of ELISA over microscopy for Giardia diagnosis when a single sample is analyzed and emphasizes the need for methods based on coproantigen detection to identify this parasite in diarrheic fecal samples.

Evaluation of milk cathelicidin for detection of bovine mastitis.

Mastitis due to intramammary infection is one of the most economically relevant diseases in dairy cows, causing reductions in milk quality and quantity. Currently, mastitis monitoring is based on somatic cell count (SCC) and bacteriologic culture (BC) of milk. Nevertheless, inflammation-specific protein markers might provide more sensitive and reliable assays, enabling immunoassay-based screening strategies. Cathelicidin is an inflammatory protein released in milk that has recently demonstrated fair reliability and diagnostic potential for ewe mastitis. To assess its performance in cows, 531 quarter milk samples from 2 herds were tested using cathelicidin ELISA, SCC, and BC. We found that 29.0% of samples were positive for cathelicidin, 18.8% had SCC >200,000 cells/mL, and 13.7% were BC-positive. Cathelicidin showed a strong positive correlation with SCC as demonstrated by receiver operating characteristics curve analysis and by the clustering of cathelicidin-negative and cathelicidin-positive samples in association with low and high SCC values, respectively. For evaluating the diagnostic performance of a novel test, BC cannot be considered a reliable gold standard for true disease status because of its known limitations. Therefore, we assessed the sensitivity (Se) and specificity (Sp) of the milk cathelicidin ELISA using a latent class analysis approach together with BC and SCC by considering different diagnostic thresholds to identify the preferred Se/Sp combination. We modeled conditional dependence of cathelicidin and SCC to account for their close association. The cathelicidin ELISA showed higher Se than SCC and BC for almost all threshold combinations. In fact, at the best-performing threshold combination, the Se of cathelicidin was 80.6%, 6.2 percentage points higher than that of SCC >200,000 cells/mL (74.4%) and similar to that of SCC >100,000 cells/mL (80.2%). Most importantly, this Se was obtained with a loss in Sp of only 1.4 percentage points compared with SCC >200,000 cells/mL (94.9% Sp for cathelicidin vs. 96.3% for SCC >200,000). The limited Se of BC (38.8%) was also confirmed in this study, and BC showed a slightly lower Sp than both cathelicidin and SCC for most of threshold combinations. This study confirmed that cathelicidin is released in the milk of cows with mastitis and that its presence is highly correlated with SCC. The measurement of cathelicidin by ELISA may hold significant potential for improving the sensitivity of mastitis detection in dairy cows while maintaining high specificity.

Investigation of Rotavirus with Various Methods in Children with Acute Gastroenteritis and Determination of Its Molecular Epidemiology in Kayseri Province, Turkey

In this study, the fresh stool samples from 254 children under 5 years of age with acute gastroenteritis which were delivered between October 2012 and December 2013 were collected. In the stool samples, rotavirus antigens were investigated using two different immunochromatographic methods which are routinely used at different times, namely the RIDA® QUICK Rotavirus/Adenovirus Combi Test (R-Biopharm AG, Germany) and the Genx® Rotavirus Test (Diamed-Lab, Turkey), in addition to the Rotavirus Ag (Stool) ELISA (DRG, Germany) kit. The results were compared with reverse transcriptase PCR (RT-PCR). When the Genx® Rotavirus Test and RIDA® QUICK Rotavirus/Adenovirus Combi Test immunochromatographic methods were compared with RT-PCR, their sensitivity and specificity were found as 97.1%, 100%, and 80.4%, 72%, respectively. As to the Rotavirus Ag (Stool) ELISA method, on the other hand, its sensitivity was found to be 95.1% and its specificity was 86.5%. The most common genotype was G9P[8] (40%), which was followed by the G1P[8] (18.7%) and G3P[8] (9.6%) genotypes. Consequently, it was revealed that the sensitivity of ELISA and immunochromatographic methods, which provide results in a short time and are used in the investigation of rotavirus antigen, was high and their specificity was low; further studies to determine the distribution of G and P genotypes will contribute to establishing strategies for vaccine development for rotavirus in the world.

