Immunoassay enzyme methods

All specimens were analyzed by EMIT (Enzyme Immunoassay Technique), an enzyme method based upon the competitive bonding of an enzyme and an antibody. This method yields a positive result with concentrations of 75 ng of PCP/ml or greater (Rubenstein et al. 

Butler, J.E. (2000b) Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. In Methods in Molecular Medicine Molecular Diagnosis of Infectious Diseases. (J. Decker, and Reischl, eds.), Vol. 94, pp. 333-372. Humana Press, Inc., Totowa, NJ. 

In addition to these research applications of fluorescence, there is a continuing use of fluorescence detection to replace analytical methods based on radioactivity, as can be judged from the recent books and conferences on fluorescence sensing methods. (7 n) These emerging applications of fluorescence can be seen by the growth and introduction of improved methods for immunoassays, enzyme-linked immunoassays... 

Brillhart, K. L., and Ngo, T. T. (1991) Use of microwell plates carrying hydrazide groups to enhance antibody immobilization in enzyme immunoassays./. Immunol. Methods 144,19— 25. 

Chlorpyrifos — Comparison of enzyme-linked immunoassay (ELISA) methods [534]... 

Pataki J, Szabo M, Lantos E, Szekvolgyi L, Molnar M, Hegedus E, Bacso Z, Kappelmayer J, Lustyik G, Szabo G. Biological microbeads for flow-cytometric immunoassays, enzyme titrations, and quantitative PCR. Cytometry Part A 2005 68A 45-52. Martins TB, Augustine NH, Hill HR. Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to group A streptococci. J. Immunol. Methods 2006 316 97-106. 

Early work demonstrated the use of enzymes coupled to antibodies or antigens as reagents in immunoassay. Enzyme activity can be measured in a variety of ways, each with certain advantages, which makes a variety of enzymes good labeling substances. Most assay methods are based on spectroscopic properties derived from an enzymatically transformed substrate. These methods are colorimetry, flurorometry, luminometry, and electrometry. 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

Methods based on radiolabels continue to hold an important place in routine analysis and in research related to clinical testing. The main techniques included in this group are radioimmunoassy (RIA), immunoradiometric assay (IRMA), and scintillation proximity assay (SPA). Many researchers in this field use short-lived radioisotopes and chelating agents in antibody labeling.139 The most popular types of immunoassay are methods that use enzymatic labels the enzyme-linked immunosorbent assay (ELISA), the enzyme-monitored immunotest (EMIT), the competitive binding enzyme immunoassay (EIA), and the immunoenzymometric assay (IEMA). 

Samples from patients receiving digoxin therapy were analyzed by the heterogeneous immunoassay LCEC method. Samples were diluted 5/1 with pooled human plasma in order to eliminate an observed antibody matrix effect. Digoxin standards in pooled human plasma were treated similarly and a standard curve constructed. Digoxin levels in patient samples were determined by reference to the standard curve. The values obtained for the samples by the electrochemical enzyme immunoassay method were compared to those obtained by radioimmunoassay. The results for the 54 samples analyzed are presented in Fig. 8. A good correlation was obtained between the two methods (r = 0.93). 

Hepler, B.R. Sutheimer, C. Sunshine, I. Sebrosky, G.F. Combined enzyme immunoassay-LCEC method for the identification, confirmation, and quantitation of opiates in biological fluids. J.Anal.Toxicol., 1984, 8, 78-90... 

Analysis of food protein Acidi metric and Enzyme immunoassay Kinetic methods... 

Enzyme immunoassay Enzymes (i) Spectrometry (ii) Fluorimetry Homogeneous methods, commercially available methods cost may be a disadvantage enzymes are labile (12), (13), (14)... 

Other methods with potential application in sensor development include certain enzyme immunoassays and sol-particle immunoassay. The enzyme methods that are potentially useful are those where the detection of the enzyme is carried out with a substrate that produces colour that may be determined colorimetrically, or a substrate that produces fluorescence. 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Enzyme Immunoassay. In EIA, antibody (or antigen) is labeled with (or conjugated to) an enzyme, and this reagent is used to complex and quantify the target antigen (or antibody) in a sample. Conjugation may utilize a variety of chemical methods. 

In the most common method for chemiluminescent immunoassay (GLIA), after the immunological reaction and any necessary separation steps, the labeled compounds or complexes react with an oxidizer, eg, hydrogen peroxide, and an enzyme, eg, peroxidase, or a chelating agent such as hemin or metal... 

P. A. Cunniff, ed.. Official Methods of Analysis of AO AC International, 16th ed., Vols. I and II, AO AC International, Arlington, Va., 1995. Vol. I includes Pesticide Formulations and Pesticide Residues. Over 2100 coUabotatively tested, approved methods for chemical and microbiological analyses, with 149 new methods, 103 revised/updated methods, methods using anibody-based test kits, enzyme immunoassay, and annual supplements containing new and revised methods chemical and common names of all dmgs and pesticides easy-to-locate references. 

The biological and medical sciences are ripe for iastmmentation advances. Whereas most immunoassays (qv) use radioactive materials, the implementation of chemiluminescent methods, enzyme techniques, and electrochemical methods is expected to become more important. New and better noninvasive methods of iavestigation are expected to become more routine. In addition, real-time measurements, whereby analyses of a number of... 

The immunochemical interaction between the antigen and antibody is very specific. By labeling either the antigen or antibody, the method s sensitivity is increased. The most frequently used labels to increase sensitivity are radionucHdes (see Radioisotopes) where the assay process is called radioimmunoassay (RIA), or en2ymes where the assay is named en2yme immunoassay (ElA) (see Enzyme applications). 

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