Cancer biomarkers detection using microstructured protein chip: implementation of customized multiplex immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins, and immobilization conditions (buffer, pH, concentration). We have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions was evaluated on the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that each antibody displays variable biological activities depending on the surface chemistry and on the immobilization procedure. Under optimized conditions, we can reach a limit of detection in tumor antigen as low as 10 pM. Our microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody on the same chip

Identification and verification of heat shock protein 60 as a potential serum marker for colorectal cancer

Colorectal cancer (CRC) is a major public health issue worldwide, and novel tumor markers may contribute to its efficient management by helping in early detection, prognosis or surveillance of disease. The aim of our study was to identify new serum biomarkers for CRC, and we followed a phased biomarker discovery and validation process to obtain an accurate preliminary assessment of potential clinical utility. We compared colonic tumors and matched normal tissue from 15 CRC patients, using two-dimensional difference gel electrophoresis (2D-DIGE), and identified 17 proteins that had significant differential expression. These results were further confirmed by western blotting for heat shock protein (HSP) 60, glutathione-S-transferase Pi, α-enolase, T-complex protein 1 subunit β, and leukocyte elastase inhibitor, and by immunohistochemistry for HSP60. Using mAbs raised against HSP60, we developed a reliable (precision of 5–15%) and sensitive (0.3 ng·mL−1) immunoassay for the detection of HSP60 in serum. Elevated levels of HSP60 were found in serum from CRC patients in two independent cohorts; the receiver-operating characteristic curve obtained in 112 patients with CRC and 90 healthy controls had an area under the curve (AUC) of 0.70, which was identical to the AUC of carcinoembryonic antigen. Combination of serum markers improved clinical performance: the AUC of a three-marker logistic regression model combining HSP60, carcinoembryonic antigen and carbohydrate antigen 19-9 reached 0.77. Serum HSP60 appeared to be more specific for late-stage CRC; therefore, future studies should evaluate its utility for determining prognosis or monitoring therapy rather than early detection

MECANISMES D'IMMUNISATION CONTRE LES MEDICAMENTS (INDUCTION D'ALLERGIE ET D'AUTO-IMMUNITE. EXEMPLES DU SULFAMETHOXAZOLE ET DE LA STREPTOZOTOCINE (DOCTORAT IMMUNOLOGIE))

LYON1-BU Santé (693882101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Cancer biomarkers detection using 3D microstructured protein chip: Implementation of customized multiplex immunoassay

Protein chips have demonstrated to be a sensitive and low cost solution to identify and detect tumor markers. However, efficient multiparametric analysis remains a challenge due to protein variability. Crucial parameters are the design of stable and reproducible surfaces which maintain biological activity of immobilized proteins. These parameters relate to surface chemistry and to immobilization conditions (printing buffers, washing etc.). In this study, we have developed and characterized various surface chemistries for the immobilization of anti-tumor antigen antibodies onto microstructured glass slides. The effect of surface properties and antibody immobilization conditions were evaluated for the detection of tumor antigens involved in colorectal cancer. Experimental results demonstrated that the biological activities of the immobilized antibodies were dependent on the surface chemistry and on the immobilization procedure. Optimal immobilization conditions were different for each antibody. Limit of detection in tumor antigen as low as 10 pM can reach under optimized conditions. Our 3D microstructured chip offers the possibility to implement a customized multiplex immunoassay combining optimal immobilization condition for each antibody-antigen system on the same chip

Usefulness of autoantigens depletion to detect autoantibody signatures by multiple affinity protein profiling

International audiencePatients with cancer produce specific autoantibodies against protein antigens present in limited amount among a large background of immunoglobulins (Igs), nonrelevant as biomarkers, including natural antibodies. Multiple affinity protein profiling (MAPPing) that combines 2-D immunoaffinity chromatography, enzymatic digestion of the isolated proteins, and identification by MS/MS, may facilitate the identification of these so far unknown patient antibodies. The first immunoaffinity chromatography is crucial, as it is used for selectively removing proteins (autoantigens) recognized by natural antibodies. Application of this depletion step to colon cancer cell proteins is specifically described along with the identification of the natural autoantigens, as well as the coupling of this depletion step with the next steps. By enabling to separate antibody-binding proteins recognized by either natural autoantibodies or patient-specific antibodies this approach may contribute significantly towards the definition of autoantibody signatures

Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer

International audienceAntibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers

Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability

International audienceObjectives: Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker. Design and methods: Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS (R) automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n = 23) or benign prostate hyperplasia (n = 31). Results: The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1 ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was <= 11% and <= 15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80 degrees C for 1 month. Conclusions: Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80 degrees C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples

Clinical Quantitation of Prostate-specific Antigen Biomarker in the Low Nanogram/Milliliter Range by Conventional Bore Liquid Chromatography-Tandem Mass Spectrometry (Multiple Reaction Monitoring) Coupling and Correlation with ELISA Tests

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low μg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 μl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma

Convergence entre l’analyse biostatistique et les méthodes d’inversion hiérarchique bayésienne pour la recherche et la validation de biomarqueurs par spectrométrie de masse

International audienceIn this communication, we are presenting an overview on the methods in biostatistical analysis and Bayesian hierarchical inversion we are studying on the BHI-PRO project for research and validation of biomarkers based on mass spectrometry in MALDI and MRM mode. We are also expliciting the performance criteria used and giving some first experimental results. In MRM mode, we are showing on a colorectal cancer cohort using the LFABP and PDI biomarkers a relevant improvement of the sensibility for a specificity satisfying the minimum requirement for a screening test.Dans cette communication, nous présentons une vue d’ensemble des méthodes d’analyse biostatistique et d’inversion hiérarchique Bayésienne que nous étudions sur le projet BHI-PRO pour la recherche et la validation de biomarqueurs par spectrométrie de masse en mode MALDI et MRM. Nous avons aussi explicité les critères de performance utilisés et présenter des premiers résultats expérimentaux. En mode MRM, nous avons montré sur une cohorte de cancer colorectal, en utilisant la LFABP et la PDI comme biomarqueurs, une amélioration significative de la sensibilité pour une spécificité correspondant aux performances demandées pour un test de dépistage