Immunoassay antigen

While antibodies display similar structures - in fact, various types of IgGs are commonly used in immunoassays, antigens and proteins show tremendous variations in size, structure and charge strongly effecting the array performance. 

Christopoulos TK, Chiu NH. Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments. Anal Chem 1995 67(23) 4290 1294. 

Enzyme-Linked Immunoassay Antigen or capture antibody 5 jig/mL in coadng buffer, which is O.IM carbonate-bicarbonate buffer, pH 9.6, brought to 0.01% sodium azide. PBS, pH 7.4. 

Polymer (2) was used as a test substance. It contains the dinitrophenyl-moiety, a classical hapten (DNP). Production of DNP-specific antibodies was measured by a radio-immunoassay (antigen-binding test). 

Immunoassays can also be classified as homogeneous or heterogeneous. In a homogeneous immunoassay, antigen, antibody, and sample are mixed in the solution phase, while in a heterogeneous assay, one constituent (typically the antibody) is immobilized on a solid surface while the other constituents are delivered via the solution phase. 

Different formulations of saponin-adjuvanted vaccines were tested for humoral and cell-mediated responses in mice. Many antigens, often including hen egg albumin (ovalbumin, OVA) as the antigen to be tested, have been used for immunizations, enzyme immunoassays, antigen-specific CTL and cellular proliferation assays [31]. 

Immunoassay is a method that identifies and quantifies unknown analytes usiag antibody—antigen reactions. Techniques are based ia immunochemistry, analytical chemistry, and biochemistry, with a history of development paralleling advances ia microbiology and immunology (see also Immunotherapeutic agents). 

There are many possible means for quantification of the antigen—antibody reaction. Immunoassays may be classified according to the technology used for detection and quantification of the analyte being detected. 

Turbidimetric Agglutination Immunoassays. Agglutination—precipitation immunoassays were among the first practical appHcations of the antigen—antibody reaction in diagnostic tests. These assays are not as widely used in the 1990s as EIA and FIA because they are either not quantitative enough or lack the sensitivity limits of RIA, EIA, and EIA. 

Latex agglutination immunoassays are easily formatted into simple kits which can provide yes/no and semiquantitative estimates of antigen (or antibody) in a sample. The first such assay was developed in 1957 for rheumatoid factor (15) and assays are on the market for the deterrnination of many species of bacteria, fungi. Mycoplasma, parasites, ckettsia, and vimses, as well as for the deterrnination of autoimmune disease, hormones (qv), dmgs (see Pharmaceuticals), and blood proteins (16). Latex agglutination is also the basis of many home pregnancy tests. 

Enzyme Immunoassay. In EIA, antibody (or antigen) is labeled with (or conjugated to) an enzyme, and this reagent is used to complex and quantify the target antigen (or antibody) in a sample. Conjugation may utilize a variety of chemical methods. 

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

Fluorescence Immunoassay. Basic FIA follows the same formats and approaches as EIA. The difference Hes in the indicator a fluotophote is used instead of an enzyme. This allows direct quantification of the indicatot—antibody—antigen complex, or free indicator-reagent, without the need for a substrate. 

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

Most immunoassay kits and many commercial immunoassay analyzers are based on heterogenous EIA or FIA. These include an immunoassay system that uses FIA linked to radial partition chromatography of the antibody—antigen complex (39) a system that uses antibody-coated tubes for enzyme immunoassay of a variety of hormones and dmgs (40) and a system that uses either a sandwich or competitive FIA format to measure a variety of analytes (41). 

The immunochemical interaction between the antigen and antibody is very specific. By labeling either the antigen or antibody, the method s sensitivity is increased. The most frequently used labels to increase sensitivity are radionucHdes (see Radioisotopes) where the assay process is called radioimmunoassay (RIA), or en2ymes where the assay is named en2yme immunoassay (ElA) (see Enzyme applications). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

The working conditions of the immunosensor (enzyme and antigen concentrations, dilutions of the antibodies, pH of the buffer solution) were found. The cholinesterase immobilized demonstrated the maximum catalytic activity in phosphate buffer solution with pH 8.0. The analytical chai acteristics of the sensor - the interval of the working concentrations and detection limit - have been obtained. The proposed approach of immunoassay made possible to detect 5T0 mg/ml of the bacterial antigen. 

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