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Combination testing for antibodies in the diagnosis of coeliac disease: comparison of multiplex immunoassay and ELISA methods.

ABSTRACT: BACKGROUND:Tissue transglutaminase (TTG) antibodies and newly developed deamidated gliadin peptide (DGP) antibodies have better accuracy than native gliadin antibodies. Multiplex immunoassay (MIA) measures multiple antibodies simultaneously providing a complete antibody phenotype with reduced turnaround time and cost. AIM:To evaluate the agreement between MIA and enzyme-linked immunosorbent assay (ELISA) test results for coeliac autibodies in biopsy-proven coeliac patients and controls and to model the diagnostic utility of combination testing. METHODS:We compared the sensitivity, specificity and accuracy of MIA and ELISA methods for TTG and DGP antibodies in mainly adult untreated coeliac patients (n = 92) and controls (n = 124). RESULTS:There was excellent agreement and a significant correlation between the results of MIA and ELISA methods (k > 0.8, r > 0.7) for all tests, except IgG. Diagnostic indices of individual and combination tests measured by the MIA method did not differ significantly from those measured by ELISA. The combination tests slightly increased sensitivity (if any test was positive) and specificity (if all tests were positive) compared to the individual tests. CONCLUSIONS:Multiplex immunoassay testing for antibodies is as accurate as ELISA for coeliac disease diagnosis and has practical advantages over ELISA method. Rational combination testing can help identify patients who need intestinal biopsy and may reduce unnecessary biopsies.

SUBMITTER: Rashtak S

PROVIDER: S-EPMC2666354 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Combination testing for antibodies in the diagnosis of coeliac disease: comparison of multiplex immunoassay and ELISA methods.

Rashtak S S Ettore M W MW Homburger H A HA Murray J A JA

Alimentary pharmacology & therapeutics 20080901 6

<h4>Background</h4>Tissue transglutaminase (TTG) antibodies and newly developed deamidated gliadin peptide (DGP) antibodies have better accuracy than native gliadin antibodies. Multiplex immunoassay (MIA) measures multiple antibodies simultaneously providing a complete antibody phenotype with reduced turnaround time and cost.<h4>Aim</h4>To evaluate the agreement between MIA and enzyme-linked immunosorbent assay (ELISA) test results for coeliac autibodies in biopsy-proven coeliac patients and con ...[more]

PMID: 19145736

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Project description:BACKGROUND: With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study. RESULTS: Of the ligation protocols tested in this study, the best results were obtained with the PPLMD I and PPLMD II protocols and no consistent differences between these two protocols were observed. Both protocols are based on padlock probe ligation combined with microarray detection. Twenty PLPs were tested for specificity and the best probes were subjected to further evaluation. Up to 13 targets were detected specifically and simultaneously. During the interlaboratory exchange study similar results were achieved by the two participating institutes (NIB, Slovenia, and RIKILT, the Netherlands). CONCLUSIONS: From the comparison of ligation protocols it can be concluded that two protocols perform equally well on the basis of the selected set of PLPs. Using the most ideal parameters the multiplicity of one of the methods was tested and 13 targets were successfully and specifically detected. In the interlaboratory exchange study it was shown that the selected method meets the 0.1% sensitivity criterion. The present study thus shows that specific and sensitive multidetection of GMO targets is now feasible.

Project description:Background: Vaccine-induced human papillomavirus (HPV) antibodies originating from cervicovaginal secretions were recently shown to be detectable in first-void (FV) urine. This presents a novel opportunity for non-invasive sampling to monitor HPV antibody status in women participating in large epidemiological studies and HPV vaccine trials. With a view towards method optimization, this study compared measurement of HPV antibodies in FV urine using a multiplex L1/L2 virus-like particles (VLP)-based ELISA (M4ELISA) with previously reported results using a glutathione S-transferase (GST)-L1-based immunoassay (GST-L1-MIA).Methods: We tested 53 paired FV urine and serum samples from 19- to 26-year-old healthy women, unvaccinated (n = 17) or vaccinated with either the bi- or quadrivalent HPV-vaccine during adolescence (n = 36). HPV6/11/16/18 antibodies were measured using M4ELISA and compared with GST-L1-MIA results. Inter-assay and inter-specimen correlations were examined using the Spearman's rank test (rs ).Findings: As expected, lower HPV antibody concentrations were found in FV urine than in serum. Vaccinated women had significantly higher HPV6/11/16/18 antibody levels in both FV urine and serum compared with those unvaccinated (M4ELISA; FV urine p = 0.0003; serum p ? 0.0001). HPV antibody levels in FV urine and serum showed a significant positive correlation (M4ELISA anti-HPV6/11/16/18, rs = 0.85/0.86/0.91/0.79, p ? 0.001). Despite assay differences, there was moderate to good correlation between M4ELISA and GST-L1-MIA (FV urine anti-HPV6/11/16/18, rs = 0.86/0.83/0.89/0.53, p ? 0.0001; serum anti-HPV6/11/16/18, rs = 0.93/0.89/0.94/0.75, p ? 0.0001).Conclusion: FV urine HPV antibody detection is comparable with both assays, further supporting this non-invasive sampling method as a possible option for HPV vaccine assessment. Approaches to improve the sensitivity and larger studies are warranted to determine the feasibility of FV urine for vaccine-induced HPV antibody detection. This article is protected by copyright. All rights reserved.

Project description:Diagnosis of coeliac disease (CD) relies on a combination of clinical, genetic, serological and duodenal morphological findings. The ESPGHAN suggested that biopsy may not be necessary in all cases. New guidelines include omission of biopsy if the concentration of CD-specific antibodies exceeds 10 times the upper limit of normal (10 ULN) and other criteria are met. We analysed the 10 ULN criterion and investigated multiple antibody-assays. Serum was collected from 1071 children with duodenal biopsy (376 CD patients, 695 disease-controls). IgA-antibodies to tissue transglutaminase (IgA-aTTG), IgG-antibodies to deamidated gliadin peptides (IgG-aDGL) and IgA-endomysium antibodies (IgA-EMA) were measured centrally. We considered 3 outcomes for antibody test procedures utilizing IgA-aTTG and/or IgG-aDGL: positive (?10 ULN, recommend gluten-free diet), negative (<1 ULN, no gluten-free diet) or unclear (perform biopsy). Positive (PPV) and negative (NPV) predictive values were based on clear test results. We required that they and their lower confidence bounds (LCB) be simultaneously very high (LCB >90% and PPV/NPV >95%). These stringent conditions were met for appropriate antibody-procedures over a prevalence range of 9-57%. By combining IgG-aDGL with IgA-aTTG, one could do without assaying total IgA. The PPV of IgG-aDGL was estimated to be extremely high, although more studies are necessary to narrow down the LCB. The proportion of patients requiring a biopsy was <11%. The procedures were either equivalent or even better in children <2 years compared to older children. All 310 of the IgA-aTTG positive children were also IgA-EMA positive. Antibody-assays could render biopsies unnecessary in most children, if experienced paediatric gastroenterologists evaluate the case. This suggestion only applies to the kits used here and should be verified for other available assays. Confirming IgA-aTTG positivity (?10 ULN) by EMA-testing is unnecessary if performed on the same blood sample. Prospective studies are needed.

Project description:BackgroundPlasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites.MethodsRecombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins.ResultsFollowing optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8-4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined.ConclusionsThe csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

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Dataset Information

Combination testing for antibodies in the diagnosis of coeliac disease: comparison of multiplex immunoassay and ELISA methods.

Publications

Combination testing for antibodies in the diagnosis of coeliac disease: comparison of multiplex immunoassay and ELISA methods.

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