Sensitive and specific serological ELISA for the detection of SARS-CoV-2 infections.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.

CD99 and polymeric immunoglobulin receptor peptides deregulation in critical COVID-19: A potential link to molecular pathophysiology?

Identification of significant changes in urinary peptides may enable improved understanding of molecular disease mechanisms. We aimed towards identifying urinary peptides associated with critical course of COVID-19 to yield hypotheses on molecular pathophysiological mechanisms in disease development. In this multicentre prospective study urine samples of PCR-confirmed COVID-19 patients were collected in different centres across Europe. The urinary peptidome of 53 patients at WHO stages 6-8 and 66 at WHO stages 1-3 COVID-19 disease was analysed using capillary electrophoresis coupled to mass spectrometry. 593 peptides were identified significantly affected by disease severity. These peptides were compared with changes associated with kidney disease or heart failure. Similarities with kidney disease were observed, indicating comparable molecular mechanisms. In contrast, convincing similarity to heart failure could not be detected. The data for the first time showed deregulation of CD99 and polymeric immunoglobulin receptor peptides and of known peptides associated with kidney disease, including collagen and alpha-1-antitrypsin. Peptidomic findings were in line with the pathophysiology of COVID-19. The clinical corollary is that COVID-19 induces specific inflammation of numerous tissues including endothelial lining. Restoring these changes, especially in CD99, PIGR and alpha-1-antitripsin, may represent a valid and effective therapeutic approach in COVID-19, targeting improvement of endothelial integrity.

Lower HbA1c in patients with type 1 diabetes and celiac disease who reached celiac-specific antibody-negativity-A multicenter DPV analysis.

OBJECTIVES: To study celiac-specific antibody status over 3 years in patients with type 1 diabetes and biopsy-proven celiac disease (T1D + CD). Furthermore, to determine clinical differences after diagnosis between patients reaching constant antibody-negativity (Ab-neg) and staying antibody-positive (Ab-pos). METHODS: A total of 608 pediatric T1D + CD patients from the multicenter DPV registry were studied longitudinally regarding their CD specific antibody-status. Differences between Ab-neg (n = 218) and Ab-pos (n = 158) patients 3 years after biopsy were assessed and compared with 26 833 T1D patients without CD by linear and logistic regression adjusted for age, gender, diabetes duration and migration background. RESULTS: Thirty-six percent of T1D + CD patients reached and sustained antibody-negativity 3 years after CD diagnosis. The median time until patients returned to Ab-neg was 0.86 (0.51;1.16) years. Three years after diagnosis, HbA1c was lowest in Ab-neg and highest in Ab-pos patients compared to T1D-only patients (adjusted mean (95%CI): 7.72 (7.51-7.92) % vs 8.44 (8.20-8.68) % vs 8.19 (8.17-8.21) %, adjusted P < 0.001, respectively). Total cholesterol, LDL-cholesterol and frequency of dyslipidemia were significantly lower in Ab-neg compared to T1D-only patients (167 (161-173) mg/dl vs 179 (178-179) mg/dl, P < .001; 90 (84-96) mg/dl vs 99 (98-99) mg/dl, P = .005; 15.7 (10.5-22.9) % vs 25.9 (25.2-26.6) %, P = .017). In longitudinal analyses over 6 years after diagnosis, a constantly higher HbA1c (P < .001) and a lower height-SDS (P = .044) was observed in Ab-pos compared to Ab-neg patients. CONCLUSION: Only one third of T1D + CD patients reached constant Ab-negativity after CD diagnosis. Achieving Ab-negativity after diagnosis seems to be associated with better metabolic control and growth, supposedly due to a higher adherence to therapy in general.

Will the Real Coeliac Disease Please Stand Up? Coeliac Disease Prevalence in the German LIFE Child Study.

