Biomedical Technology and Devices Handbook

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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 determined 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

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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 concentration 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.

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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

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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. The antigen-binding sites appear to reside on the H and L chains of the IgG molecule. While the titer, or concentration of the antibody is important, the main criterion for establishing a suitable antiserum is the energy of interaction between the antigen and antibody, or the specificity and affinity for the antigen being assayed. For most clinical chemists who are interested in performing radioimmunoassay procedures, it would be more advantageous to procure the specific antiserum from a laboratory or commercial supplier that has the facilities required for the generation and evaluation of antibodies. The important limiting factors for the development of highly sensitive radioimmunoassays are antibody affinity, avidity, specificity, and cross reactivity. Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody. Affinity is the equilibrium constant that describes the Ag-Ab reaction as illustrated below. Most antibodies have a high affinity for their antigens.

[]Ag- Ab Keq = []Ag¥[] Ab Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities. Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in Ag-Ab reactions. Antibodies can distinguish differences in (1) the primary structure of an antigen, (2) isomeric forms of an antigen, and (3) secondary and tertiary structure of an antigen. Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one

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antigen. Cross reactions arise because the cross-reacting antigen shares an epitope in common with the immunizing antigen or because it has an epitope that is structurally similar to one on the immunizing antigen (multispeciﬁcity). The general method of inducing antibody formation is to inject into a number of animals the pure antigen mixed with “Freund’s adjuvant.”28,29 Freund’s adjuvant is a mixture of mineral oil, waxes, and killed bacilli that enhances and prolongs the antigenic response. Small peptides (mol wt 1000 to 5000) or nonpeptidal substances which are not of themselves antigenic may be rendered so by coupling to a large protein. A variety of methods may be employed to bind the small molecules to immunogenic carriers.30-32 The antisera can be stored for long periods of time under proper conditions. Repeated freezing and thawing should be avoided and all antisera should be stored properly diluted. Reports vary as to the best temperature for antiserum storage; some researchers prefer –80˚C, others prefer –20 or –15˚C.33 Once thawed, the sera are best kept at 4˚C.

17.3.3 Separation Methods The third requirement for a radioimmunoassay is a suitable method for complete and rapid separation of the bound antigen from the free antigen. In addition, a separation procedure that permits further association and dissociation of the reactants will seriously impair the effectiveness of the assay. Regardless of the method of separation chosen, it must be reproducible, simple to perform, and economically feasible. Table 17.2 lists a variety of techniques that have been used for separation of antibody-bound and free- labeled antigens. The use of so many methods is a tribute to the imagination and versatility of investigators in the ﬁeld, and is also due to the recognition that no single method has proved completely satisfactory for all antigens. Following are some examples of the separation method. 17.3.3.1 Precipitation with Ammonium Sulfate Ammonium sulfate (33 to 50% ﬁnal concentration) will precipitate immunoglobulins, but not many antigens. Thus, this can be used to separate the immune complexes from free antigen. This has been called the Farr technique.

TABLE 17.2 Methods and Materials for the Separation of Antibody-Bound and Free Antigen

Separating Action Method/Material

Precipitation of the Ag-Ab complex Ammonium sulfate Sodium hydrogen sulfate Zirconyl phosphate Ethanol 2-Propanol Dioxane Polyethylene glycol (PEG) Partition of the components due to their Chromatography different mobility and molecular size Electrophoresis Gel ﬁltration Ultracentrifugation Adsorption of free antigen to solid-phase Charcoal (also bound to dextran or albumin) materials Cellulose Sephadex (Cross-linked dextrans) Sepharose (Beaded agarose) Silicates Iron-exchange resins Polymerized antibodies Binding of the antibody to a solid phase Antibody chemically bound to polymer carrier Coated tubes or beads Immunological complex formation with a Double-antibody technique second antibody Second antibody chemically bound, e.g., to activated cellulose

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FIGURE 17.3 Separation of antibody-bound and free antigen.

