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Biomedical Technology and Devices Handbook 1140_C17.fm Page 1 Monday, July 14, 2003 9:24 AM 17 Radioimmunoassay: Technical Background CONTENTS 17.1 Introduction 17.2. Principle 17.3. Radioimmunoassay Techniques The Radioactive Marker • Specific Antibody • Separation Methods Eiji Kawasaki 17.4. Validation of a Radioimmunoassay Procedure Nagasaki University School 17.5 Applications of Medicine References 17.1 Introduction The technique of radioimmunoassay (RIA), first developed in 1960 by Berson and Yalow for the measurement of insulin, has expanded to include the detection of other biological agents.1-8 Radioimmunosassays are based on the ability of an unlabeled antigen (Ag) to inhibit the binding of labeled antigen (Ag*) by antibody (Ab). Ag Ag* Ag — Ab Ab Ag* • Ab The process may be viewed as a simple competition in which Ag reduces the amount of free Ab, decreasing the availability of Ab to Ag*. When the assay is performed, Ag* and Ab are incubated together in the presence and absence of samples containing unlabeled Ag. After equilibration, free Ag* and Ag*•Ab are separated. Commonly used separation procedures include solid-phase absorption, precipitation of Ag*Ab complexes with either a second antibody or a salt, and chromato-electrophoresis. Ag*•Ab (or free Ag*) is then deter- mined by comparing the diminished Ag* binding of the sample to that of a standard curve obtained by adding graded, known amounts of Ag to Ag* and Ab. A new standard curve is determined in each assay to allow for variation in antigen binding from assay to assay. Radioimmunological methods combine the extreme sensitivity of detection of isotopically labeled compounds with the high specificity of immunological reactions. Thus, upon use of radioisotopes the detection limit is improved up to 107-fold over physicochem- ical analytical methods. Recently, in some fields of internal medicine, especially in autoimmune disease, radioimmunoassay to measure autoantibodies associated with disease prediction, diagnosis, and progression has been improved and simplified using a small amount of serum.9,10 17.2 Principle Radioimmunoassay is a general method by which the concentration of virtually any substance can be determined. The principle on which it is based is summarized in the competing reactions shown in Figure © 2004 by CRC Press LLC 1140_C17.fm Page 2 Monday, July 14, 2003 9:24 AM 17-2 Biomedical Technology and Devices Handbook FIGURE 17.1 Principles of radioimmunoassay. 17.1. The concentration of unlabeled antigen in the unknown sample is obtained by comparing its inhibitory effect on the binding of labeled antigen to a limited amount of antibody with the inhibitory effect of known standards. A typical RIA is performed by the simultaneous preparation of standard and unknown mixtures in test tubes. To these tubes are added a fixed amount of labeled antigen and a fixed amount of antiserum. After an appropriate reaction time, the antibody-bound (B) and free (F) fractions of the labeled antigen are separated by one of many different techniques. The B/F ratios in the standards are plotted as a function of the concentration of unlabeled antigen (“standard curve”), and the concen- tration of antigen in the unknown sample is determined by comparing the observed B/F ratio with the standard curve (Figure 17.2). Radioactive isotopes most frequently used for labeling are 3H, 14C, 35S, 57Co, 75Se, 125I, and 131I (Table 17.1). Of these, 125I offers useful characteristics for labeling and is very widely used. The RIA principle is not limited to immune systems, but can be extended to systems in which in place of the specific antibody there is a specific reactor (that is, a binding substance) that might be, for instance, a specific binding protein in plasma,11 an autoantibody,12 an enzyme,13 or a tissue receptor site.14 For FIGURE 17.2 Standard curve for the assay of antigen. Concentration of antigen in unknown sample is determined by comparing the observed B/F ratio as shown. © 2004 by CRC Press LLC 1140_C17.fm Page 3 Monday, July 14, 2003 9:24 AM Radioimmunoassay: Technical Background 17-3 TABLE 17.1 Radioactive Isotopes Used for Labeling in Radioimunoassay Radioisotope Half-life Energy Detection Method 3H 12.3 years b Liquid scintillation 14C 5730 years b Liquid scintillation 35S 87.4 days b Liquid scintillation 57Co 270 days g Scintillation crystal 75Se 120.4 days g Scintillation crystal 125I 60 days g Scintillation crystal 131I8 daysb, g Scintillation crystal example, concentration of antibody in an unknown sample can be obtained by measuring the binding to an appropriate labeled antigen. Recently, for in vitro assays, the quantity of samples and the number of items to be measured are rapidly increasing. To meet this trend, the equipment for radioimmunoassay has been semiautomated or automated. 17.3 Radioimmunoassay Techniques The essential requirements for RIA include suitable reactants (labeled antigen and specific antibody) and some technique for separating the antibody-bound antigen from the free-labeled antigen, since under the usual conditions of assay, the antigen-antibody complexes do not spontaneously precipitate. 