Radioimmunoassay labeled antigen

Labeled antigen radioimmunoassays involve competition between a labeled antigen and an unlabeled... 

Immunoassays based on fluorescence polarization have become very popular because, in contrast to radioimmunoassays, they require no steps to separate free and bound tracer. In a fluoroimmunoassay, the fluorescently labeled antigen is... 

The specific activity of proteins assayed by direct immunochemical methods or those used as standards in radioimmunoassays profoundly affects the resolution attainable by these techniques. Therefore, the nature of the radioactive label to be used in such experiments must be considered carefully. Table 8-6 lists the isotopes available for this purpose along with the number of atoms of each isotope that must be incorporated to produce an arbitrary counting rate. As can be seen here, 557 atoms of H and 261,672 atoms of C must be incorporated into every molecule of protein to yield the same number of disintegrations per minute as only one I or 11 S molecules. S-methionine is often the isotope of choice for many direct immunochemical procedures since it is relatively inexpensive to prepare at high specific activity. On the other hand, the relative ease with which radioactive iodine may be incorporated into a purified antigen makes it the isotope of choice for radioimmunoassay methods. Of the two iodine isotopes available, is most often used because of its longer half-life. This is an important consideration since it usually takes more than 1 week to prepare and test a labeled antigen prior to its experimental use. 

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. 

Efforts have been made to develop liquid scintillation cocktails that can be utilized to count low energy gamma emitters in a liquid scintillation counter, but these require careful preparation, have been generally inefficient with volumes greater than 3 cc, require quench correction, and are expensive (3) (4) (5) (6)(7). Since both beta and gamma emitters are commonly employed to label antigens in radioimmunoassay procedures, the ability to utilize both isotopes has obvious economic advantages. 

Radioimmunoassay (RIA). RIA is a procedure used to detect or determine an antigen in a sample based on immunology. A radioisotope-labelled antigen is mixed into a sample, an antigen specific antibody is added to the mixture, and the amount of antibody bound with radiolabeled antigen is then measured by scintillation counter. The amount of antigen in the sample is calculated from the radioactivity. RIA is more sensitive than ELISA, but ELISA has replaced RIA because the latter requires the use of radioactive compounds. 

A solid-phase binding assay similar to ELISA can be adapted to radioactive detection when a radioactively labeled antigen or antibody is used. The radioimmunoassay (RIA) is more sensitive and reliable than ELISA but is usually more time consuming and more expensive because of the cost of the radioactive label. 

A technique which is similar to radioimmunoassay, the difference being that the antigen is labelled with a free radical such as nitroxide. When the antibody combines with the labelled antigen, the free radical is immobilized and broad spectral peaks are observed on an electron spin resonance spectrometer. When the labelled antigen is displaced by unlabelled antigen from the test sample, a sharp peak is produced. In this way, the amount of antigen in the patient s sample can be measured. 

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). 

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. 

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. 

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 . ... 

Choice of Radioligand. A 11+C radiolabel will probably exist for most pesticides which will be considered for radioimmunoassay development. Such an intrinsic radiolabel will prove very valuable in titering antisera and possibly in numerous other steps from antigen synthesis through assay development. Unfortunately, for the actual assay, the commonly available 11+C radiolabels may not be of high enough specific activity. The theoretical limit on the specific activity of a single carbon atom is 63 mCi/mmole, and few pesticides have a specific activity of over 50 mCi/mmole even when they are labeled in... 

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. 

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