Nonisotopic immunoassay labeled methods

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. 

Ishikawa, E., Hashida, S., Kohno, T. and Tanaka, K. Methods for enzyme-labeling of antigens, antibodies and their fragments , in Ngo, T. T. (ed.), Nonisotopic Immunoassay. Plenum Press, New York, 1988, p. 37. 

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. 

The essential criteria for a useful analytical technique are specificity, sensitivity, accuracy, precision, simplicity, rapidity, economy, wide applicability, and freedom from hazard. It is well known that radioimmunoassay (RIA) was developed in 1959 by Yalow and Berson (Yl). Since then the radioimmunoassay method has been widely used in the field of clinical chemistry. Radioimmunoassay has inherent in it the advantages listed above. However, this method always requires special facilities for use and disposal of radioisotopes and consideration must be given to the fact that the labeled substances have short half-lives. Immunoassay methods are explosively increasing in use and development as an analytic technique in basic science as well as in clinical laboratory medicine (L1-L3, VI). With these points as background, efforts have been made to develop nonisotopic immunoassay methods or alternative immunoassay methods that are based on antigen-antibody reactions but do not involve use of a radioisotope. 

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. ... 

Nonisotopic immunoassays Use of nonisotopic immunoassays has, in the last few years, become increasingly common in both research and clinical laboratories. Although these immunoassays do not employ radioactive labels, they may have sensitivities similar to those of classic RIA. The immunochemical mechanism and principles are the same as for RIA only the label and the method of measurement are different. The most common labels are enzymes and fluorescent conjugates, which may be covalently bound to antibodies or haptens. 

Based on solid phase technologies numerous fully automated immunoassay systems which allow the determination of a broad spectrum of analytes are nowadays commercially available. While very successful in the case of nonisotopic methods, attempts for automation in the RIA field more or less failed due to the properties of the radioactive label. 

Immunoassays employing antibodies are widely used to quantify hormones (see Chapter 9). Currently, labeled-antibody (immunometric) assays with nonisotopic labels are the method of choice for measuring most hormones, especially peptides and proteins. Immunometric assays use saturating concentrations of two or more antibodies (often... 

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 sequential two-step immunoassay for FT4 was first introduced commercially in 1979. Subsequently, a number of manual and automated procedures have become available for FT4 and FT3. The earliest kit methods used radioactive labels and antibody-coated tubes or microbeads, but automated systems are now available that use nonisotopic tracers and a variety of sofid-phase formats. One two-step FT4 method involves the application of serum to glass-fiber... 

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. 

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