OBJECTIVES: Assessing the seroprevalence and the prevalence of definite coeliac disease (CD) in the German LIFE Child Health study cohort including immunoglobulin A (IgA) antibodies against tissue transglutaminase (IgA-TTG) in addition to IgG antibodies against deamidated gliadin peptides (IgG-DGP) and human leukocyte antigen (HLA)-DQ2/8 genotyping. METHODS: Samples from children and adolescents were first screened for IgA-TTG and IgG-DGP. If IgA-TTG was above 0.5 times the upper limit of normal and/or IgG-DGP was positive, IgA antibodies against endomysium (IgA-EmA) were measured, and HLA was genotyped. In patients with only IgG-DGP positivity, total IgA was assayed. Subjects with suspicious results were followed up serologically and, in case of repeatedly positive antibody results, invited for a personal interview. Further diagnostic data were obtained independent from our study. RESULTS: We screened 2363 children's blood samples collected from 2011 to 2015. The seroprevalence, that is, IgA-TTG and/or IgA-EmA positivity or IgG-DGP positivity with IgA <0.05 g/L, was 1.57% (95% confidence interval [CI95%] 1.14-2.15). The prevalence of suspected CD, that is, seroprevalence and compatible HLA genotype with hitherto unknown mucosal damage, was 1.35% (CI95% 0.96-1.91). Definite CD, that is, seropositivity accompanied by positive intestinal biopsy or IgA-TTG ≥ 10 × upper limit of normal, was found in 0.42% (CI95% 0.22-0.80). Seven children claimed to have CD. The HLA haplotype, however, matched in only 4 of them resulting in an overall CD prevalence of at least 0.59% (CI95% 0.34-1.02). Thirteen unclear cases remained; therefore, the prevalence may even be higher. CONCLUSIONS: The prevalence of definite CD in a population-representative German cohort is higher than previously described. HLA-DQ typing is helpful to identify false-positive IgA-TTG patients negative for IgA-EmA and/or IgG-DGP under screening conditions and unmasks possible misdiagnoses of CD.

Antibody Concentrations Decrease 14-Fold in Children With Celiac Disease on a Gluten-Free Diet but Remain High at 3 Months.

BACKGROUND & AIMS: Celiac disease can be identified by a serologic test for IgA against tissue transglutaminase (IgA-TTG) in a large proportion of children. However, the increased concentrations of antibody rarely normalize within the months after children are placed on a gluten-free diet (GFD). Early serologic predictors of sufficient adherence to GFD are required for optimal treatment. METHODS: In a prospective study, we observed the response to a GFD in 345 pediatric patients (67% girls; mean age, 8.4 y) who underwent duodenal biopsy to confirm or refute celiac disease from October 2012 through December 2015. Baseline serum samples were tested centrally for IgA-TTG and IgG against deamidated gliadin. Follow-up serologic analyses of children on a GFD were performed about 3 months later. RESULTS: The geometric mean concentration of IgA-TTG decreased from 72.4-fold to 5.2-fold the upper limit of normal (ULN), or by a factor of 14.0 (95% CI, 12.0-16.4). A substantial response (defined as a larger change than the typical variation in patients not on a GFD) was observed in 80.6% of the children. Only 28.1% of patients had a substantial response in the concentration of IgG against deamidated gliadin. Concentration of IgA-TTG remained above 1-fold the ULN in 83.8% of patients, and above 10-fold the ULN in 26.6% of patients with a substantial response. CONCLUSIONS: Serum concentration of IgA-TTG decreases substantially in most children with celiac disease within 3 months after they are placed on a GFD, but does not normalize in most. This information on changes in antibody concentrations can be used to assess patient response to the diet at short-term follow-up evaluations. Patients with a substantial response to a GFD often still have high antibody levels after 3 months. German Clinical Trials Registry no. DRKS00003854.

Hemolysis and IgA-antibodies against tissue transglutaminase: When are antibody test results no longer reliable?

BACKGROUND: Antibodies against tissue transglutaminase (TTG) of isotype IgA (IgA-aTTG) represent reliable diagnostic markers to confirm or exclude celiac disease (CD). Hemolysis (HL) is an important pre-analytical factor. HL can be quantified as HL index (HI) correlating with the concentration of free hemoglobin. TTG is abundant in erythrocytes and released upon HL. In immunoassays, the released TTG may interfere with binding of IgA-aTTG to the coated TTG. METHODS: We selected 17 HL-free sera from children with biopsy-confirmed CD: 7 with low-positive (1-5 multiples of upper limit of normal [×ULN]), 5 with intermediate (5-10 × ULN) and 5 with high IgA-aTTG (10-15 × ULN). Sera were spiked with hemolysates resulting in HIs ranging from 12.5 to 800 (12.5-800 mg/dL free hemoglobin). RESULTS: IgA-aTTG values were significantly decreased (>10%) after addition of hemolysates even if HL was invisible (HI <50). This effect is diagnosis-relevant if IgA-aTTG values are measured just below the cut-offs: (i) 0.4-1 × ULN at HI ≥25 (CD not excludable) and (ii) 8.5-10 × ULN at HI ≥200 (diagnosis of CD without biopsy not possible). Antibodies against deamidated gliadin were not influenced by HL. CONCLUSIONS: IgA-aTTG results in sera with HI ≥25 can yield inconclusive results. Therefore, those antibody results should be assessed only under consideration of the HI.

