Littérature scientifique sur le sujet « Immuno-enzymatic assays »

Abstract Objective The interference of the hemoglobin variant (Hb J-Bangkok) was evaluated on 4 different glycated hemoglobin assays and compared with a reference immuno assay. Methods An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of Hb J-Bangkok caused a statistically significant difference in HbA1c results compared with a reference immuno assay. Statistical analysis was performed on the difference of the estimated average glucose calculated from HbA1c values and fasting plasma glucose in the Hb J-Bangkok variant group using the different detection systems. Deming regression analysis was used to determinate whether Hb J-Bangkok had a significant interference on HbA1c results using an HbA1c±10% relative bias at 6% and 9% HbA1c as evaluation limits. Results Turbidimetric inhibition immunoassay method, and enzymatic methods were not affected by Hb J-Bangkok. However, Hb J-Bangkok showed statistically significant interference to the two ion-exchange high-performance liquid chromatography methods. Conclusion When performing HbA1c tests, clinical laboratory personnel should identify the Hb variant and select the appropriate methods or use alternative indicators.

SummaryAs part of a European multicentre prospective study involving the measurement of a number of haemostatic factors, a quality assessment (QA) scheme was organized. This paper describes the preparation, design and results of the first Qa exercise, involving 16 European laboratories and 10 haemostatic assays. The design allowed the investigation, for each assay, of the variability between duplicates and the variability between days within each centre, and of the agreement between centres. A graphical presentation of each centre’s performance in comparison to that of others was adopted, which preserved the confidentiality of each centre’s results. The factor VIII clotting activity assay (VIII: C) and the rocket immuno-electrophoresis assays of von Willebrand factor related antigen (vWF R:Ag), antithrombin III, protein C and histidine-rich glycoprotein showed the highest betweenduplicate and between-day coefficients of variation (CVs), whereas the clotting assays of activated partial thromboplastin time and fibrinogen had the lowest CVs. CVs for the enzymatic assays using synthetic substrates of antithrombin III, plasminogen and alpha-2-antiplasmin were between these extremes. The between-centre CVs were high for both the VIII:C and vWFR:Ag assays. The QA exercise showed that, in multicentre studies involving the measurement of haemostatic factors, it is feasible to undertake analysis locally at each centre.

Heterotis niloticus is an African species of Osteoglossiformes that presents biological peculiarities and zootechnical performances favorable for fish farming. However, the absence of a sexual dimorphism hinders the optimization of its reproduction in captivity and limits the understanding of its reproductive behavior. This study is aimed at developing a minimally invasive and reliable sexing method to detect vitellogenin (Vtg) in female plasma. A commercial sexing kit (Acobium, Montpellier, France) for Arapaima gigas—a phylogenetically sister species of H. niloticus—successfully identified only 20% of mature H. niloticus females. Enzyme-linked immunosorbent assays (ELISA) were carried out using three Vtg antibodies. The A. gigas Vtg1 antibody cross-reacted significantly with plasma dilutions of female H. niloticus ranging from 1:1000 to 1:10,000, but with relatively low intensity. The Vtg antibody from Osteoglossum bicirrhosum, another species of Osteoglossiformes, showed non-specific binding with the Vtg of H. niloticus female plasma. Finally, an antibody for H. niloticus Vtg developed in this study allowed us to differentiate the two sexes with plasma coating dilutions ranging from 1:1000 to 1:10,000. The results of the assay were validated by a proteomic approach showing that Vtg-targeted mass spectrometry analysis of H. niloticus blood protein extracts could be used to accurately determine the presence of Vtg in the plasma of mature females. The final validation of the ELISA technique using the H. niloticus Vtg antibody was confirmed by visual sexing of a significant number of blood-sampled fish gonads; 100% of the fish were correctly sexed by the ELISA method.

