Immunoradiometric assay

Processing for Radioimmunoassays and Immunoradiometric Assays. Clin. Chem. (1974), 1255. 

K.L. Hoffman, G.H. Parsons, L.J. Allerdt, J.M. Brooks, and L.E. Miles, Elimination of hook-effect in two-site immunoradiometric assays by kinetic rate analysis. Clin. Chem. 30, 1499-1501 (1984). 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Immunoradiometric assays (IRMAs) are like RIAs in that a radiolabeled substance is used in an antibody-antigen reaction, except that the radioactive label is attached to the antibody instead of the hormone. Furthermore, excess of antibody, rather than limited quantity, is present in the assay. All the unknown antigen becomes bound in an IRMA rather than just a portion, as in a RIA IRMAs are more sensitive. In the one-site assay, the excess antibody that is not bound to the sample is removed by addition of a precipitating binder. In a two-site assay, a molecule with at least two antibody-binding sites is adsorbed onto a solid phase, to which one of the antibodies is attached. After binding to this antibody is completed, a second antibody labeled with 125I is added to the assay. This antibody reacts with the second antibody-binding site to form a sandwich, composed of antibody-hormone-labeled antibody. The amount of hormone present is proportional to the amount of radioactivity measured in the assay. 

Induction ofmRNA for pS2 and secretion of cell-type-specific proteins. pS2 was measured in the culture medium of MCF7 cells with the ELSA-pS2 immunoradiometric assay (CIS Bio International, Gif-sur-Yvette, France). Cells were subcultured in 24-well plates for 144 h in 10% CDHuS. The culture medium was centrifuged at 1200g for 10 min to eliminate floating and detached cells. Results are expressed as ng of secreted protein per million cells. The relative-induced protein potency (RIPP) was calculated as 100 X the ratio between the dose of E2 and that of the chemical needed to produce maximal expression of cell-type-specific proteins (pS2). 

M15. Marz, W., Siekmeier, R., Gross, E., and Gross, W., Determination of lipoprotein(a) Enzyme immunoassay and immunoradiometric assay compared. Clin. Chim. Acta 214, 153-163 (1993). 

Martino E, Bambini G, Bartalena L, Mammoli C, Aghini-Lombardi F, Baschieri L, Pinchera A. Human serum thyro-trophin measurement by ultrasensitive immunoradiometric assay as a first-line test in the evaluation of thyroid function. Clin Endocrinol (Oxf) 1986 24(2) 141-8. 

Competitive binding immunoassay (e.g., RIA) Enzyme-linked immunosorbent assay (ELISA) Immunoradiometric assay (IRMA) — dual monoclonal antibody assay Receptor binding assay Cell binding assay... 

Fukata J (1989) Two-site immunoradiometric assay for adreno-corticotrophin a cautionary study about the reactivity to its precursor molecules. Endocrinol Jpn 36 155-61 Ganong WF (1974) ACTH and the regulation of adrenocorticol secretion. N Engl J Med 290 1006 Genazzani AR (1975) Immunoreactive ACTH and cortisol plasma levels during pregnancy. Detection and partial purification of corticotrophin-like placental hormone the human chorionic corticotrophin (HCC). din Endocrinol (Oxf) 4 1-14... 

Hodgkinson SC (1984) Development of a non-extracted twosite immunoradiometric assay for corticotropin utilizing extreme amino and carboxy-terminally directed antibodies. Biochem J 218 703-711... 

Meeran K (1989) Venepuncture causes rapid rise in plasma ACTH. Br J din Pract 1993 47 246-7 Raff H (1989) A new immunoradiometric assay for corticotropin evaluated in normal subjects and patients with Cushing s syndrome, din Chem 35 596-600 Raff H (1995) Intraoperative measurement of adrenocorticotropin (ACTH) during removal of ACTH-secreting bronchial carcinoid tumors. J Clin Endocrinol Metab 80 1036-9... 

Zahradnik R (1989) Immunoradiometric assay of corticotropin with use of avidin-biotin separation. Clin Chem 35 804-807... 

Immunoradiometric Assay (IRMA) of Ferritin with Bead Separation... 

The RIA-gnost Ferritin kit (no more commercially available) from formerly Behringwerke AG, Radiochemical Laboratory described below uses the principle of an immunoradiometric assay (IRMA). It is a two-site solid phase assay of the sandwich type, based on a plastic bead as solid phase to which the antiferritin antibody adheres. The antibody-solid phase is incubated with standards or serum samples containing ferritin and in this process the ferritin in the solution is bound quantitatively to the solid phase via the antibody. The amount of ferritin bound to the solid phase is then determined by a reaction with 125I-labeled anti-ferritin antibody. An antibody-ferritin-125I-antibody complex is thus formed. 

It is typical for an immunoradiometric assay that a high dose hook effect occurs in the region of very high concentrations. Therefore, sera with concentration above the highest standard S7 have to be diluted with the dilution serum included in the kit. 

Gl. Garnero, P., and Delmas, P. D., Assessment of the serum alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease. J. Clin. Endocrinol. Metab. 77, 1046-1053 (1993). 

P2. Pandian, M. R., Morgan, C. H., Carlton, E., and Serge, G. V., Modified immunoradiometric assay of parathyroid hormone-related protein Clinical application in the differential diagnosis of hypercalcemia. Clin. Chem. 38, 282-288 (1992). 

P3. Panigrahi, K., Delmas, P. D., Singer, E., Ryan, W., Reiss, O., et al., Characteristics of a two-site immunoradiometric assay for human skeletal alkaline phosphatase in serum. Clin. Chem. 40, 822-828 (1994). 

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... 

Eiizyme-Iinked immunosorbent assay Radioimmunoassay Immunoradiometric assay Radioreceptor assay... 

The theory and procedure for several immunoradiometric assays are given in the chapters by Hales and Woodhead and by the Merretts." I-labeled protein A is rapidly becoming a valuable tracer in immunoassays. It can bind to the F,. region of IgG molecules without inhibiting the reaction between antigen and antibody. Procedures in which an antibody or protein A are iodinated are particularly useful when the antigen cannot be labeled. 

RADIOIMMUNOASSAYS AND IMMUNORADIOMETRIC ASSAYS [13] Other lodination Reagents... 

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