title: Development of a microarray detection method for galectin cancer proteins based on ligand binding.
abstract: In this article, we describe the development of a novel detection method for the visualization of ligand-binding proteins. Current proteomic tools, such as the enzyme-linked immunosorbent assay (ELISA), are based on protein abundance rather than protein activity and can result in conflicting data. To address this issue, we developed an assay in which ligand binding is detected using a microarray approach with immobilized antibodies on a porous aluminum oxide matrix. The galectin family of proteins was used as a model system to evaluate the performance of this approach. Galectins selectively bind galactosides and are linked to cancer progression. Our assay employed antibodies directed against different galectins. The antibodies were immobilized on the microarray surface by use of protein A/G. In our example, galectin-1 and galectin-9 were then detected in cell lysates. Lysates were exposed to the anti-galectin surface, followed by washing and quantification with a general fluorescent galectin ligand. The optimal galectin ligand allowed detection of nanogram amounts of galectin using only 1 μg of antibody. Galectin-1 was visualized in HeLa and tumor cell lysates, indicating the potential of the method for a clinical setting.
authors.affiliation: Medicinal Chemistry and Chemical Biology, Utrecht University, 3585 CG Utrecht, The Netherlands.
authors.forename: Hilde
authors.initials: H
authors.lastname: van Hattum
authors.forename: Nathaniel I
authors.initials: NI
authors.lastname: Martin
authors.forename: Rob
authors.initials: R
authors.lastname: Ruijtenbeek
authors.forename: Roland J
authors.initials: RJ
authors.lastname: Pieters
mesh_terms: D000537:Aluminum Oxide / Q000737:chemistry; D000818:Animals; D055910:Antibodies, Immobilized / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D037483:Galectin 1 / Q000032:analysis*; D037161:Galectins / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D016062:Porosity; D040081:Protein Array Analysis; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism
chemical_list: D055910:Antibodies, Immobilized; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D037483:Galectin 1; D037161:Galectins; C034284:IgG Fc-binding protein, Streptococcus; C105032:LGALS9 protein, human; D008024:Ligands; D013205:Staphylococcal Protein A; D000537:Aluminum Oxide