Antibody Microarrays.ppt

Antibody (Ab) Microarray A complete microarray-based system for profiling protein expression in biological samples; used to compare two biological samples to measure the relative differences in protein expression. The microarray consists of hundreds of monoclonal antibodies covalently bound in an ordered layout to a glass slide. A protein which can be synthesized in pure form by a single clone (population) of cells. These antibodies can be made in large quantities and have a specific affinity for certain target molecules called antigens which can be found on the surface of cells and those that are malignant. The array can be used as a means to correlate specific proteins with physiological or pathological process of interest, by comparing hundreds of proteins at a time. It is used for toxicity testing, disease investigation, and drug discovery. Antigens Antibodies Antigens: Molecules that stimulate the production of specific antibodies and combine specifically with the antibodies produced. Most antigens are foreign to the blood and other bodily fluids. Antibodies: Antibody proteins (immunoglobulins) are found in the gamma globulin class of plasma proteins. There are five main subclasses : IgG, IgA, IgM, IgD, and IgE. (ex. Most antibodies in serum are from the class IgG). Antibody Structure Consists of four interconnected polypeptide chains. Two heavy chains (H-chains) and joined to two shorter chains (L-chains). These four chains are arranged in the form of a ‘Y’ ; with the stalk of the Y is called the “crystallizable fragment” and the top of the Y is known as the “antigen-binding fragment”. Ab Array Procedure Extraction of total cellular protein from biological samples of interest (eg. Serum samples). Labeling of extracted protein with fluorescent dyes Cy5 and Cy3 (direct labeling, direct labeling with hapten tag, paired Ab sandwich assay). Removal of unbound dye. Incubation of labeled protein with the array. Scanning of the ar