Abstract Background With the worldwide increase of diabetes mellitus prevalence, ensuring that HbA1c assays are accurate is essential. External quality assessment (EQA) programs enable laboratories to verify that analytical methods perform according to the manufacturers’ specifications. However, assessing trueness requires commutable materials, a property that is rarely characterized for EQA materials. Methods The difference in bias approach was used to assess commutability of 26 processed quality control materials for 17 of the most frequently used HbA1c assays. Involved assays included immuno-assays, enzymatic assays, affinity, ion-exchange HPLC boronate affinity HPLC and capillary electrophoresis. The measurements were performed at manufacturers or expert laboratories. Assay trueness was additionally assessed against the IFCC reference measurement procedure using fresh clinical specimens that were distributed to 450 medical laboratories. Results Commutability of processed EQA materials was highly heterogeneous and globally insufficient to rigorously assess the trueness of HbA1c assays. Using fresh clinical specimens, mean bias was −0.13 mmol/mol for low HbA1c (34 mmol/mol), between +1.0 and +1.3 mmol/mol for intermediate HbA1c (49 and 58 mmol/mol) and +1.2 mmol/mol for elevated HbA1c (90 mmol/mol). Conclusions This study demonstrates that due to insufficient commutability, most processed EQA materials are unsuitable to assess trueness of HbA1c assays and agreement between the different assays. These materials can only provide information on comparability of individual laboratory results with its peers and on assay precision. Using fresh whole blood samples, this study additionally shows that most HbA1c assays are fairly accurate and meet the total allowable error quality target of 5 mmol/mol.

ABSTRACT Diagnosing chronic Chagas disease (CD) requires antibody–antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi -specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti- T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania , a pathogen with high similarity to T. cruzi , showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland–Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

The patho-mechanism of changes in the thyroid gland, including carcinogenesis, is a complex process, which involves oxidative stress. The goal of our investigation was to verify the extent of stress in the thyroid gland related to glycation. The study samples were comprised of blood sera, thyroid, and adipose tissue sections probed from 37 patients diagnosed with thyroid cancers and goiter. Using immuno-enzymatic and fluorometric assays we analyzed the content of advanced glycation end-products (AGEs), pentosidine, receptors for advanced glycation end-products (RAGE), scavenger receptor class (SR)-A, SR-B, glutathione, malondialdehyde and nitric oxide synthase. In addition to classic AGEs, a recent study detected the melibiose-derived glycation (MAGE) product. We demonstrated the presence of AGEs, MAGE and their receptors of the RAGE and SR-A. In addition, in the control samples of thyroid glands SR-B groups were detected as well as of pathological groups without noticeable tendency to antigen concentration in the area of carcinogenesis. Fluorescent AGEs correlate positively with glutathione, which supports the assumption that glycation stress leads to augmentation of oxidative stress and increase of the intensity of antioxidant mechanisms.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which is highly inflammatory. Compared to a healthy control group, SLE patients exhibit a higher concentration of advanced glycation end products (AGEs) and a lower concentration of receptors for AGEs (RAGE) in serum, however, the exact aetiology is still unclear. In the present study, non-enzymatic glycation induced modification of human serum albumin (HSA) has been studied by biophysical techniques. Glycated HSA (G-HSA) was used as an antigen, and serum autoantibody levels were estimated in SLE and normal humans (NH) against it, using direct binding ELISA and competitive inhibition ELISA. Compared to N-HSA, remarkable structural modifications were observed in G-HSA. Modified HSA also showed increased pentosidine fluorescence (213.7 ± 13.4 AU). Glycation of HSA induced a conversion of α-helix and random coil to β-sheet and β-turns. Serum immuno assays results exhibited significantly (p < 0.001) higher binding of G-HSA with serum autoantibodies from SLE patients when compared with native HSA (N-HSA). Furthermore, competitive ELISA results showed significantly (p < 0.001) high percent inhibition of serum IgG from SLE patients with modified antigen. Chronic inflammation with excessive oxidative stress in SLE patients could be a possible reason for structural alterations in blood proteins, generating highly immunogenic unique new-epitopes. These in turn induce the generation of specific autoantibodies against G-HSA that may serve as a potential biomarker for SLE pathogenesis.

