Nonisotopic immunoassay enzyme assay

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

Immunoassays are used to measure PSA and are commercially available. Most of them use nonisotopic labels, such as enzyme, fluorescence, or chemiluminescence. The majority of these assays are automated on an immunoassay system. Different assays and even the same assay with different lots of reagent may produce different results. The reasons for such differences are due to changes in assay calibration, production lot variation, assay reaction time, reagent matrices, assay sensitivity, and imprecision. Antibodies react with different PSA epitopes therefore, some antibodies react dissimilarly with the various molecular forms of PSA. Assays are classified as equimolar if they bind to free and cPSA equally and nonequimolar if they bind to free or cPSA differently. Examples of equunolar assays are the ACCESS... 

Considerable effort was expended in the development of alternative technologies that did not require the use and measurement of radioactivity. A number of nonisotopic assays for T4 were subsequently developed commercially for use on fuUy automated immunoassay systems or for use with existing chemistry analyzers. According to a 2002 College of American Pathologists Ligand Assay Survey, more than 95% of laboratories now use a nonisotopic T4 method. A variety of different labels were used to construct these nonisotopic assays. Enzymes such as horseradish peroxidase, alkaline phosphatase, and [3-D-galactosidase were the most... 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

Enzyme immunoassay The tendency to avoid the use of radioisotopes in immunoassays has led to the increasing use of nonisotopic labels in steroid hormone assays. Of these, enzyme immunoassays are most widely used. 

The immunometric-type assay has also been adapted for use with nonisotopic labels and is typically carried out in a heterogeneous format in which the antibody is immobilized on a solid support, such as a microtiter dish, membrane, or collection of beads. The canonical clinical immunoassay format in toady s laboratories is the enzyme-linked immunosorbent sandwich assay, which employs two antibodies, one to capture the analyte and the other to detect and quantify it. More details of the principles of these and other immunoassay techniques are given elsewhere in this encyclopedia. 

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