Rabbit Anti-Human CEACAM5 (a.a. 301-400) Polyclonal antibody

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Purpose: To detect anti-CEACAM5 targeted superparamagnetic iron oxide (SPIO) particles in vitro on the cell surface by quantitative magnetic resonance (MR) imaging and to compare with flow cytometry. Materials and Methods: The monoclonal mouse antibody T84.1 and an appropriate IgG isotype antibody were conjugated to dextran-coated SPIO particles. HT29 cells expressing carcinoembryonic antigen (CEACAM5) were treated with antibody-conjugated SPIO particles. Purified cell samples were examined on a 3.0-T MR scanner using a multi-echo spin-echo sequence for MR relaxometry. Aliquots of the cell samples were further treated with a fluorescein isothiocyanate (FITC) anti-dextran antibody and an Alexa Fluor 488 anti-mouse antibody for the corresponding flow cytometry. Results: MR relaxometry revealed a dose-dependent binding of T84.1-conjugated SPIO particles with a positive correlation between R(2) relaxation rate of cell samples and SPIO particle concentration during incubation (r=0.993, P<.01). Positive correlations were also observed between R(2) relaxation rate and flow cytometry (geometric mean) with both fluorescent antibodies (r=0.972 and r=0.953, both P<.01), respectively. Conclusion: The study revealed the feasibility of quantitative MR imaging of targeted SPIO particles on the cell surface comparable to flow cytometry. (C) 2010 Elsevier Inc. All rights reserved.