Discovery of Lung Cancer Biomarkers by Profiling the Plasma Proteome with Monoclonal Antibody Libraries
ABSTRACT A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.
- SourceAvailable from: Laszlo TakacsAdvances in Nanoparticles 01/2013; 02(02):133-144. DOI:10.4236/anp.2013.22022
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ABSTRACT: The analysis and polyclonal antibody response for newly synthesized maltose - BSA conjugate neoglycoproteins is described. In this first, proof of concept study a simple carbohydrate antigen, maltose, was linked to bovine serum albumin by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization. The neoglycoproteins were investigated by capillary gel electrophoresis and the number of conjugated maltose residues was determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The neoglycoproteins were then evaluated by immunization of BALB/c mice and the polyclonal antibody response was tested by enzyme-linked immunosorbent assay as evidence for the presence of sugar-containing epitope specific antibodies. Selective antibody binding was demonstrated to the synthesized neoglycoproteins with different (low and high) glycosylation degree suggesting the possible use of this approach to generate antibodies. Moreover, the polyclonal antibody response was not inhibited by maltose or other simple carbohydrates to confirm presence of the neoglycoprotein-specific antibodies. This article is protected by copyright. All rights reserved.Electrophoresis 08/2013; 34(16). DOI:10.1002/elps.201300052 · 3.16 Impact Factor
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ABSTRACT: Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.BMC Genomics 10/2013; DOI:10.1186/1471-2164-14-S6-S5 · 4.04 Impact Factor