Antibodies analytical reagent

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

Common Procedures. The general analytical scheme for immunochemical methods is rather simple. The analyte of interest, the antigen (Ag), reacts with the analytical reagent, the corresponding antibody (Ah), forming an immunochemical antigen—antibody complex ... 

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

The principle approach to immunoassay is illustrated in Figure 1, which shows a basic sandwich immunoassay. In this type of assay, an antibody to the analyte to be measured is immobilized onto a solid surface, such as a bead or a plastic (microtiter) plate. The test sample suspected of containing the analyte is mixed with the antibody beads or placed in the plastic plate, resulting in the formation of the antibody—analyte complex. A second antibody which carries an indicator reagent is then added to the mixture. This indicator may be a radioisotope, for RIA an enzyme, for EIA or a fluorophore, for fluorescence immunoassay (FIA). The antibody-indicator binds to the first antibody—analyte complex, free second antibody-indicator is washed away, and the two-antibody—analyte complex is quantified using a method compatible with the indicator reagent, such as quantifying radioactivity or enzyme-mediated color formation (see Automated instrumentation, clinical chemistry). 

Pure PA is available commercially from Pharmacia Fine Chemicals, (Piscataway, New Jersey), who also produce PA bound covalently to Se-pharose 4B, and from Sigma Chemical Co. (St. Louis, Missouri). PA has been used mainly as an indicator of antibody bound to cell-surface anti-ggn. 14.15 por this purpose, it has been labeled with a fluorescent. tag (fluorescein 1 ), with ferritin, or with radionuclides. 1-, and H-labeled PA have been prepared, and their suitability as analytical reagents has been demonstrated. 

Immunochemical procedures provide an important supplement to the battery of available chemical and instrumental methods and can often yield new information not readily obtainable in other ways. Antibodies are extraordinary analytical reagents since they can have specificity for macromolecules (proteins, nucleic acids, and polysaccharides) as well as for small molecules belonging to almost every chemical class. In a field that is moving so rapidly, an exhaustive compilation of immunochemical techniques is neither possible nor practical. Our purpose is to provide the investigator with significant examples and sufficient background information so that he can properly assess and adapt these techniques to his research. 

Immunoassay Analytical technique that uses antibodies as reagents for binding of analyte(s) in the sample... 

Since immunoassays utilize antibodies as analytical reagents, the challenge is to obtain antibodies specific for an individual compound that may be present in a milieu of structurally related and unrelated compounds. Antibodies can be produced by in vivo immunization (polyclonal antibodies), hybridoma technology (monoclonal antibodies) and by genetically engineered bacteria (antibody fragments with affinities for specific molecules). Some caveats pertaining to the production and use of such antibodies in immunoassay procedures will be presented. 

The analyte concentration differs significantly before and after the use of commercially available heterophilic antibody blocking reagents. 

Antibodies can be used as exquisitely specific analytic reagents to quantify the amount of a protein or other antigen. The technique is the enzyme-linked... 

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