Quantitative enzyme immunoassay techniques

This chapter deals with the operational details of EIA. Many different designs can be found in the hundreds of publications which appear annually. The assays described here are general methods and might need modifications for individual systems. 

The two bases of EIA, the immune reaction and the measurement of the enzyme activity, are discussed in two separate sections since 

In the schemes in this chapter, x and y denote different animal species, H hapten, Ab antibody in general and AB antibody to Ig (anti-Ig), Ag antigen, E enzyme, ] = is the symbol for solid-phase immobilization, covalent links are indicated by a stop (Ab E, H E), immunological or other non-covalent links (biotin-avidin, Ab-H, Ab-Ag, Ab-AB, SpA-Ab) are indicated by long hyphens and links in immune complexes, prepared prior to addition, by a colon (Ab E). 

In solid-phase EIA, one of the immunoreactants or some other molecule (complement factors, receptors, capture, etc.) is immobilized on the solid phase (Chapter 13), the sample is added, and the test substance retained is reacted with the corresponding immunoreactants, one of which carries an enzyme label. By measuring the extent of the enzymatic reaction the activity or concentration of the immu-noreactant in the test sample is determined. 

Antibodies to the IgG of one species quite often cross-react with IgG of other species (Section 17.3.4.1). A powerful method to prevent 

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Ternyck TH and Avrameas S (eds.) (1990) Quantitative enzyme immunoassays. In Immunoenzymatic Techniques. Vol. 1 - Techniques in Immunology, pp. 43-68. Amsterdam Elsevier. 

The introduction of enzyme immunoassay (EIA) and similar allied immunoassay techniques in early eighties showed, in fact, a brighter path towards quantitative analysis. 

Benzimidazole carbamates have generally been determined by HPLC (20,124-126), although other methods have been reported based on enzyme immunoassay (147) or in gas chromatography (124). The commercial availability of high-performance thin-layer chromatography (HPTLC) plates with layers of very different polarities has improved the sensitivity, selectivity, and analytical precision of TLC, which has led to a reconsideration of the use of this technique for qualitative and quantitative analysis (54). 

If the fluorescent emission spectrum of the bound labeled ligand is sufficiently displaced, enhanced or decreased in intensity (quenched) relative to that of the free labeled ligand, the resulting spectroscopic measurements can be used for quantitation without a separation step. Additionally, the techniques previously described in enzyme immunoassays, such as reactant-labeled immimoassay, can form the bases of fluorescent... 

Blecka, L.J. Shaffar, M. Dworschack, R. Inhibitor enzyme immunoassays for quantitation of various haptens a review. In Immunoenzymatic Techniques Avrameas, S., Dmet, P., Mosseyeff, R., Feldman, G., Eds. Elsevier Amsterdam, 1983 207. 

Enzyme-Multiplied Immunoassay Technique. EMIT is a homogeneous method for the quantitation of haptens, especially hormones, therapeutic drugs, and drugs of abuse. This method is a competitive assay, in which hapten and enzyme-labeled hapten compete for a fixed, insufficient quantity of antibody (Eq. 6.16). 

Several diagnostic tests are available to detect acute HCV infection through detection of antibodies or viral target amplification. Antibody detection methods include enzyme immunoassay (EIA) and the recombinant immunoblot assay (RIBA). Specific antibodies to HCV by EIA are positive in only 50% to 70% of patients during the initial onset of symptoms, but 90% of patients have HCV antibodies after 3 months. A number of viral antigens are included in the current version of EIA, resulting in a 99% sensitivity and specificity for detection of HCV antibodies in immunocompetent patients. Patients with autoimmune disorders may have a false-positive EIA and no detectable HCV RNA, in which case the RIBA may be used as a supplemental test to rule out HCV. Viral target amplification techniques are used to detect HCV RNA either qualitatively or quantitatively. Qualitative tests are more sensitive with a detection limit of up to 100 copies/mL and should be reserved to determine spontaneous clearance of acute infection. Spontaneous clearance of HCV can occur in 50% of patients within 3 months of the acute onset of symptoms. ... 

M.F. Katmeh, A.J.M. Godfrey, D. Stevenson, G.W. Aherne, Enzyme Immunoaffinity Chromatography - A Rapid Semi-quantitative Immunoassay Technique for Screening the Presence of Isoproturon in Water Samples , Analyst, 122, 481-486 (1997). 

Enzyme immunoassay methods offer a biological-based detection technique which is complementary to that of traditional residue analysis. Potential advantages must be evaluated in light of the intended application— use in a nationwide program for quantitatively measuring pesticide residues in foods. Ideally, each EIA method would contain simple, rapid extraction and cleanup (if required) steps, require minimal or portable equipment, be usable in the field by individuals with little scientific training, and produce quantitative results. 

See also Bioluminescence. Chemiluminescence Liquid-Phase. Electrophoresis Blotting Techniques. Enzymes Immobilized Enzymes Enzyme Assays. Fluorescence Quantitative Analysis. Forensic Sciences Blood Analysis. Immunoassays Overview. Immunoassays, Applications Clinical Forensic. Immunoassays, Techniques Radioimmunoassays. 

The platelet enzymes involved (90), the complexity of their interactions and of others (91), and their relationships, both physiologically and perhaps philogenetically, with the reactions of certain white blood cells (90, 92) help us to see platelets as living particles. Their aggregation is used as a quantitative aid in immunoassay (93) in a technique that may well be applicable to quantitation of heat aggregatable gammaglobulins (94) and of specific immunoglobulin G (95). 

The enzymatic methods described in Chapters 2-4 are important bioanalytical tools for the quantitation of those biochemicals participating in enzyme-catalyzed reactions. A vast number of biochemicals that are of analytical interest, however, are not amenable to enzymatic assay techniques. These species are not involved in metabolic processes for which a sufficiently selective enzyme exists, and may not even be species that are commonly found in living systems. For many of these analytes, whether macromolecular or of relatively low molecular weight, immunoassays are the methods of choice. 

Immunochemical methods are rapidly gaining acceptance as analytical techniques for pesticide residue analysis. Unlike most quantitative methods for measuring pesticides, they are simple, rapid, precise, cost effective, and adaptable to laboratory or field situations. The technique centers around the development of an antibody for the pesticide or environmental contaminant of interest. The work hinges on the synthesis of a hapten which contains the functional groups necessary for recognition by the antibody. Once this aspect is complete, immunochemical detection methods may take many forms. The enzyme-linked immunosorbent assay (ELISA) is one form that has been found useful in residue applications. This technique will be illustrated by examples from this laboratory, particularly molinate, a thiocarbamate herbicide used in rice culture. Immunoassay development will be traced from hapten synthesis to validation and field testing of the final assay. 

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