Y-box protein-1/p18 as novel serum marker for ovarian cancer diagnosis: A study by the Tumor Bank Ovarian Cancer (TOC)

IntroductionThe cold shock Y-box binding protein-1 (YB-1) fulfills important roles in regulating cell proliferation and differentiation. Overexpression occurs in various tumor cells. Given the existence of extracellular YB-1 we set out to determine the diagnostic, predictive and prognostic role of serum YB-1/p18 for patients with primary epithelial ovarian cancer (EOC). MethodsThe protein fragment YB-1/p18 was quantified by sandwich ELISA in serum samples from 132 healthy female volunteers and 206 patients with histological diagnosis of primary EOC. The ELISA sensitivity and specificity to detect EOC were calculated using receiver operating curves. Survival data were calculated using Kaplan Maier curves. ResultsMedian age at the time of diagnosis was 60years and follow-up ended with a mean of 44.8month. 188 (91%) patients were diagnosed at advanced stages (FIGO III/IV) and 188 patients (91%) suffered from high-grade serous ovarian carcinoma. YB-1/p18 levels were significantly decreased in older patients (p=0.021). Significantly lower serum levels of YB-1/p18 were detected in the EOC cohort when compared to the control group (p<0.0001, AUC=0.827; 95% CI, 0.787–0.867). Using the expression of serum YB-1/p18 in early stages I and II cases these could be differentiated from control cases (p<0.0001, AUC=0.816; 95% CI 0.704–0.929). No other significant associations between clinical prognostic factors and YB-1/p18 serum levels were detected. Immunoblotting results with serum samples suggest that masking of epitopes by the YB-1/p18 fragment in multiprotein-complexes under non reducing conditions leads to the observed reduced ELISA readings in the EOC cohort. ConclusionsThe quantification of fragment YB-1/p18 derived from cold shock protein YB-1 in serum samples could be useful for the early diagnosis of EOC.

Development of a Novel PmpD-N ELISA for Chlamydia psittaci Infection

ObjectiveChlamydia psittaci is an avian respiratory pathogen and zoonotic agent. The wide prevalence of C. psittaci poses a threat to the poultry industry and its employees. However, few commercial kits are available for detecting avian antibodies excluding the in-house ELISA kit. In this study, we developed a novel ELISA kit for detecting antibodies against C. psittaci based on the N-terminal fragment of polymorphic outer membrane protein D (PmpD-N) as the coating antigen. MethodsThe antigen concentrations, primary antibody, and cut-off value were determined and optimized. The ELISA, designated PmpD-N ELISA, was assessed for sensitivity, specificity, and concordance using sera samples from 48 experimentally infected and 168 uninfected SPF chickens. ResultsThe sensitivity and specificity of PmpD-N ELISA were 97.9%, 100%, respectively, while the concordance was 98.1% as compared to that of MOMP-ELISA. No cross-reaction with positive sera for other avian pathogens was found. Using PmpD-N ELISA, 799/836 clinical samples were positive, including 93.0% and 98.1% positivity in layers and broilers, respectively. ConclusionThese data indicate that indirect ELISA with PmpD-N as the antigen candidate is a promising approach for the surveillance of C. psittaci infection.