17.3.3.2 Anti-Immunoglobulin Antibody The addition of a second antibody directed against the first antibody can result in the precipitation of the immune complexes and thus the separation of the complexes from free antigens. This has been called the double antibody method (Figure 17.3). This method is widely applicable and gives highly satisfactory results provided careful attention is given to possible pitfalls. The diluted antiserum containing the first antibody must contain sufficient g -globulin to give a macroscopic precipitate with an excess of second antibody (usually 2 to 5 mg/ml). To minimize the unnecessary expenditure of second antibody and to avoid nonspecific binding of radiolabeled antigen to bulky precipitate, the quantity of precipitate is adjusted just enough to give good precipitation of radioactivity. In the event that the first antibody has a low titer, the expenditure of the second antibody will be prohibitive and other methods must be used. 17.3.3.3 Immobilization of the Antibody The antibody can be immobilized onto the surface of a plastic bead or coated onto the surface of a plastic plate, and thus the immune complexes can easily be separated from the other components by simply washing the beads or plate.34 This is the most common method used today and is referred to as solid- phase RIA (Figure 17.3). A number of methods have been used for attaching antibody to the solid phase. Formation of a stable resin-protein complex may require an initial chemical reaction to activate the resin followed by exposure to the antibody. In conjunctions involving antibodies, ideally g-globulin fractions rather than whole serum are used in the second step in order to minimize binding of extraneous proteins to the resin. Activation of the resin is commonly accomplished with bromoacetyl bromide or cyanogens bromide.35 Alternatively, stable azide or aromatic amine residues can be introduced into a resin and the activation accomplished just prior to the introduction of the protein. Resins containing primary aliphatic amino or carboxyl groups combine with antibody in the presence of carboxyl activating reagents. Com- monly used resins include agarose, Sephadex, and cellulose. Solid phase systems have the advantage of simplicity and rapidity. However, specific problems may arise, such as difficulties in dispension of the resin. The establishment of equilibrium, although rapid in some systems, may require continuous mixing over a period of many hours in others.

17.4 Validation of a Radioimmunoassay Procedure

The radioimmunoassay differs from the traditional bioassay in that it is an immunochemical procedure, which is not affected by biological variability of the test system. The measurement depends only upon the interaction of chemical reagents in accordance with the low of mass action. However, nonspecific factors do interfere in chemical reactions, and cross-reacting prohormones, molecular fragments, and related hormonal antigens can alter the specificity of the immune reaction. In order to ensure reliability and the results obtained, and thus to guarantee the quality of a radioimmunoassay, it is necessary to know a number of characteristic data. These include the specificity and sensitivity,

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and above all, the accuracy, reproducibility, and precision of an assay. The specificity of a radioimmunoassay is essentially determined by the antibody. It can be impaired by cross reactions with similar substances and fragments thereof, and also by the separating step. Serum factors, pH value, and additives such as albumin, buffer, heparin, or preservatives can also lower the specificity. The sensitivity depends on the specificity of the antibody and the adequate specific radioactivity of the tracer. The sensitivity can be regarded as the special case of the accuracy at zero concentration. The sensitivity of assays in which the immunological reaction is irreversible or measurement is performed at nonequilibrium can occasionally be raised by delayed addition of the radioactively labeled substance.36 The accuracy attainable in a determination depends on three errors of various magnitudes. Apart from experimental errors arising in several pipetting steps and interference of the reaction, errors also occur in measuring the radioactivity and on evaluation of the various samples. Owing to the different nature of these errors, it is clear that the accuracy cannot be the same in each portion of the curve. However, multiple determinations and long counting time can do much to reduce random errors and thus lead to a more accurate result. Reproducibility is another criterion of quality, including intra- and interassay reproducibility. Dupli- cate or multiple determinations on samples of an assay and the resulting deviations from the average value yield the intraassay reproducibility. It normally lies below 5%. The interassay reproducibility is the coefficient of variation obtained on determination of the same sample in several different assay runs using different reagents. Its value lies below 10% for accurate and reproducible radioimmunoassay.37

17.5 Applications

In the years since the development of radioimmunoassay, the application of the technique has brought profound changes to medicine and biology. Figure 17.4 shows the schematic methods for radioligand binding assay currently used to detect autoantibodies against recombinant antigen in sera.38 In the measurement of autoantibodies, the cDNA of autoantigens are required to prepare the radiolabeled autoantigens. Such cDNA are usually obtained from an appropriate cDNA library using plaque hybrid- ization techniques or appropriate mRNA using the reverse-transcriptase (RT)-PCR method. The cloned cDNA are then subcloned into the plasmid vectors suitable for an in vitro transcription/translation system. Radiolabeled autoantigens are prepared using the in vitro transcription/translation system with reticu-

FIGURE 17.4 Schematic methods for radioligand binding assay.

