Immunoassay methods

Immunoassay methods correlate total petroleum hydrocarbons with the response of antibodies to specific petroleum constituents. Many methods measure only aromatics that have an affinity for the antibody, benzene-toluene-ethylbenzene-xylene, and PAH analysis (EPA 4030, Petroleum Hydrocarbons by Immunoassay). 

The principle behind the test method(s) is that antibodies are made of proteins that recognize and bind with foreign substances (antigens) that invade host animals. Synthetic antibodies have been developed to complex with petroleum constituents. The antibodies are immobilized on the walls of a special ceU or filter membrane. Water samples are added directly to the cell, while soils must be extracted before analysis. A known amount of labeled analyte (typically, an enzyme with an affinity for the antibody) is added after the sample. The sample analytes compete with the enzyme-labeled analytes for sites on the antibodies. After equilibrium is established, the cell is washed to remove any um-eacted sample or labeled enzyme. Color development reagents that react with the labeled enzyme are added. A solution that stops color development is added at a specified time, and the optical density (color intensity) is measured. Because the coloring agent reacts with the labeled enzyme, samples with high optical density contain low concentrations of analytes. Concentration is inversely proportional to optical density. 

The antibodies used in immunoassay kits are generally designed to bond with selected compounds. A correction factor supplied by the manufacturer must be used to calculate the concentration of total petroleum hydrocarbons. The correction factor can vary depending on product type because it attempts to correlate total petroleum hydrocarbons with the surrogates measured. 

Immunoassay tests do not identify specific fuel types and are best used as screening tools. The tests are dependent on soil type and homogeneity. In particular, for clay and other cohesive soils, the tests are limited by a low capacity to extract hydrocarbons from the sample. 

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Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

In spite of these drawbacks, RIA remains a principal immunoassay method and it is expected to continue to be used extensively in many clinical and research laboratories for the foreseeable future. 

A number of soHd-phase automated immunoassay analyzers have been used for performing immunoassays. Table 5 (96) provides usefiil information on maximum tests that can be mn per hour, as well as the maximum number of analytes per sample. A number of immunoassay methods have been found usefiil for environmental analysis (see AUTOMATED INSTRUMENTATION). 

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

To understand how immunoassay-based analytical methods can be constructed to comply with tolerance enforcement requirements, a brief examination of those requirements is in order. This discussion is not intended to be comprehensive but to highlight aspects of special significance to immunoassay method development. The reader is urged to consult the literature " for further details. 

These requirements have special implications with regard to immunoassay methods. Eirst, the lack of commercial availability of reagents precludes preparing antibody-coated tubes or plates on-site, which may require knowledge of special skills. Commercial availability also ensures the analyst access to a reproducibly manufactured product. Therefore, the method must be based on an immunoassay that is a commercial product. Method developers may choose to introduce an in-house assay to the marketplace by partnering with a manufacturer, although this approach is costly and time-consuming. 

The only published immunoassay method submitted to date to EPA OPPTS as an enforcement method for a range of substrates (water, sediment, crops, processed crop fractions, and animal tissues) is the spinosad method, developed by Young et al. This method uses the spinosad RaPID Assay (Strategic Diagnostics) for determination of total spinosad residues (TSR). This discussion will be limited to crop and animal tissues, because the water and soil analyses are analogous to the triasulfuron method. The extraction, cleanup, and method parameters are summarized in Table 2. 

The methylxanthines can be determined in foods and biological systems by the chromatographic methods of TLC, GC, HPLC, or CE. Ultraviolet spectroscopy following a separation procedure can also be used. More recently, immunoassay methods have been developed. There is no single best method the analyst must balance the features of each assay with the final requirements for data precision and reproducibility. 

A competitive fluorescence-polarization immunoassay method was described for the monitoring of 12 drugs including valproic acid [18]. Samples (serum or plasma) were deproteinated. Fluorescence from the fluorescein-labeled analyte used as tracer was excited at 488 nm and polarization of light emitted at 531 nm was measured. The calibration was stable for 4 weeks and the coefficient of variation was below 10%. A single measurement took 8-10 min. 

Volume 92. Immunochemical Techniques (Part E Monoclonal Antibodies and General Immunoassay Methods)... 

Butler, J.E. (2000a) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods 22, 4-23. 

J.H. Skerritt and B.E.A. Rani, in Residue Analysis in Food Safety Applications of Immunoassay Methods (R.C. Berier and L.H. Stanker, eds), ACS Symposium Series, Washington (1996). 

Immunoaffinity chromatography (IAC), 6 400—402 12 137, 145 Immunoanalyzers, automated, 14 150 Immunoassay(s), 14 135-159. See also Immunoassay- DNA probe hybrid assays Immunoassay methods Immuno(bio)sensors antibody-antigen reaction, 14 136-138 basic technology in, 14 138-140 chemiluminescent, 14 150-151 classification of, 14 140-153 design of, 14 139-140 enzyme, 14 143-148 fluorescence, 14 148-150 highly specific, 14 153 historical perspective on, 14 136 microarrays and, 14 156—157 microfluidics in, 26 968—969 monoclonal versus polyclonal antibodies in, 14 152-153... 

Immunoassay kits, 14 152 Immunoassay methods alternative, 14 151 comparison of, 14 151-153 Immunoassay technology, 12 97 Immunoblotting, 9 756 Immunochromatographic assay, 14 141-142... 

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. 

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