Evaluation of milk cathelicidin for detection of dairy sheep mastitis

Mastitis due to intramammary infections is one of the most detrimental diseases in dairy sheep farming, representing a major cause of reduced milk productions and quality losses. In particular, subclinical mastitis presents significant detection and control problems, and the availability of tools enabling its timely, sensitive, and specific detection is therefore crucial. We have previously demonstrated that cathelicidins, small proteins implicated in the innate immune defense of the host, are specifically released in milk of mastitic animals by both epithelial cells and neutrophils. Here, we describe the development of an ELISA for milk cathelicidin and assess its value against somatic cell counts (SCC) and bacteriological culture for detection of ewe mastitis. Evaluation of the cathelicidin ELISA was carried out on 705 half-udder milk samples from 3 sheep flocks enrolled in a project for improvement of mammary health. Cathelicidin was detected in 35.3% of milk samples (249/705), and its amount increased with rising SCC values. The cathelicidin-negative (n=456) and cathelicidin-positive (n=249) sample groups showed a clear separation in relation to SCC, with median values of 149,500 and 3,300,000 cells/mL, respectively. Upon bacteriological culture, 20.6% (145/705) of the milk samples showed microbial growth, with coagulase-negative staphylococci being by far the most frequent finding. A significant proportion of all bacteriologically positive milk samples were positive for cathelicidin (110/145, 75.9%). Given the lack of a reliable gold standard for defining the true disease status, sensitivity (Se) and specificity (Sp) of the cathelicidin ELISA were assessed by latent class analysis against 2 SCC thresholds and against bacteriological culture results. At an SCC threshold of 500,000 cells/mL, Se and Sp were 92.3 and 92.3% for cathelicidin ELISA, 89.0 and 94.9% for SCC, and 39.4 and 93.6% for bacteriological culture, respectively. At an SCC threshold of 1,000,000 cells/mL, Se and Sp were 93.3 and 91.9% for cathelicidin ELISA, 80.0 and 97.1% for SCC, and 39.4 and 93.5% for bacteriology, respectively. In view of the results obtained in this study, the measurement of cathelicidin in milk by ELISA can provide added Se while maintaining a high Sp and may therefore improve detection of subclinical mastitis.

Study on detection and identification of some Mycobacterium species by using some bacteriological and immunological techniques

This study aimed to compare and analyze the results of microbiological and immunological laboratory investigations of pulmonary tuberculosis and non-tuberculosis patients from Hospitals at Saudi Arabia, and also to evaluate the serodiagnosis detection of tuberculosis promptly using immunological assays in comparison with the traditional Ziehl Neelsen microbiological techniques. This is to develop reliable, effective assay, shorter duration and more specific diagnosis of active tuberculosis. The sensitivity, specificity and efficiency of Ziehl-Neelsen stain were 48 %, 100 % and 64 %, respectively. An identified antigen in serum samples of infected persons with pulmonary tuberculosis was detected using TB-Spot ELISA technique. The sensitivity, specificity and efficiency of TB-Spot ELISA for the detection of antigen were 97 %, 95 % and 98 %, respectively. The serum levels of TNF-α reach statistical difference (P< 0.0001) in patients positive for TB antigen in comparison with the levels in negative individuals for TB.

STUDY ON DIAGNOSIS TECHNIQUES INFECTED FUNGIS, TRICHOMONAS VAGINALIS AT WOMEN IN THE CLINIC THE HUE UNIVERSITY HOSPITAL

Introduction: Trichomonas vaginalis and Candida sp is one of the pathogens common vaginal infection in the world. This study aimed to assess the value of diagnostic techniques fungus and Trichomonas vaginalis. Materials and method: Using laboratory direct test, culturing fungi, Trichomonas vaginalis and compared the results of both technique, thereby determining the sensitivity, specifi city of direct test from that culturing. Through culture techniques, identify Candida albicans and Candida non albicans infection rate. Perform ELISA technique to determine the rate of IgG antibodies Trichomonas vaginalis simultaneously using ROC curves assessed the sensitivity and specifi city of ELISA with the ratio of serum diluted 1/50, 1/100 and 1/200. Results: The study on 201 patients with vaginal infections, with 44.77%, infections caused by fungi and / or Trichomonas vaginalis including 2 types mixed infection is 0.99%. In 63 cases of fungal, 12 cases of Candida albicans, accounting for 19.05% ratio, remaining Candida non albicans remaining. With Trichomonas vaginalis, direct technique detected 27 cases, culture technique detected 31 cases. ELISA technique detects antibodies T.vaginalis 77.40% sensitivity, specifi city of 89.00% with a serum dilution 1/100. Conclude: Direct techinique has sensitivity and high specifi city, useful for diagnosis fungal, Trichomonas vaginalis. Elisa technique has sensitivity and high specifi city, useful for diagnosing trichomonas vaginalis, especially in epidemiological study. Key words: vagina, fungus, Candida albicans, Candida non albicans, Trichomonas vaginalis, culture, direct technique, ELISA