Dark-field imaging in coronary atherosclerosis.

OBJECTIVES: Dark-field imaging based on small angle X-ray scattering has been shown to be highly sensitive for microcalcifications, e.g. in breast tissue. We hypothesized (i) that high signal areas in dark-field imaging of atherosclerotic plaque are associated with microcalcifications and (ii) that dark-field imaging is more sensitive for microcalcifications than attenuation-based imaging. METHODS: Fifteen coronary artery specimens were examined at an experimental set-up consisting of X-ray tube (40kV), grating-interferometer and detector. Tomographic dark-field-, attenuation-, and phase-contrast data were simultaneously acquired. Histopathology served as standard of reference. To explore the potential of dark field imaging in a full-body CT system, simulations were carried out with spherical calcifications of different sizes to simulate small and intermediate microcalcifications. RESULTS: Microcalcifications were present in 10/10 (100%) cross-sections with high dark-field signal and without evidence of calcifications in attenuation- or phase contrast. In positive controls with high signal areas in all three modalities, 10/10 (100%) cross-sections showed macrocalcifications. In negative controls without high signal areas, no calcifications were detected. Simulations showed that the microcalcifications generate substantially higher dark-field than attenuation signal. CONCLUSIONS: Dark-field imaging is highly sensitive for microcalcifications in coronary atherosclerotic plaque and might provide complementary information in the assessment of plaque instability.

Validation of Antibody-Based Strategies for Diagnosis of Pediatric Celiac Disease Without Biopsy.

BACKGROUND & AIMS: A diagnosis of celiac disease is made based on clinical, genetic, serologic, and duodenal morphology features. Recent pediatric guidelines, based largely on retrospective data, propose omitting biopsy analysis for patients with concentrations of IgA against tissue transglutaminase (IgA-TTG) >10-fold the upper limit of normal (ULN) and if further criteria are met. A retrospective study concluded that measurements of IgA-TTG and total IgA, or IgA-TTG and IgG against deamidated gliadin (IgG-DGL) could identify patients with and without celiac disease. Patients were assigned to categories of no celiac disease, celiac disease, or biopsy required, based entirely on antibody assays. We aimed to validate the positive and negative predictive values (PPV and NPV) of these diagnostic procedures. METHODS: We performed a prospective study of 898 children undergoing duodenal biopsy analysis to confirm or rule out celiac disease at 13 centers in Europe. We compared findings from serologic analysis with findings from biopsy analyses, follow-up data, and diagnoses made by the pediatric gastroenterologists (celiac disease, no celiac disease, or no final diagnosis). Assays to measure IgA-TTG, IgG-DGL, and endomysium antibodies were performed by blinded researchers, and tissue sections were analyzed by local and blinded reference pathologists. We validated 2 procedures for diagnosis: total-IgA and IgA-TTG (the TTG-IgA procedure), as well as IgG-DGL with IgA-TTG (TTG-DGL procedure). Patients were assigned to categories of no celiac disease if all assays found antibody concentrations <1-fold the ULN, or celiac disease if at least 1 assay measured antibody concentrations >10-fold the ULN. All other cases were considered to require biopsy analysis. ULN values were calculated using the cutoff levels suggested by the test kit manufacturers. HLA typing was performed for 449 participants. We used models that considered how specificity values change with prevalence to extrapolate the PPV and NPV to populations with lower prevalence of celiac disease. RESULTS: Of the participants, 592 were found to have celiac disease, 345 were found not to have celiac disease, and 24 had no final diagnosis. The TTG-IgA procedure identified patients with celiac disease with a PPV of 0.988 and an NPV of 0.934; the TTG-DGL procedure identified patients with celiac disease with a PPV of 0.988 and an NPV of 0.958. Based on our extrapolation model, we estimated that the PPV and NPV would remain >0.95 even at a disease prevalence as low as 4%. Tests for endomysium antibodies and HLA type did not increase the PPV of samples with levels of IgA-TTG ≥10-fold the ULN. Notably, 4.2% of pathologists disagreed in their analyses of duodenal morphology-a rate comparable to the error rate for serologic assays. CONCLUSIONS: In a prospective study, we validated the TTG-IgA procedure and the TTG-DGL procedure in identification of pediatric patients with or without celiac disease, without biopsy. German Clinical Trials Registry no.: DRKS00003854.

