Antibody Microarrays.ppt
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1、Antibody Microarrays,Merrill Birkner ph296December 1, 2003,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
2、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 antigen
3、s 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
4、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 g
5、lobulin 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
6、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”.,Antigen/Antibody Interaction,Ab Array Procedure,Extraction of total cellular protein from biological samples of interest (eg.
7、 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 array and the analysis of the results.,This procedure i
8、s a fluorescence-based analysis; covalently immobilized antibodies are used to capture fluorescently labeled antigens. They do not measure absolute concentrations- instead they provide a relative measure of protein abundance i.e. the abundance of protein in one sample as compared to another sample.
9、As part of array development, all antibodies are printed and tested against their specific purified antigen (when available) and against cell lines and tissues samples (for quality control). A reference pool is also used, and similar to the gene expression microarrays, a pool of equal aliquots from
10、each sample to be measured is used, thus ensuring that all proteins from the samples are represented in the reference.,Direct LabelingDirect Labeling with a hapten tag.Paired Ab sandwich assays.,Direct Labeling (w/ hapten tag),A convenient method to measure multiple proteins in a complex mixture. Al
11、l proteins are labeled with either a fluorophore or a hapten tag such as biotin. Advantages: Only one captured antibody per target is required, as compared to the next method- easier to expand detection to new targets for which matched antibody pairs may not be available. Can label different samples
12、 with different tags and to co-incubate the samples on the same arrays. Disadvantages: Potential for a high background: all proteins are labeled from the sample, including high concentration proteins such as albumin in serum; nonspecific binding or adsorption of these proteins to Ab could cause inte
13、rference reduce detection sensitivity or data accuracy. Potential for disruption of antibody-antigen interactions if the labeling reaction severely alters an antigens binding site.,Dual Antibody Sandwich,Antibodies spotted onto microarray substrates capture specific antigens, and a “cocktail” of det
14、ection antibodies, each antibody matched to one of the spotted antibodies, is incubated on the arrays. Advantages: Quantification of the bound detection antibodies provides a measure of each antigens abundance. Sandwich assays are more sensitive than the direct labeling method because background is
15、reduced through the specific detection of two antibodies instead of one. Disadvantages: The development and validation of assays measuring many targets in parallel is difficult because of the cross reactivity and precipitation when using many detection antibodies.,ELISA as a validation method,The En
16、zyme-Linked Immunoabsorbent Assay is serologic test used as a general screening tool for the detection of antibodies or antigens in a sample. ELISA technology links a measurable enzyme to either an antigen or antibody. These tests are often used to validate the microarray results,Ab/Ag interaction i
17、n ELISA wells,Gene Expression vs. Ab Microarray,Gene expression, in most cases, does not necessarily correlate with changes in protein expression. In cases when there is a correlation between mRNA and protein abundance, the correlation is often time shifted. This time shift is likely to be different
18、 for each mRNA-protein pair. With these arrays it is now possible to compare changes in gene expression with changes in protein expression using similar technologies. There are also many reasons for merely studying protein abundance.,Ab Microarrays & Cancer Research,Information from protein profilin
19、g experiments may reveal associations between proteins or groups of proteins and disease states or experimental conditions. Biomarkers in cancer are potentially valuable for early detection, staging of patients, classification of patients, or as surrogate markers for drug response.These microarrays
20、increase the number of proteins that can be conveniently measured, therefore taking advantage of the benefit of using combined markers in diagnostics.,Important in this field because there is a low volume requirement and the multiplex detection capability of microarrays make optimal use of precious
21、clinical samples. Work continues on the optimization of various aspects of the protocols, such as substrates for Ab attachment, the methods of Ab attachment, Ab buffers and concentrations, wash conditions, etc.,Antibody microarray profiling of human prostate cancer sera: Antibody screening and ident
22、ification of potential biomarkers. Proteomics 2003, 3, 56-63.,Miller, J., Zhou, H., Kwekel, J., Cavallo, R., Burke, J., Butler, E.B., Teh, B., Haab, B.,Background,Protein Biomarkers in the serum hold great promise for noninvasive disease detection and classification. Ab & protein microarrays can hav
23、e many applications including protein profiling of cancer tissue, autoimmune diagnostics, protein interaction screening, and Ab-based detection of multiple antigens. Certain parts of the Ab microarray technology have not been perfected: An optimized protein immobilization method is needed that retai
24、ns native structure and reactivity and decreases nonspecific protein adsorption. Ab can be immobilized by adsorption to poly-L-lysin membranes, by chemical crosslinking to derivatized glass surfaces. Hydrogels recently have also been introduced as a protein microarray substrate.,Another important is
25、sue is to create an efficient method of validating antibody performance in the microarray assay. Previous work in the development of the antibody microarray methods made use of solutions of known target antigen concentrations to characterize antibody performance. This is often very expensive and the
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