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FIGURE 17.5 High-throughput radioimmunoassay for autoantibodies to recombinant autoantigens. (From Kawasaki, E. and Eisenbarth, G. S., Front. Biosci., 5, E181, 2000. With permission.)

locyte lysates and a radioactive amino acid (e.g., 35S-methionine, 3H-leucine). For detection of autoantibodies to recombinat autoantigens, we use a 96-well plate ﬁltration technology and a microplate direct b counter (Figure 17.5).10,38 After determining the incorporation rate of radioisotope by trichloroacetic acid (TCA) precipitation method, in vitro-translated radiolabeled protein (20,000 cpm of TCA precipi- table protein) is incubated with patients’ sera (5 ml for duplicate) at a 1:25 dilution overnight at 4∞C, and the resulting immunocomplexes are precipitated with 25 ml of protein A-Sepharose in the 96-well plate. After a washing step utilizing a vacuum-operated 96-well plate washer, radioactivity is determined directly in the 96-well plate with 96-well plate b counter. The adaptation of the assay to a 96-well plate and semiautomated 96-well counting allows a single person to analyze more than 40,000 samples per year. Table 17.3 lists in vitro transcribed and translated recombinant autoantigens which we have suc- cessfully utilized for disease diagnosis and prediction using radioligand binding assay. A similar assay format can be used to measure several autoantibodies at the same time using recombinat autoantigens labeled with different radioisotopes. Eisenbarth and co-workers have recently developed a combined IA-2-GAD65 autoantibody radioassay utilizing [3H]-labeled GAD65 and [35S]-labeled IA-2 that allows simultaneous detection and discrimination of both autoantibody speciﬁcities.39 In vitro-translated

TABLE 17.3 Autoantibody Radioassays Based on the In Vitro Transcription/Translation of Autoantigens

Worked Didn’t work

GAD65 Proinsulin ICA512/IA-2 ICA69 Phogrin/IA-2b H,+ K+-ATPase Carboxypeptidase H 21-hydroxylase CYP2D6 Transglutaminase

Source: Kawasaki, E. and Eisenbarth, G.S., High-throughput radioassays for autoantibodies to recombinant autoantigens, Front. Biosci., 5, E181, 2000. With permission.

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[3H]-GAD65 and [35S]-IA-2 were mixed and incubated with serum in a tube and the radioactivity was counted in a 96-well plate with channel windows set for each radionucleotide after protein-A Sepharose precipitation. The combined assay gave essentially identical results to those obtained in the single radioassays. The simplicity of this assay with dual determination of the two antibodies utilizing 5 ml of sera, 96-well membrane separation of autoantibody-bound labeled autoantigen, and 96-well b counting facilitates the rapid screening of thousands of samples.

References 1. Berson, S.A. et al., Insulin-I131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects, J. Clin. Invest., 35, 170, 1956. 2. Berson, S.A. and Yalow, R.S., Kinetics of reaction between insulin and insulin-binding antibody, J. Clin. Invest., 36(Abstr.), 873, 1957. 3. Berson, S.A. and Yalow, R.S., Isotopic tracers in the study of diabetes, Adv. Biol. Med. Phys., 6, 349, 1958. 4. Berson, S.A. and Yalow, R.S., Recent studies on insulin-binding antibodies, Ann. N.Y. Acad. Sci., 82, 338, 1959. 5. Yalow, R.S. and Berson, S.A., Assay of plasma insulin in human subjects by immunological methods, Nature, 184, 1648, 1959. 6. Yalow, R.S. and Berson, S.A., Immunoassay of endogenous plasma insulin in man, J. Clin. Invest., 39, 1157, 1960. 7. Yalow, R.S. and Berson, S.A., Introduction and General Considerations, J.B. Lippincott, Philadel- phia, 1971, 1. 8. Yalow, R.S. and Berson, S.A., Fundamental Principles of Radioimmunoassay Techniques in Measure- ment of Hormones, Excepta Medica, Amsterdam, 1971, 16. 9. Kawasaki, E. and Eisenbarth, G.S., Multiple autoantigens in the prediction and pathogenesis of type I diabetes, Diab. Nutr. Metab., 9, 188, 1996. 10. Kawasaki, E. and Eisenbarth, G.S., High-throughput radioassays for autoantibodies to recombinant autoantigens, Front. Biosci., 5, E181, 2000. 11. Murphy, B.E.P., Engelberg, W., and Pattee, C.J., Simple method for the determination of plasma corticoids, J. Clin. Endocr., 23, 293, 1963. 12. Carr, R.I., Wold, R.T., and Farr, R.S., Antibodies to bovine gamma globulin (BGG) and the occurrence of a BGG-like substance in systemic lupus erythematosus sera, J. Allergy Clin. Immu- nol., 50, 18, 1972. 13. Rothenberg, S.P., A radio-enzymatic assay for folic acid, Nature, 206, 1154, 1965. 14. Lefkowitz, R.J. et al., ACTH receptors in the adrenal: specific binding of ACTH-125I and its relation to adenyl cyclase, Proc. Natl. Acad. Sci. U.S.A., 65, 745, 1970. 15. Yalow, R.S. and Berson, S.A., Labelling of proteins-problems and practices, Trans. N.Y. Acad. Sci., 28, 1033, 1966. 16. Berson, S.A. and Yalow, R.S., Recent Advances in Immunoassay of Peptide Hormones in Plasma, Excepta Medica, Amsterdam, 1969, 50. 17. Berson, S.A. and Yalow, R.S., Iodoinsulin used to determine specific activity of iodine-131, Science, 152, 205, 1966. 18. Jackson, R.J. and Hunt, T., Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA, Methods Enzymol., 96, 50, 1983. 19. Berson, S.A. and Yalow, R.S., Preparation and purification of human insulin-I131; binding to human insulin-binding antibodies, J. Clin. Invest., 40, 1803, 1961. 20. Desranleau, R., Gilardeau, C., and Chretien, M., Radioimmunoassay of ovine beta-lipotropic hormone, Endocrinology, 91, 1004, 1972. 21. Catt, K.J. and Cain, M.C., Measurement of angiotensin II in blood, Lancet, 2, 1005, 1967.