Improved Diagnostics by Assessing the Micromorphology of Breast Calcifications via X-Ray Dark-Field Radiography.

Breast microcalcifications play an essential role in the detection and evaluation of early breast cancer in clinical diagnostics. However, in digital mammography, microcalcifications are merely graded with respect to their global appearance within the mammogram, while their interior microstructure remains spatially unresolved and therefore not considered in cancer risk stratification. In this article, we exploit the sub-pixel resolution sensitivity of X-ray dark-field contrast for clinical microcalcification assessment. We demonstrate that the micromorphology, rather than chemical composition of microcalcification clusters (as hypothesised by recent literature), determines their absorption and small-angle scattering characteristics. We show that a quantitative classification of the inherent microstructure as ultra-fine, fine, pleomorphic and coarse textured is possible. Insights underlying the micromorphological nature of breast calcifications are verified by comprehensive high-resolution micro-CT measurements. We test the determined microtexture of microcalcifications as an indicator for malignancy and demonstrate its potential to improve breast cancer diagnosis, by providing a non-invasive tool for sub-resolution microcalcification assessment. Our results indicate that dark-field imaging of microcalcifications may enhance the diagnostic validity of current microcalcification analysis and reduce the number of invasive procedures.

Primate liver tissue as an alternative substrate for endomysium antibody immunofluorescence testing in diagnostics of paediatric coeliac disease.

BACKGROUND: Immunofluorescence assays of antibodies against endomysium (EmA) on primate oesophagus sections represent the gold standard in serological testing for coeliac disease (CD). As alternative immunofluorescence technique, staining of primate liver tissue is in use. We compared performance and predictive power of IgA- and IgG-EmA on primate oesophagus and primate liver sections. METHODS: Sera of 298 paediatric biopsy-proven CD patients under gluten-containing diet and 574 disease controls were investigated. Samples were collected between 2004 and 2013 in six children's hospitals. The antibodies were assayed blinded to diagnoses and histological data. Sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) were calculated for different assays. RESULTS: (Oesophagus vs liver): For IgA-EmA, sensitivity (0.953 vs 0.956) and specificity (0.981 vs 0.972) as well as PPV (0.963 vs 0.947) and NPV (0.976 vs 0.979) were comparable on both tissues. IgG-EmA on liver showed significantly higher sensitivity (0.520 vs 0.631; p=0.006) but significantly lower specificity (0.995 vs 0.963; p=0.002) and PPV (0.981 vs 0.899; p=0.0002) than on oesophagus. NPV on liver was higher than NPV on oesophagus, however, the difference was not statistically significant (0.799 vs 0.834; p=0.099). CONCLUSION: Primate liver can be used as alternative, equally well functioning substrate for IgA-EmA testing.

Vascular risk factors in children, adolescents, and young adults with type 1 diabetes complicated by celiac disease: results from the DPV initiative.