Cavity-Enhanced Immunoassay Measurements in Microtiter Plates Using BBCEAS.

We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorized XY stage, which functioned as a receptacle for 96-well microtiter plates. The custom-built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30-fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10(-5) cm(-1) Hz(-1/2). This corresponded to a 39-fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115-fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests.

Science with the space-based interferometer eLISA. II: gravitational waves from cosmological phase transitions

We investigate the potential for the eLISA space-based interferometer to detect the stochastic gravitational wave background produced by strong first-order cosmological phase transitions. We discuss the resulting contributions from bubble collisions, magnetohydrodynamic turbulence, and sound waves to the stochastic background, and estimate the total corresponding signal predicted in gravitational waves. The projected sensitivity of eLISA to cosmological phase transitions is computed in a model-independent way for various detector designs and configurations. By applying these results to several specific models, we demonstrate that eLISA is able to probe many well-motivated scenarios beyond the Standard Model of particle physics predicting strong first-order cosmological phase transitions in the early Universe.

Dengue outbreak in Swat and Mansehra, Pakistan 2013: An epidemiological and diagnostic perspective.

To high light some epidemiological, clinical and diagnostic features of dengue fever during an outbreak and the role of different diagnostic techniques to achieve the highest level of accuracy in results. Blood samples (n=323) were collected along with epidemiological and clinical data from suspected dengue patients who visited different hospitals in Swat and Mansehra district of Pakistan between May-November 2013 during a dengue outbreak. Samples were tested for the detection of viral nucleic acid by real-time PCR, non structural protein-1 (NS1) antigen and IgM antibodies by ELISA. Out of 323 cases with clinical dengue infection, 304 were positive by one or more diagnostic parameter; 201 samples were positive by real-time PCR, 209 were positive by NS1 ELISA and 190 were positive by IgM antibodies. Sensitivities of real-time PCR and NS1 ELISA were comparable for early diagnosis of dengue virus infection, IgM antibody detection assay was found useful for the diagnosis in the samples collected later than day 5 of onset. The use of real-time PCR or detection of non structural protein NS1 by ELISA followed by IgM antibodies detection can be recommended for early diagnosis of dengue virus infection with a high level of accuracy.

Investigation of the cause of geographic disparities in IDEXX ELISA sensitivity in serum samples from Mycobacterium bovis-infected cattle.

Accurately identifying Mycobacterium bovis-infected cattle is critical for bovine tuberculosis prevention and control. One method for identifying infected cattle is an ELISA developed by IDEXX laboratories, which detects antibodies to two M. bovis proteins, MPB70 and MPB83. The assay’s sensitivity varies by geographic region, with sensitivities of 77%, 45%, and 9% in bovine serum samples from the United Kingdom (n = 126), the United States (n = 146), and Mexico (n = 128), respectively. We hypothesized that geographically-biased sequence variation in mpb70 and mpb83, or in the genes that regulate their expression (sigK and rskA), may explain these differing sensitivities. This hypothesis was tested by comparing the sequences of these four genes in 455 M. bovis strains isolated from cattle in the aforementioned countries. For each gene, a single, common sequence was identified in most genomes of the M. bovis strains collected in all three countries. Twelve of the 455 strains were isolated from infected cattle for which the IDEXX ELISA was also performed. Five of the seven ELISA-positive genomes and three of the five ELISA-negative genomes contained the most common sequence of all four genes. Thus, sequence variation in mpb70, mpb83, sigK, and rskA does not explain the geographic disparities in IDEXX ELISA sensitivity.