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22. Midgley, A.R., Jr., Radioimmunoassay: a method for human chorionic gonadotropin and human luteinizing hormone, Endocrinology, 79, 10, 1966. 23. Saxena, B.B. et al., Radioimmunoassay of human follicle stimulating and luteinizing hormones in plasma, J. Clin. Endocrinol. Metab., 28, 519, 1968. 24. Wide, L., Bennich, H., and Johansson, S.G., Diagnosis of allergy by an in vitro test for allergen antibodies, Lancet, 2, 1105, 1967. 25. Miles, L.E. and Hales, C.N., Labelled antibodies and immunological assay systems, Nature, 219, 186, 1968. 26. Miles, L.E. and Hales, C.N., The preparation and properties of purified 125-I-labelled antibodies to insulin, Biochem. J., 108, 611, 1968. 27. Hubacek, J., Kubicek, R., and Vojacek, K., Immunoassay of human luteinizing hormone using univalent radioactive antibodies, J. Endocrinol., 51, 91, 1971. 28. Freund, J., The effect of peraffin oil and mycobacteria on antibody formation and sensitization, Am. J. Clin. Pathol., 21, 645, 1951. 29. Freund, J., Some aspects of active immunization, Annu. Rev. Microbiol., 1, 291, 1947. 30. Erlanger, B.F. and Beiser, S., Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA, Proc. Natl. Acad. Sci. U.S.A., 52, 68, 1964. 31. Talamo, R.C., Haber, E., and Austen, K.F., Antibody to bradykinin: effect of carrier and method of coupling on specificity and affinity, J. Immunol., 101, 333, 1968. 32. Richards, F.M. and Knowles, J.R., Glutaraldehyde as a protein cross-linkage reagent, J. Mol. Biol., 37, 231, 1968. 33. Thoeneycroft, I.H. et al., Preparation and Purification of Antibodies to Steroids, Appleton-Century- Crofts, New York, 1970, 63. 34. Wide, L., Radioimmunoassays employing immunosorbents, Acta Endocrinol. Suppl. (Copen- hagen), 142, 207, 1969. 35. Robbins, J.B., Haimovich, J., and Sela, M., Purification of antibodies with immunoadsorbents prepared using bromoacetyl cellulose, Immunochemistry, 4, 11, 1967. 36. Rodbard, D. et al., Mathematical analysis of kinetics of radioligand assays: improved sensitivity obtained by delayed addition of labeled ligand, J. Clin. Endocrinol. Metab., 33, 343, 1971. 37. Rodbard, D., Statistical quality control and routine data processing for radioimmunoassays and immunoradiometric assays, Clin. Chem., 20, 1255, 1974. 38. Sera, Y. et al., Autoantibodies to multiple islet autoantigens in patients with abrupt onset type 1 diabetes and diabetes diagnosed with urinary glucose screening, J. Autoimmun., 13, 257, 1999. 39. Kawasaki, E. et al., Evaluation of islet cell antigen (ICA) 512/IA-2 autoantibody radioassays using overlapping ICA512/IA-2 constructs, J. Clin. Endocrinol. Metab., 82, 375, 1997.