OBJECTIVE: Celiac disease (CD) is a common comorbidity of type 1 diabetes (T1D). Long-term consequences of CD are not completely understood, and adhering to a gluten-free diet is a burden for many patients. We investigated the effect of CD on vascular risk factors in a large cohort of T1D patients aged <20 yr. RESEARCH DESIGN AND METHODS: Within the longitudinal Diabetes Patienten Verlaufsdokumentation (DPV)-diabetes registry, data were analyzed from 59,909 < 20-yr-old T1D patients treated at 392 centers in Germany and Austria. A total of 974 patients with biopsy-proven celiac disease (BPCD) were compared with 28,398 patients without CD with respect to blood pressure (BP), lipids, glycohemoglobin (HbA1c ), body mass index (BMI), and reported smoking behavior. RESULTS: Patients with T1D and BPCD showed significantly lower high-density lipoprotein (HDL) cholesterol levels [median (interquartile range): 53.0 (43.0-62.6) mg/dL] than patients without CD [55.0 (45.0-66.0) mg/dL; p < 0.01; p < 0.001 after adjustment for confounding variables]. Systolic BP was lower in patients with CD [105.5 (100.0-112.5) mmHg] than in patients without CD [110.0 (102.0-117.0) mmHg; p < 0.0001; p < 0.001 after adjustment]. There were no significant differences regarding smoking behavior, BMI, or HbA1c . In a subgroup of 335 patients with BPCD, HDL cholesterol was measured 1 yr after diagnosis of CD:HDL increased by 8% (p < 0.01). CONCLUSION: Young people with T1D and CD have lower HDL cholesterol values than patients without CD. As low HDL cholesterol is associated with vascular risk, our findings support screening for CD and monitoring of HDL cholesterol in young people with T1D.

Microvascular Complications in Childhood-Onset Type 1 Diabetes and Celiac Disease: A Multicenter Longitudinal Analysis of 56,514 Patients From the German-Austrian DPV Database.

OBJECTIVE: To investigate whether celiac disease (CD) associated with type 1 diabetes increases the risk of microvascular complications. RESEARCH DESIGN AND METHODS: Patients (n = 56,514) aged >10 years with diabetes duration <20 years from 392 centers in Germany and Austria were assigned to one of three categories (n): no CD (50,933), biopsy-confirmed CD (812), or suspected CD (4,769; clinical diagnosis or positive antibodies). The confirmed and suspected groups were combined and analyzed for retinopathy or nephropathy. Cox proportional hazards regression was used to adjust for potential confounders (glycated hemoglobin [HbA1c], age at diabetes onset, sex, smoking, dyslipidemia, and hypertension). RESULTS: Kaplan-Meier analysis revealed that retinopathy and nephropathy occurred earlier in the presence versus absence of CD: retinopathy at age 26.7 years (95% CI 23.7-30.2) in 25% of patients with CD vs. age 33.7 years (33.2-34.4) in 25% without CD and microalbuminuria at age 32.8 years (29.7-42.5) vs. 42.4 years (41.4-43.3). The adjusted risk for both retinopathy (hazard ratio 1.263 [95% CI 1.078-1.481]) and nephropathy (1.359 [1.228-1.504]) was higher in patients with diabetes and CD versus those without CD. Cox regression revealed CD as an independent risk factor for microvascular complications after adjustment for confounders. CONCLUSIONS: CD is an independent risk factor for retinopathy and nephropathy in patients with type 1 diabetes. Our study therefore supports the recommendation for regular serologic testing for CD, even in the absence of clinical CD. Further prospective studies are required to investigate whether a gluten-free diet might reduce the risk of microvascular disorders in patients with diabetes and CD.

Fast one-dimensional wave-front propagation for x-ray differential phase-contrast imaging.

Numerical wave-optical simulations of X-ray differential phase-contrast imaging using grating interferometry require the oversampling of gratings and object structures in the range of few micrometers. Consequently, fields of view of few millimeters already use large amounts of a computer's main memory to store the propagating wave front, limiting the scope of the investigations to only small-scale problems. In this study, we apply an approximation to the Fresnel-Kirchhoff diffraction theory to overcome these restrictions by dividing the two-dimensional wave front up into 1D lines, which are processed separately. The approach enables simulations with samples of clinically relevant dimensions by significantly reducing the memory footprint and the execution time and, thus, allows the qualitative comparison of different setup configurations. We analyze advantages as well as limitations and present the simulation of a virtual mammography phantom of several centimeters of size.

The influence of IL-10 and TNFα on chondrogenesis of human mesenchymal stromal cells in three-dimensional cultures.

Chondrogenic differentiated mesenchymal stromal cells (MSCs) are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D) culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL)-10 and Tumor Necrosis Factor (TNF)α on chondrogenesis by MSCs in 3D high-density (H-D) culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA) scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls.

Antibodies in the diagnosis of coeliac disease: a biopsy-controlled, international, multicentre study of 376 children with coeliac disease and 695 controls.

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.

Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.