Sensitivity of IgG ELISA for diagnosing cysticercosis in high risk group in and around Aligarh District of Uttar Pradesh, India

ObjectivesTo study the seroprevalence of cysticercosis and compare immunological, radiological and clinical features in patients of cysticercosis in high risk group of community. MethodsThe study was conducted in the Department of Microbiology and concerning departments of Jawaharlal Nehru Medical College Hospital, Aligarh from January 2012 to June 2013. The study population included 27 participants from high risk group in community like those involved in pig rearing, slaughtering of pigs and consumption of pork in and around Aligarh. The kit used to detect antibodies against Taenia solium (T. solium) was NovaTec T. solium IgG-ELISA. ResultsMajority of participants [12 (44.4%)] belonged to age-group 30–44 years. Majority of participants [25 (92.6%)] were males. Maximum number of participants [16 (59.3%)] belonged to socio-economic Class IV, followed by Class V. Only 2 (7.4%) participants showed positive for immunoglobulin G antibodies against T. solium. ConclusionsCysticercosis is a health problem in and around Aligarh with higher risk in males. Seroprevalence of cysticercosis in high risk community of Aligarh is 7.4%.

Nanospherical Brush as Catalase Container for Enhancing the Detection Sensitivity of Competitive Plasmonic ELISA.

Plasmonic enzyme-linked immunosorbent assay (pELISA) based on catalase (CAT)-mediated gold nanoparticle growth shows great potential for the determination of disease-related biomarkers at ultralow concentrations by using sandwich formats. However, the relatively low sensitivity of this strategy using competitive formats limits its adoption for hapten detection. Herein, we present an improved competitive pELISA for ultrasensitive detection of ochratoxin A (OTA), where silica nanoparticles carrying poly(acrylic acid) brushes (SiO2@PAA) were used to decrease the affinity of competing antigens to anti-OTA monoclonal antibodies and amplify the signal as a "CAT container" (SiO2@PAA@CAT). The developed competitive pELISA exhibits extremely high sensitivity for OTA with detection limits of 10(-18) and 5 × 10(-20) g/mL by the naked eye and microplate reader, respectively. These values are at least 7 orders of magnitude lower than that of competitive CAT-based pELISA (10(-11) g/mL by the naked eye) and 8 orders of magnitude lower than that of horseradish peroxidase-based conventional ELISA (10(-11) g/mL by the microplate reader), respectively. Reliability and robustness of the proposed method were evaluated using actual agricultural products and human serum samples. This study demonstrated the potential of this modified method in practical applications involving the ultrasensitive detection of mycotoxins or other haptens.

A Diagnostic Algorithm for the Detection of Clostridium difficile-Associated Diarrhea.