Abstract Introduction Our group and others have reported the murine SHIP1-deficient mouse model of MDS/CMML is characterized by expansion of immunosuppressive, arginase 1 (Arg1)-positive M2-macrophages and myeloid-derived suppressor cells (MDSC) (Rauh et al., Immunity, 2005). Moreover, translational studies confirmed increased Arg1 in bone marrow aspirate cells of subsets of MDS and CMML patients (Rauh et al., Blood Abstract 2010: 1855), although involving impractical Arg1 enzymatic assays and Western blots. Herein, our goals were to 1) confirm this Arg1 immune signature in an independent cohort of patients, using more clinically applicable immunohistochemistry (IHC), 2) determine the associated risk profile with recent prognostic scoring systems, and 3) begin to connect this Arg1 immune signature with recurring MDS/CMML-acquired mutations. Methods With ethics approval, 29 BM biopsies (decalcified, FFPE) and clinical parameters were retrieved from the archives of Kingston General Hospital: 6 controls (4 negative lymphoma staging BM and 2 mild anemia NYD; mean age +/- std = 57 +/- 13 y), 13 MDS (1 RA, 1 RARS, 1 del(5q), 5 RCMD, 3 RAEB-1, 2 RAEB-2; 76 +/- 14 y), and 10 CMML patients (7 CMML-1, 3 CMML-2; 72 +/- 10 y). H&E and anti-human Arg1 IHC (1/2500 dilution, clone HPA003595, Sigma) were conducted under optimized, automated conditions (Ventana). IHC scoring was recorded independently by 2 blinded Hematopathologists. IPSS, IPSS-R (Greenberg), WPSS (Malcovati), CMML PSS (Such) and Mayo CMML (Patnaik) scores were calculated. Floxed TET2 and Vav-Cre mice were obtained from JAX and used according to approved Queen's University Animal Care protocols. TET2 sequences were obtained from genomic DNA using custom AmpliSeq primer pools and the Ion Torrent PGM platform (LifeTech). Linear regression and student's t-tests were conducted with Prism software (GraphPad). Results 1) We demonstrated increased BM biopsy Arg1 IHC expression in CMML (22 +/- 17% Arg1-positive cells; n = 10; p = 0.0093) and low-grade MDS, particularly RCMD (14 +/- 11%; n = 5; p = 0.012) relative to control subjects (0.4 +/- 1%; n = 6) (Figure 1). Significantly increased mean Arg1 expression was not seen in other MDS subtypes (n = 8). These findings were consistent with our previously reported 40-subject (Toronto) BM aspirate Arg1 assay/Western blot cohort (Rauh et al. Blood Abstract 2010:1855), suggesting the reproducibility of these findings and the clinical utility of IHC-based assessment. Parallel Arg1 IHC is underway on the Toronto cohort, to determine correlations with enzymatic assays/Westerns. 2) We previously reported Arg1 over-expression in the Toronto cohort was significantly associated with the lowest IPSS and WPSS MDS clinical risk categories and now extend this to IPSS-R. In our Kingston cohort, only trends to lower MDS risk were observed. In contrast, increased Arg1 expression was not associated with clinical risk (CPSS and Mayo scores) in either CMML cohort. Thus, Arg1 expression associated with neutral risk in CMML and neutral to lower risk in MDS patients. Both the clinical risk profiles and proportions of Arg1 over-expressing MDS/CMML patients were reminiscent of reported TET2 mutation risk profiles/percentages. 3) TET2-deficient mice demonstrated increased monocytes, macrophages and CD11b+Gr1+ splenocytes (immuno-phenotypically consistent with MDSC), phenocopying SHIP1-deficient mice. We are currently determining whether TET2-/- mouse macrophages are similarly M2-skewed. In parallel, were are obtaining TET2 genomic sequences (along with other recurring mutated genes) for our Toronto and Kingston cohorts, in order to determine if the Arg1 immune signature associates with a particular mutation(s). These findings will be discussed. Conclusions 1) Using two independent patient cohorts, we demonstrated significant Arg1 over-expression in CMML and low-grade (RCMD) MDS. Arg1 IHC warrants further investigation as an ancillary diagnostic test. 2) Increased Arg1 expression had neutral prognostic significance in CMML and neutral to low-risk MDS associations. 3) Increased Arg1 expression in MDS/CMML may be driven by mutant TET2, impacting the epigenetics of MDSC and M2-macrophage expansion, controlled by SHIP1 and related signaling networks. Confirmatory studies are in progress. Disclosures: No relevant conflicts of interest to declare.

