Immunoassay quantitative

ST28 Goodnow, T., LeBlanc, M., Koski, K., Welsh, M., Leung, K., Charie, L. and Evans, S. (1987). Radial partition immunoassay Quantitating CKMB levels in human serum. Clin. Chem. 33, 988, Abstr. 528. 

Turbidimetric Agglutination Immunoassays. Agglutination—precipitation immunoassays were among the first practical appHcations of the antigen—antibody reaction in diagnostic tests. These assays are not as widely used in the 1990s as EIA and FIA because they are either not quantitative enough or lack the sensitivity limits of RIA, EIA, and EIA. 

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). 

Typically, quantitative protein determination is done on the one hand by colorimetric or nephelometric methods, on the other hand for more difficult analytical problems by more sophisticated techniques such as high performance liquid chromatography (HPLC), gel-electrophoresis and immunoassay. However, these methods are tedious, time-consuming and expensive. 

Technology providers use quantitative immunoassays to determine expression data of field material for regulatory submissions. Regulatory authorities require that expression levels of introduced proteins in various plant parts be determined by quantitative, validated methods. Immunoassays are also used to generate product characterization data, to assess food, feed and environmental characteristics, to calculate concentrations for toxicology studies and to obtain tolerance exemption or establish tolerances for pesticidal proteins. 

Quantitative immunoassays have also been used as screening devices to determine whether drug residues exceed established maximum residue limits (MRLs) or tolerances in edible tissues. " For these applications, a cut-off value is set at the tolerance or MRL samples detected above this level are positive , and samples below this level are negative. ... 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Shelver and Smittf confirmed fhaf commercial clenbuterol immunoassays cross-react with some, but not all, clenbuterol metabolites. As a result, quantitative clenbuterol immunoassays may differ from determinative methods if substantial concentrations of metabolites are present. For clenbuterol, the parent clenbuterol level is... 

Kennedy et developed an immunoassay for salinomycin and utilized the lA to check the depletion of salinomycin in chicken muscle and liver. The limit of quantitation was 0.31 pgkg for muscle and 0.29 pgkg for liver these levels were based on the assay response of negative samples -I- 6 SD. The antibody cross-reacted with narasin but did not recognize lasalocid, maduramicin, and monensin. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

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