Clostridium difficile is a common cause of hospital-acquired diarrhea, which is usually associated with previous antibiotic use. The clinical manifestations of C. difficile infection (CDI) may range from mild diarrhea to fulminant colitis. Clostridium difficile should be considered in diarrhea cases with a history of antibiotic use within the last 8 weeks (community-associated CDI) or with a hospital stay of at least 3 days, regardless of the duration of antibiotic use (hospital-acquired CDI). This study investigated the frequency of CDI in diarrheic patients and evaluated the efficacy of the triple diagnostic algorithm that is proposed here for C. difficile detection. Cross-sectional study. In this study, we compared three methods currently employed for C. difficile detection using 95 patient stool samples: an enzyme immunoassay (EIA) for toxin A/B (C. diff Toxin A+B; Diagnostic Automation Inc.; Calabasas, CA, USA), an EIA for glutamate dehydrogenase (GDH) (C. DIFF CHEK-60TM, TechLab Inc.; Blacksburg, VA, USA), and a polymerase chain reaction (PCR)-based assay (GeneXpert(®) C. difficile; Cepheid, Sunnyvale, CA, USA) that detects C. difficile toxin genes and conventional methods as well. In this study, 50.5% of the patients were male, 50 patients were outpatients, 32 were from inpatient clinics and 13 patients were from the intensive care unit. Of the 95 stool samples tested for GDH, 28 were positive. Six samples were positive by PCR, while nine samples were positive for toxin A/B. The hypervirulent strain NAP-1 and binary toxin was not detected. The rate of occurrence of toxigenic C. difficile was 5.1% in the samples. Cefaclor, ampicillin-sulbactam, ertapenem, and piperacillin-tazobactam were the most commonly used antibiotics by patients preceding the onset of diarrhea. Among the patients who were hospitalized in an intensive care unit for more than 7 days, 83.3% were positive for CDI by PCR screening. If the PCR test is accepted as the reference: C. difficile Toxin A/B ELISA sensitivity and specificity were 67% and 94%, respectively, and GDH sensitivity and specificity were 100% and 75%, respectively. Tests targeting C. difficile toxins are frequently applied for the purpose of diagnosing CDI in a clinical setting. However, changes in the temperature and reductant composition of the feces may affect toxin stability, potentially yielding false-negative test results. Therefore, employment of a GDH EIA, which has high sensitivity, as a screening test for the detection of toxigenic strains, may prevent false-negative results, and its adoption as part of a multistep diagnostic algorithm may increase accuracy in the diagnosis of CDIs.

A Study on the Importance of Anti Cyclic Cytrullinated Peptide Antibodies (Anti-CCP) and Rheumatoid Factor (RF) Assay in Detection of Patients with Rheumatoid Arthritis in North Eastern Part of India

<i>Objective:</i> Rheumatoid arthritis (RA) is a polyarticular and systemic chronic inflammatory autoimmune disease, occurring throughout the world. The aim of the present study is to observe the role of anti-CCP antibody and Rheumatoid Factor (RF) assay in diagnosing patients of rheumatoid arthritis in north-eastern part of India, in the state of Assam and also to evaluate the individual and combined sensitivity and specificity of anti-CCP antibody and RF assay in diagnosing RA. <i>Methods:</i> A total of 88 cases presenting with history of polyarthritis were included in the study of which the study group comprised of 57 clinically diagnosed cases of rheumatoid arthritis and control group comprised of 31 patients of non-RA rheumatic diseases having joint pain. Individual sensitivity and specificity of the above tests, and also the combined specificity of the two tests and also the three tests were calculated and the results of the tests were compared with each other to examine the correlation between them. <i>Results:</i> Sensitivity of anti-CCP ELISA, RF IgM ELISA and RF Latex test was 91.23%, 75.44% and 40.35% with specificity of 70.97%, 67.74% and 90.32% respectively. If the three tests are combined then the combined specificity of the three tests is 99.59%. One important observation was that out of 9 anti-CCP positive cases in the control group(n = 31) 5 cases were weakly positive (titer 6.25 – 20 U/ml), considering cut-off value 6.25 U/ml as mentioned by the kit.If these weakly positive cases in the control group are considered as negative, then specificity of anti-CCP ELISA increases from 70.97% to 87.1% which is more acceptable. <i>Conclusion:</i> From this study it is observed that anti-CCP antibodies are highly sensitive and specific for diagnosing RA. If this test is combined with RF IgM ELISA and Latex test then combined specificity can be increased more than 99%. However, weakly positive titers of anti-CCP antibodies are more prevalent in other non-RA rheumatic diseases in North-Eastern part of India.