Autoimmune disorders (AID) represent a group of chronic and heterogeneous diseases, whose common trait is the immune system reaction against self-components of the organism. Most of the AID have unknown etiology, but it was demonstrated that both genetic and environmental factors are involved in triggering the pathologic mechanism. Because of their chronicity and their debilitating complications, AID have high medical and socioeconomic costs, leading to the crucial necessity to perform an early diagnosis and to monitor the disease follow up. Unfortunately, the available diagnostic and prognostic instruments are often complicated and invasive. In order to develop diagnostic and/or prognostic tools marked by low-invasivity, low-cost, and easy execution, it is crucial to detect trustworthy biomarkers (BM). The BM characterization has a significant importance also because it represents a powerful instrument to disclose the molecular mechanisms involved in the ethiopathogenesis of the disorder of interest. In this context, the main goal of this work is to identify the target(s) of the humoral autoimmune response using the chemical reverse approach, which involves the screening of focused Ag libraries with pts serum. Indeed, in the case of autoimmunity, an easily detectable and reliable BM may be represented by the titer of a specific auto-Ab. A key feature of this study is the evaluation of the role of aberrant post-translational modifications (PTM) in autoimmunity, as it was hypothesized that environmental agents may induce the occurrence of non-natural PTM on self-proteins, uncovering neo-epitopes and triggering the autoimmune response. In particular, the attention was focused on two main topics, which will be treated separately within this presentation: the role of Myelin Oligodendrocyte Glycoprotein (MOG) as putative auto-Ag in central nervous system AID (Chapter 2); investigation of the involvement of aberrant PTM in the ethiopathogenesis of Primary Biliary Cirrhosis (Chapter 3).

The protein C activator in Southern Copperhead (Agkistrodon Contortrix Contortrix) venom was isolated by sequential chromatographies on SP�Sephadex, Con A Sepharose and hydroxylapatite. It was found to be a single chain glycoprotein with an apparent molecular weight of 36,000 and an enzymatic specificity on chromogenic substrates resembling kallikein.This "contortrix activator" was used in a solid-phase immunochromometric assay (ICMA) for functional protein C in which heterologous antibody against protein C was passively coated onto microtitre wells and used to immoblize protein C. This was then activated, easily freed of excess activator by washing and assessed by its subsequent overnight cleavage of chromogenic substrates sensitive to activated protein C.Correlation between protein C results obtained by ICMA and immunoradiometric assay (IRMA) on a variety of patient samples was excellent when relatively high concentrations of the venom activator was used. However with lower concentration of activator plasmas from patients deficient in vitamin K gave lower protein C values by ICMA then obtained by IRMA.Normal protein C and "acarboxy" protein C from a patient on oral anticoagulant therapy were immuno-immobilized and studied by the ICMA technique using varying concentrations of the venom activator. The acarboxy-protein C, although completely activatable by high concentrationa of activator, was found to activate much more slowly than normal protein C at low concentrations of the contortrix activator. Thus by reducing the intensity of the activation step, the ICMA protein C results were increased in their sensitivity for functional protein C.