Nonisotopic labeling

Reagents. Nonisotopically labelled hydrocarbon gases used were Phillips research grade and were further purified by trap-to-trap distillation. 

Obviously, this approach cannot be used for selecting the nonisotope-labeled components. In the following we will consider isotope filtering/editing techniques that do not use heteronuclear chemical shift evolution. 

We have developed a simple method of nonisotopically labeling sample nucleic acids, which are then hybridized simultaneously to an array of unlabeled, immobilized probes. This "reversed hybridization" procedure thus provides identification results after a single hybridization reaction. 

Incorporation studies with isotopes showed that when anthranijate was converted to tryptophan, the carboxyl group df anthranilate was lost as carbon dioxide, but the nitrogen was retained. Because the enzymes in the tryptophan biosynthetic pathway have only a limited specificity, it was possible to substitute 4-methyl-anthranilate in E. coli extracts that could convert anthranilate to indole. This nonisotope label was conserved during the conversion to yield 6-methyl indole. 

The commercially available Enzygnost HBe monoclonal kit (distributed by Dade Behring) can be considered as an example for an immunoassay which combines many favorable features, e.g. nonisotopic label, sandwich immunoassay principle, use of monoclonal antibodies, determination of antigen and anti-antigen antibodies in a single test, microtiter plate format, possibility for semi- and fully automation. 

There is increasing interest in the use of nonisotopic labels in immunoassay. The main advantages of such labels in labeled antibody techniques are the expected longer shelf life of the reagent and the ability to use a detection apparatus that may for one reason or another prove to be more convenient—e.g., for automation or cheapness. Some alternatives that are being explored are bacteriophages, spin labels, enzymes, fluorescent compounds, and luminescent compounds. ... 

Takeuchi T, Dobashi A, Kimura K (2000) Molecular imprinting of biotin derivatives and its application to competitive binding assay using nonisotopic labeled ligands. Anal Chem 72 2418... 

Irrespective of the enzyme employed a variety of labels could be used, including radioactive nucleotide triphosphates. However, methods based on the use of nonisotopic labels have been developed and are superior for a variety of reasons, including ease of use, stability, simplicity, and speed of detection, and the increased resolution obtained. Using this approach, it has been... 

Amplified DNA is identified by solution hybridization of two nonisotopically labeled oligonucleotides to one strand of the amplified DNA. Following hybrid formation DNA is bound to a solid phase and detected by enzyme-labeled specific antibodies. Because only one strand is used for detection, the other one is washed off and constitutes the main source of laboratory contamination and carryover. Take precautions to avoid the spread of these molecules to other rooms (e.g., work in a chemical fume hood, decontaminate used wash buffer with acid or sodium hypochloride, and so on). 

With the development of radioimmunoassays (RIAs) in the 1960s that used radioactive isotopes as labels (see Chapter 9), the measurement of radioactivity became a common and important practice in clinical laboratories. However, concerns about, and problems with, the safe handhng and disposal of radioactive reagents and waste have led to the development of immunoassays that use nonisotopic labels (see Chapter 9). The rapid acceptance and extensive use of nonisotopic immunoassays by the clinical laboratory have resulted in a decreased use of RIA and ultimately a decreased requirement for them to measure radioactivity. Because of this deemphasis on the necessity to measure radioactivity, only a brief discussion of the topic is presented here. Readers requiring more detail on this topic are referred to the chapter entitled Basic Principles of Radioactivity and Its Measurement that is included in a prior edition of this textbook, ... 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

Immunoassays are used to measure PSA and are commercially available. Most of them use nonisotopic labels, such as enzyme, fluorescence, or chemiluminescence. The majority of these assays are automated on an immunoassay system. Different assays and even the same assay with different lots of reagent may produce different results. The reasons for such differences are due to changes in assay calibration, production lot variation, assay reaction time, reagent matrices, assay sensitivity, and imprecision. Antibodies react with different PSA epitopes therefore, some antibodies react dissimilarly with the various molecular forms of PSA. Assays are classified as equimolar if they bind to free and cPSA equally and nonequimolar if they bind to free or cPSA differently. Examples of equunolar assays are the ACCESS... 

Immunoassays employing antibodies are widely used to quantify hormones (see Chapter 9). Currently, labeled-antibody (immunometric) assays with nonisotopic labels are the method of choice for measuring most hormones, especially peptides and proteins. Immunometric assays use saturating concentrations of two or more antibodies (often... 

Besides commercial kits, many immunochemical reagents are available (191,192) anti-drug antibodies, anti-enzyme antibodies, radioisotopic labels, nonisotopic labels, secondary antibodies, and biotinylated conjugates. Because of the flexibility in IA designs, investigators can consider several options from the availability of the commercial materials in addition to in-house resources in preparing the IA reagent components. 

In the early stages of immunoassay technology, radiolabels were employed as tracers. Isotopic labels were popular due to their high sensitivity, selectivity, and unobtrusive nature. The use of radioactive labels does have drawbacks, such as health and safety issues, short shelf life, cost, long exposure times, and disposal problems. Due to these drawbacks, there has been considerable growth in the development of nonisotopic labels for use in immunoassays. Consequently, radioactive labels are now rarely used in immunoassays. Several criteria needed to be met in order for nonisotopic labels to become a viable alternative to radioactive labels 1. Nonisoto-... 

Enzyme immunoassay The tendency to avoid the use of radioisotopes in immunoassays has led to the increasing use of nonisotopic labels in steroid hormone assays. Of these, enzyme immunoassays are most widely used. 

The immunometric-type assay has also been adapted for use with nonisotopic labels and is typically carried out in a heterogeneous format in which the antibody is immobilized on a solid support, such as a microtiter dish, membrane, or collection of beads. The canonical clinical immunoassay format in toady s laboratories is the enzyme-linked immunosorbent sandwich assay, which employs two antibodies, one to capture the analyte and the other to detect and quantify it. More details of the principles of these and other immunoassay techniques are given elsewhere in this encyclopedia. 

In RIAs, a radioactive label is used for detection of the formation of an antibody -antigen complex. Thus, a simple, specific, sensitive, and precise determination of the radioactive isotope is available. At present, however, immunoassays are increasingly carried out with nonisotopically labeled antigens or antibodies because of the legal restrictions on the use of radioactive material. 

The most common type of immunoassay is the nonisotopically labeled heterogeneous test [1]. Either the antibody or the hapten (analyte) to be detected is fixed to a solid interface via a covalent bond or by adsorption. Tlie other significant difference lies in the fact that no radioactive labeling is used for signal production. 

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