Radioimmunoassay labeled antibody

Radioimmunoassay (RIA), like ELISA, is based on the radioactive labelling of the antibody molecules. The labelled antibody reacts with the antigen present in the tube the amount of radioactivity present in the bound complex is directly proportional to the amount of antigen added to the tube . ... 

The most common types of assays employed to quantitate protein concentrations in biological matrices are listed in Table 32.4. Enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunoradiometric assays (IRMAs) require protein-specific antibodies, labeled proteins, or labeled antibodies as reagents, and are generally competitive inhibition assays. Radioimmunoassays measure concentrations by displacing ligands from cell-bound receptors. The most common assay, the... 

In immunoradiometric assays the use of labeled antibody as the reagent for antigen measurement imposes the need for a very high degree of purity of the antibody. This situation is the converse of that posed by specificity in a radioimmunoassay. In the latter the antiserum used may be markedly heterogeneous provided only that the purity of the labeled antigen selects from the mixture of antibodies present an immune reaction that has the specificity required. Therefore, in an immunoradiometric assay in order to achieve sufficient purity of the antibody it is essential to prepare immunoadsorbent from a highly purified polypeptide. 

It is now common practice in our laboratory to assay parathyroid hormone by a fully automated system in batches of 60 samples with appropriate controls at intervals of 20 samples. Patient samples are input at the appropriate dilution, this process taking approximately 1 hr. On the reagent addition unit, labeled antibody is added, as well as plasma to standards and buffer to plasma samples. Reagent addition takes 10-15 min. After incubation at 4 for 72 hr, the assay tubes are allowed to adjust to room temperature (the Kemtek 3000 does not have means of refrigeration), and immunoadsorbent is added, with a 10 sec delay between each addition. When addition is complete (30-40 min), filtration is commenced with a cycle that takes 50 sec for its five simultaneous transfers plus washing. The filters are dried and counted. It should be noted that this procedure differs from the standard immunoradiometric technique in that it is the radioactivity not bound to antigen that is counted. The data therefore resemble those derived from a classical radioimmunoassay and can be processed by the Kemtek computer using a polynomial cubic analysis. ... 

D Rodbard, DM Hutt. Statistical analysis of radioimmunoassays and immunoradio-metric (labelled antibody) assays A generalized weighted, iterative, least-squares method for logistic curve fitting. In Radioimmunoassay and Related Procedures in Medicine, Vol I. Vienna International Atomic Energy Agency, 1974, p 165. 

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). 

Radioimmunoassay is a competitive protein binding assay which utilizes an antibody as the binding protein. This assay also employs a highly purified antigen which has been radio-labeled (tagged). 

The first application of immunologically based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selectively bound malathion. A few years later, radioimmunoassays were developed for aldrin and dieldrin and for parathion. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked... 

Nowadays, antibodies are utilized in numerous immunoanalytical methods. Those widely used in practice, such as radioimmunoassays, fluoroimmunoassays and enzyme-linked immunosorbent assays (ELISA), require labelled reagents. Millions of ELISA tests for diagnostics of various diseases are daily performed in clinical laboratories. The detection of analytes by two-antibody "sandwich" ELISA, is schematically outlined in Figure 3. 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

Monodisperse microspheres imprinted with theophylline or 17 (3-estradiol were used in competitive radioimmunoassays showing the MIP s high selectivity for the template molecule. In this case the assay is based on the competition of the target molecule with its radioactively labeled analogue for a limited number of antibody binding sites [77,118]. Figure 15 demonstrates that displacing the radioactively marked theophylline from the imprinted polymer was only possible with theophylline as competitor. Structurally related molecules showed effects solely at elevated concentrations [77]. 

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

All radioimmunoassays published thereafter, except those described by Hock and Liemann (38), Freebairn and Crosby (39), and Pohlschmidt et al. (40), were based on a similar procedure (Table 28.2). However, Hock and Liemann (38) applied a more simplified extraction/cleanup procedure for the analysis of chloramphenicol residues in animal tissues, milk, urine, and plasma. In this assay, competitive inhibition between chloramphenicol labeled with " C and antibody has been demonstrated. 

FIGURE 23-3 Radioimmunoassay (RIA). (a) A low concentration of radiolabeled hormone (red) is incubated with (T) a fixed amount of antibody specific for that hormone or (2) a fixed amount of antibody and various concentrations of unlabeled hormone (blue). In the latter case, unlabeled hormone competes with labeled hormone for binding to the antibody the amount of labeled hormone bound varies inversely with the concentration of unlabeled hormone present, (b) A radioimmunoassay for adrenocorticotropic hormone (ACTH). A standard curve of the ratio [bound] to [unbound radiolabeled ACTH] vs. [unlabeled ACTH added] is constructed and used to determine the amount of (unlabeled) ACTH in an unknown sample. If an aliquot containing an unknown quantity of unlabeled hormone gives, say, a value of 0.4 for the ratio [bound]/[unbound] (see arrow), the aliquot must contain about 20 pg of ACTH. 

Sheep, rabbit, or goat antibodies to rat or mouse F[ab ]2, IgG, IgA, and IgM for labeling with l25I to carry out radioimmunoassay (RIA), or conjugated to alkaline phosphatase or biotin for an enzyme-linked immunosorbent assay (ELISA)... 

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