Bayesian estimation of sensitivity and specificity of a commercial serum/milk ELISA against the Mycobacterium avium subsp. Paratuberculosis (MAP) antibody response for each lactation stage in Greek dairy sheep

A total of 854 paired milk and blood samples were collected from ewes of a Greek flock and used to estimate the sensitivity and specificity of a commercial ELISA for detection of Mycobacterium avium subsp. paratuberculosis (MAP) specific antibodies in each stage of lactation. We implemented Bayesian mixture models to derive the distributions of the test response for the healthy and the infected ewes. In the colostrum period, early, mid and late lactation stage the median values of the area under the curves (AUC) were 0.61 (95% credible interval: 0.50; 0.84), 0.61 (0.51;0.84), 0.65 (0.51;0.91), 0.65(0.51;0.89) for the serum ELISA and and 0.60 (0.50; 0.84), 0.61 (0.50; 0.84), 0.67(0.51; 0.91), 0.66(0.50; 0.90) for the milk ELISA, respectively. Both serum and milk ELISA had low to average overall discriminatory ability as measured by the area under the curves and comparable sensitivities and specificities at the recommended cutoffs.

Bayesian estimation of diagnostic sensitivity and specificity of a nervous necrosis virus antibody ELISA

Diagnosis of nervous necrosis virus (NNV) infection in susceptible fish species is mostly performed post-mortem due to the neurotropism of the causative agent and the only validated diagnostic assays require samples from brain and retinal tissue. However, a non-lethal alternative to test for exposure of fish to NNV is needed. An indirect ELISA for the detection of anti-NNV antibodies in was recently developed and evaluated to detect responses in the sera from immunized fish. For this study, we assessed the accuracy of the assay at detecting specific antibodies from naturally exposed fish using field samples from populations with differing infection status. We applied a Bayesian model, using RTqPCR as a second test. Median estimates of the diagnostic sensitivity and specificity of the VNN ELISA were 81.8% and 86.7%, respectively. We concluded that the assay was fit for the purpose of identifying animals in naturally exposed populations. With further evaluation in larger populations the test might be used to inform implementation of control measures, and for estimating infection prevalence to facilitate risk analysis. To our knowledge this is the first report on the diagnostic accuracy of an antibody ELISA for an infectious disease in finfish.

Development of recombinant BgP12 based enzyme linked immunosorbent assays for serodiagnosis of Babesia gibsoni infection in dogs

Indirect ELISA and dot-ELISA using recombinant BgP12 (rBgP12) were developed for the diagnosis of Babesia gibsoni infected dogs. The complete open reading frame of BgP12 gene (378bp) was cloned in pET-32a(+) expression vector and expressed in Escherichia coli as a soluble thioredoxin (Trx) fusion protein. The purified rBgP12 was used for production of anti-rBgP12 rabbit serum, which recognized a native 12-kDa protein in B. gibsoni infected erythrocyte by Western blot analysis. To evaluate the potential of rBgP12 for the serodiagnosis of B. gibsoni, a panel of serum/plasma samples from dogs infected with B. gibsoni (n=13), uninfected sera (n=13) and sera from dogs infected with other haemoparasites viz., Babesia canis vogeli (n=3), Ehrlichia canis (n=3), Hepatozoon canis (n=1) and Dirofilaria immitis (n=1) were used in ELISA formats. In addition, the performance of rBgP12 based indirect ELISA and dot-ELISA were evaluated using 75 serum/plasma samples collected from suspected dogs, in respect to the nested PCR as reference test. The diagnostic sensitivities of indirect ELISA and dot-ELISA were 94.59% and 89.18%, respectively, while their specificities were 84.21% and 81.57%, respectively. Moreover, both the assays using rBgP12 showed no cross reaction with sera from dogs infected with other common haemoparasites indicating their high specificity. High kappa values of indirect ELISA and dot-ELISA indicated the potentials of these assays with substantial agreement at 95% confidence level. It is concluded that indirect ELISA and dot ELISA using rBgP12 might be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs.