Radiolabeled immunoassays

Radiotracers have also been used extensively for the quantitative rnicrodeterrnination of blood semm levels of hormones (qv), proteins, neurotransmitters, and other physiologically important compounds. Radioimmunoassay, which involves the competition of a known quantity of radiolabeled tracer, usually I or H, with the unknown quantity of semm component for binding to a specific antibody that has been raised against the component to be deterrnined, is used in the rnicro deterrnination of physiologically active materials in biological samples (see Immunoassay). 

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. 

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

Immunoassay Methods. Radioimmunoassay (RIA) allows measurement of biologically active materials which are not detectable by traditional cold chemistry techniques. RIAs can be used to measure molecules that cannot be radiolabeled to detectable levels in vivo. They also are used for molecules unable to fix complement when bound to antibodies, or they can be used to identify cross-reacting antigens that compete and bind with the antibody. 

With enzyme-multiplied immunoassay technique (EMIT) assays, enzyme tags are used instead of radiolabels. The antibody binding alters the enzyme characteristics,... 

MEGX is readily detected by HPLC and fluorescence polarization immunoassay techniques [14,21,25,40,41]. The test is simple, normally requiring a onetime blood sampling, and informative because it depends on the capacity of the hepatic enzymes to metabolize lidocaine. While the analysis of lidocaine metabolites is rapid, this method has not been adapted for continuous hepatic function monitoring, which may be possible with the radiolabeled analogues such as Tc-Sn-lidocaine iminodiacetic acid [42]. 

The most common, but by no means the only or even the most promising, immunochemical assay for small molecules is radioimmunoassay (R1A). As an overview, an immunoassay involves chemically attaching the small molecule of interest (or a derivative of it) to a carrier protein and raising specific antibody titers to it in the serum of an animal. Very dilute antibody solutions are then used to bind the small molecule which has been radiolabeled. The competition of varying known concentrations of unlabeled material is measured and the resulting standard curve used to determine unknown concentrations (Table 1). The steps leading to the development of an R1A are outlined below followed by a description of other immunochemical procedures and an analysis of the attributes and limitations of immunoassay. 

Due to the high specificity and sensitivity of immunoassays, there are bioanalytical methods for the measurement of an analyte of interest, with little or without preconcentration or purification of the samples. The principle behind immunoassays is based on an interaction between an antibody and a corresponding antigen, and the detection of the specific interaction using radiolabels (247), enzyme, fluorescent and luminescent compounds (178, 179,181,183), electroactive markers (177,180, 228, 248), or nanomaterials (249-251). 

Antigens, haptens, and antibodies radiolabeled with or are commonly used as tracers in immunoassay. These nuclides can be introduced directly into functional groups normally present in proteins and other macromolecules or into suitable derivatives that can be synthesized by a variety of chemical procedures. The most widely used iodination methods have been direct chemical or enzymic substitution of hydrogen in tyrosine or related groups using chloramine-T or lactoperoxidase, respectively. These methods are described in separate chapters in this volume. 

This report will describe the preparation of radiolabeled PA and emphasize recent advances in the use of the I-labeled compound in immunoassays of fluid-phase and cell-bound antibody and antigen. For a general discussion of the properties of PA and a summary of earlier applications of labeled PA, the reader is referred to the review by Coding. ... 

Use of [ I]PA as a tracer for IgG antibody is independent of antibody specificity. Thus immunoassays can be developed for substances for which stable, immunoreactive radiolabeled derivatives of high specific activity are not readily available. Use of a single tracer obviates the need to prepare, purify, and store large numbers of radioactive ligands in laboratories where radioimmunoassay is a routine analytical method. 

The earliest immunoassays made use of radioactively labeled antigens or antibodies. These analytes are referred to as radiolabeled immunoassays. Antibody binding sites are extremely specific and to retain this specifity the best option would be to replace a non-radioactive isotope in the tracer molecule by its radioisotope (e.g., replace hydrogen by H). However when the substitution is made in a part of the molecule away from the antibody binding site, the choice of radioisotope can be governed by other considerations, such as half-life, availability, high activity, and radiochemical purity. 

Signal antibodies have most often been radiolabeled (for immunoradiometric assays [IRMA]) with ( 25j) 7,62,i%,344,4i4 labeled with a chemiluminescent (for immunochemilummo-metric assays [ICMA]) compound, such as acridinium ester, or an enzyme (enzyme-linked immunosorbent assay [ELISA] or enzyme immunoassay [EIA]), such as ALP, converting a substrate (1,2-dioxetane phosphate) to a chemiluminescent product. 

As of 2004, the College of American Pathologists Interlaboratory Survey listed five manufacturers as providing first generation noncompetitive immunoassays for intact PTH. Most of these commercially available methods are on fully automated immunoassay analyzers using chemiluminescence detection including ALP with 1,2-dioxetane phosphate, acridinium ester, or electrochemiluminescence (ruthenium chelate). Signal antibody is less commonly radiolabeled with L... 

More sensitive and noncompetitive immunoassays (IRMAs) have also been developed. Currently available assays use antibodies against PTHrP (sequences 37-74,1-40, i-40, 1-34, 1-40, and so on) as capture antibodies. Their radiolabeled signal antibodies are against PTHrP (sequences 1-36, 60-72, 57-80, 37-67, 50-83, and so on), respectively. The limit of detection for these assays is reported to be from 0,1 to 1.0 pmol/L. 

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