17.3.1 The Radioactive Marker 17.3.1.1 Radiolabeled Antigen The first requirement for a radioimmunoassay is the preparation of a highly purified antigen that can be radiolabeled or “tagged” without producing any loss of immunoreactivity. Since most polypeptide hormones contain at least one tyrosine residue, they can be labeled with a radioisotope of iodine (e.g., 125I or 131I). The radioiodine usually substitutes onto a tyrosine residue. The radioisotopes of iodine have the advantage of higher specific activities than can be found with 3H or 14C. Because the isotopic abundance of 125I is close to 100%, and the isotopic abundance of 131I is not more than 15 to 30% at the time of receipt into the laboratory,15 the shorter half-life of 131I confers no advantage, and 125I has been the radioiodine isotope of choice. The specific activity of a 125I-labeled hormone may be increased by increasing the number of radioiodine substitutions. However, it has been shown that the more highly iodinated molecules have diminished immunoreactivity as well as increased susceptibility to damage.16,17 The latter appears to arise from radiation self-damage within the molecule. Isotopes 3H and 14C can be used for labeling; however, because they emit extremely low-energy b rays, a liquid scintillation counter is used to make measurements with these two isotopes. Recent advances of molecular biology techniques allow developing the cell-free protein synthesizing and labeling system.18 With an in vitro transcription/translation system using reticulocyte lysate, wheat germ extract, or E. coli extract, one can directly prepare the labeled antigen from the plasmid-containing antigen cDNA using a radioactive amino acid (e.g., 35S-methionine, 3H-leucine). Labeled antigen often needs to be purified for separating from the free isotope. There are various techniques available for purification. Adsorption column chromatography on powdered cellulose is a rapid assay.6,19 For more extensive purification, one must resort to a separation involving dialysis, gel filtration (using a molecular sieve), or ion-exchange chromatography.20-22 Inorganic iodine resin has also been used to absorb the unreacted 131I.23 After purification, one should determine the absolute quality of antigen required in a particular assay for high sensitivity. It is important that this quantity be kept at a minimum. Therefore, it is desirable to produce high specific activity of the radiolabeled antigen. If the labeled antigen is to be stored for a considerable time, it is usually kept at 2 to 4∞C (e.g., steroids) or it may be quickly frozen for storage at –20∞C (e.g., polypeptides). After storage, the © 2004 by CRC Press LLC 1140_C17.fm Page 4 Monday, July 14, 2003 9:24 AM 17-4 Biomedical Technology and Devices Handbook antigen must be checked for changes in immunoreactivity before use in an assay. The actual conditions for storing the labeled antigen depend on the particular antigen. 17.3.1.2 Radiolabeled Antibody Wide and co-workers24 and Miles and Hales25,26 have pointed out the possible advantage of radioiodinating the antibody instead of antigen. The larger molecular weight of immunoglubulin and the presence of multiple tyrosines on the molecule permit the introduction of multiple-radioiodine molecules without detrimental effects on antibody activity. Iodination of antibody rather than antigen may be especially advantageous if the antigen is easily damaged during iodination or lacks readily iodinatable tyrosines. The major difficulty of the radioimmunoassay using radioiodinated antibody is likely to come in the strong propensity of iodinated antibodies to adhere nonspecifically to glassware and insoluble resin. Thus, the selection of the immunoadsorbent is likely to be critical if this method is to be made to work. Nonspecific binding of the iodinated antibody to the resin can be diminished by preparing Fab fragments of the labeled antibody,27 but this will reduce the functional avidity of the antibody for the resin and may adversely affect assay sensitivity. Another problem is the requirement for substantial amounts of antigen to prepare the resin, which includes the use of this approach for antigens that are in short supply. 17.3.2 Specific Antibody The second prerequisite for a radioimmunoassay is the production of a suitable antiserum. The antibodies are a group of serum proteins that are also referred to as g-globulins or immunoglobulins. Most of these immunoglobulins belong to the IgG class, while the other classes are termed IgA, IgM, IgD, and IgE. Because these immunoglobulins possess not only antibody-reaction sites, but also antigenic determinant sites, the immunoglubulins themselves can serve as antigens when injected into a “foreign” animal. The labeled antigen must, of course, be highly purified to avoid interaction of labeled contaminants with nonspecific antibody.
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