Enzyme-linked immunosorbent assay antigen measurement

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

Enzyme-Linked Immunosorbent Assays (ELISAs) These assays are performed in the wells of a plastic microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an ezyme, which will produce a colored product when exposed to its substrate. The amount of color produced can be used to determine the amount of protein (or antibody) in the sample to be tested. 

Friguet, B., Chaffotte, A. F., Djavadi-Ohamance, L, and Goldberg, M. E (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent-assay. J Immunol Methods 77,305-319... 

The enzyme-labeled antibody can be evaluated by enzyme-linked immunosorbent assay. Immobilize the appropriate antigen on the wells of a microtiter plate or strip (at a concentration of 2-10 ]Xg/ mL), incubate various dilutions of the conjugate for a few hours, wash the wells, add substrate, and measure the amount of product formed (see Chapter 15). This approach may also be used for monitoring conjugate purification in chromatography fractions. 

The dual-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA Fig. 1) is a sensitive and specific technique for quantifying molecules in solution. The technique is dependent upon the availability of two antibodies recognizing separate epitopes upon the antigen to be measured such that they are able to bind to the molecule simultaneously (1,2). The capture antibody specific to the substance to be measured is first coated onto a high-capacity... 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

The titer of an antibody describes the immunoglobulin concentration in serum and is a measure of the highest dilution that will still give a visible antibody-antigen precipitation. Higher antibody titers are usually obtained after repeated antigen boosts. Antibody titers can be determined by double-diffusion assays in gels, enzyme-linked immunosorbent assays... 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

After more than two decades, advances in IHC have provided a feasible approach to performing immunostain-ing on routinely processed tissues, such that this method is now routine for the performance of IHC special stains in surgical pathology laboratories using EEPE tissues (see Appendix lA). However, demands for improved reproducibility and for quantification have led to a growing recognition that IHC has the potential to be more than just a special stain. If properly controlled in all aspects of its performance, IHC can provide a tissue-based immunoassay with the reproducibility and quantitative characteristics of an ELISA (enzyme-linked immunosorbent assay) test, which not only detects the presence of an analyte (protein or antigen) but also provides an accurate and reliable measure of its relative or real amount (see Quality Control and Standardization section). 

The most prominent of these assays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). In the case of RIA, the analyte sample is either extracted or used directly, and mixed with a constant amount of antibody and radiolabeled analyte [tracer mostly fi-emit-ters (e.g., H, " C) or y-emitters (e.g., I)]. After equilibration and separation of the free and bound antigen, radioactivity is measured for quantification of the analyte. In the case of sandwich-RIA, two antibody preparations are used the first serves as the binding partner of the analyte, whilst the second - which is radiolabeled - is directed either against the analyte or the first... 

Muromonab-CD3 is an immunosuppressive agent that blocks T-cell function, which plays a major role in graft rejection, by reacting with and blocking the T3 (CD3) molecule on the membrane of human T-cells associated with antigen recognition. Serum levels are measured with an enzyme-linked immunosorbent assay (ELISA). It is indicated... 

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