Enzyme immunoassay technology

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Ishikawa, E., Imagawa, M., and Hashida, S. (1983b) Ultra sensitive enzyme immunoassay using fluoro-genic, luminogenic, radioactive and related substances and factors to limit the sensitivity. Proceedings of the 2nd International Symposium on Immunoenzymatic Technology. 

Examples exist of different types of immunobased technology being interfaced to produce an effective analytical system. In a variety of recent methods, immunoaffinity chromatography has been employed for purification, chemiluminescence enzyme immunoassay has been used for quantification of salbutamol and clenbuterol in tissue and plasma from calves and pigs (122), clenbuterol in cattle hair (123), and monensin in chicken tissues (124). In these methods, quantification at sub-ppb levels has been demonstrated. 

An ingenious method of decreasing detection limits in biosensors that is frequently employed is enzyme amplification. An example is enzyme multiplied immunoassay technology (emit). 

Immunosensors have been designed which use both direct and indirect immunoassay technology to detect specific analytes within a minute or less in a variety of matrices (see Fig. 9). Indirect immunosensors may employ ELA, FLA, or CLIA principles whereby enzyme-, fluorophore- or chemiluminescent-labeled analyte competes with the target (nonlabeled) analyte for binding sites on the immobilized antibody. Unbound (free) labeled analyte is then quantitated using an electrochemical, optical, or electromechanical transducer and compared to the amount of target analyte in the sample. 

In the homogeneous immunoassays there is no separation step because they are based on a changing signal by formation of the immune complex. The first assay of this type was the EMIT method (enzyme-modulated immunoassay technology) described by Engvall et al. (1970). The main disadvantages of homogeneous assays are low sensitivity and a more pronounced susceptibility to interferences. 

MODERN TECHNOLOGICAL ADVANCES RELATED TO ENZYME IMMUNOASSAY... 

A growing application of the strip type of technology for immunoassay is for infectious disease testing and the rapid detection of various infectious antigens and antibodies including Chlamydia,group A streptococci, " Helicobacter pylori, ° infectious mononucleosis, and human immunodeficiency virus. These tests commonly use a lateral-flow format for urine, serum, plasma, or whole blood samples in conjunction with analytical techniques, such as enzyme immunoassay and immunochromatography. An alternative... 

Franken N, Lenz H, Maier L, Kessler A. Sensitive, fuUy mechanized TSH enzyme immunoassay using strepavidin/biotin technology. CHn Chem 1991 37 1035. 

Enzyme Immunoassay. Enzyme immunoassay or ElA is probably the immunoassay technology most widely applied today U2). In an EIA, either the antibody or the antigen is tagged with an enzyme. After the assay is performed, the presence or absence of the enzyme-labeled component is detected by the addition of an appropriate substrate. EIA s are quite sensitive, reagents are relatively inexpensive and widely available, and the reagents are relatively stable. Quantitative results can be obtained using instrumentation and EIA is easily automated. 

The tests used most frequently for speciation of animal products are the enzyme-hnked immunosorbent assay (ELISA), including immunosticks and, more recently, lateral flow immunoassays (LFIAs), the latter of which now use latex particles or colloidal gold for detection rather than the older enzyme immunoassay (EIA) technology (seeTable 13.1). 

Winkler ME, Blaber M, Bennett GL et al. (1985) Purification and characterization of recombinant urokinase from Escherichia coli. Bio/Technology 3 990-1000 Wisdom GB (1976) Enzyme-immunoassay. Qin Chem 22 1243—1255... 

Solvent Extraction Principles. Forensic Sciences Drug Screening in Sport Systematic Drug Identification. Gas Chromatography Mass Spectrometry Chiral Separations. Immunoassays, Techniques Enzyme Immunoassays Luminescence Immunoassays. