Radio Immunoassay of Drugs and Hormones

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About Authors:
Dhiren Shah
Seth G.L.Bihani S.D.College of Pharmacy, R.U.H.S.
Sri Ganganagar, Rajasthan, INDIA

A method has been developed for immobilisation of antisera on fresh plastic tubes through an immunochemical bridge. This type of immobilisation has been shown to be more consistent than direct adsorption on plastic. Such immunochemically coated antisera on plastic tube has been used in the development of a noncentrifugation radioimmunoassay. This assay system has been found to be technically as sound as the conventional method.

Reference Id: PHARMATUTOR-ART-1489

Radioimmunoassay (RIA) is a laboratory method that measures, with relative accuracy, minute amounts of substances present in the body. When it was developed in the 1950’s, radioimmunoassay was considered a revolution in medical investigations. As of 2009, it is still considered revolutionary because it is the blueprint for more advanced methods of laboratory techniques.

First discovered by Rosalyn Yalow and Solomon Berson, radioimmunoassay was used to investigate blood volume, iodine metabolism, and hormones like insulin. Radioimmunoassay has expanded its viability by being able to measure trace amounts of substances using sensitive laboratory techniques. Drugs, antigens and hormones are some of the substances measured by radioimmunoassay.

Radioimmunoassay is a specific scientific process. First, laboratory technicians must obtain a substance that contains the antigen they are testing for. This antigen is then injected with radioactive chemicals, such as a gamma-radioactive isotope made from iodine or some other substance. The radioactive chemicals cause the antigen to become radioactive, so it can be observed.

The radioactive antigen is then mixed with a set amount of antibodies that scientists have determined are appropriate. The antigens and antibodies bind to each other and become one substance. This provides the benchmark or basis for testing. Then, an unknown substance which contains some tiny amount of the antigen is added. This new substance is the substance being tested.(

Immunoanalytical methods that are based on the selective, reversible binding of small molecules (drugs) or macromolecules by biologically derived antibodies have revolutionized the ?eld of biomedical analysis. Immunoassays have allowed the determinaion of very small amounts of analytes that were previously unassayable in biological matrices by other techniques. Since the origin al work on the analys is of insulin by Berson and Yalow.

Immunoassay methods have been developed for the determination of a wide variety of drugs, pesticides, hormones, and biological proteins. Immunoassays are relatively simple procedurally. This has led to the development of many ‘‘kit-type’’ immunoassay systems that are used routinely for home diagnostics. Anti bodies that are used as reagents in immunoassays are, in general, molecules of the immunoglobulin G (IgG) type. They are produced by white blood cells in response to foreign substances introduced in mammalian species. Million s of years of vertebrate evolution have developed immunoglobulins into exquisitely discriminating devices capable of recognizing subtle differences between molecules; for example, a mouse can generate millions of different immunoglobulin speci?cities. These immunoglobulins combine speci?cally with the substances (antigens) that elicited their formation. This then triggers processes by which the foreign antigens are cleared from the organism, which is the ultimate goal of the immune process.(Dr.Arora.D.R)

These molecules are heterogeneous, bifunctional glycoproteins in which the variable amino acid sequence in the polypeptide component provides its biologic activity. This polypeptide component is made up of two heavy or H chains (50,00 0 Da) and two light or L chains (20,000 Da), held together by disul?de bonds . The two binding sites of the antibody molecule appear to reside on the NH terminal ends of the polypeptide chains.

Antibodies produced by injection of foreign antigens into a host animal are structurally heterogeneous and respond to different aspects of the same antigen with different binding strengths and speci?cities. If the antibody -producing blood cell can be isolated in a pure cell culture, however, only one particular antibody structure will be produced.

Antibodies harvested from pure cell cultures are referred to as monoclonal antibodies, are homogeneous and react with only one or a few closely related anti- gens. The antibody producing cells which can be grown in culture are produced by cell fusion techniques and provide superior antibodies for use in immunoassays because of their identical speci?cities and binding strengths. Antigens must have molecular weights in excess of 104 Da in order to evoke antibody production. However, small molecules, such as drugs, can be bound to macromolecular carriers and some of the antibodies produced from these will respond to the drug (hapten).

All immunoassay procedures take advantage of the speci?c reactions between antibodies an d antigens. They involve measurement, directly or indirectly, of the extent of binding between antibodies (reagents) and antigens (analytes). Labels are used in conjunction with the antigens or antibodies so that the concentrations of molecular species can be measured instrumentally. Labels are entities that impart some measurable signal, such as radioactivity, ?uorescence, chemiluminescence, on electrochemical or enzyme activity to the antibody or antigen to which it is attached. The determination of the extent of antibody binding requires measurements of the amounts of labeled antigen or antibody in the complexed (bound) and in the free forms. This is generally expressed as the bound/ free (b/f ) concentration ratio, which is related to the concentration of analyte. The measured signal can be either directly or inversely proportional to the b/f ratio depending on the chemistry of the system used. There are two ways to determine the amount of antigen present: one using a limited amount of reagent (competitive assays), the other using an excess amount of reagent (non-competitive assays).

The competitive assay uses a limited amount of antibody, which is insuf?cient to bind all of the antigen. The antigen competes with a ?xed amount of labeled antigen for the limited number of antibody binding sites. From the proportion of bound (or free) labeled antigen, the concentration of unlabeled antigen can be determined.

The non-competitive assay uses an excess of anti-body. Different approaches to detect the bound antigen have been developed, the most common use an antibody , in excess , coupled to a solid phase. The bound antigen is then detected with a second antibody labeled in a way that aids detection (e.g., radioactive , ?uorophore, etc.) . The amount of antigen in the sample is then directly proportional to the amount of labeled antibody captured on the solid phase. Both assays require differentiation of bound label from free label. This can be achieved by two methods: heterogeneous assay or homogeneous assay. Heterogeneous assay separates bound label from free label using a means of removing the antibody. Homogenous assay compares the signal of the label when antigen is bound to antibody compared to when the antigen is free.(

The principle involves competitive binding of radiolabeled Ag and unlabeled Ag to the limited supply of a high affinity Ab.

Immunoanalytical methods that are based on the selective, reversible binding of small molecules (drugs) or macromolecules by biologically derived antibodies have revolutionized the ?eld of biomedical analysis.(Kamboj.P.C)



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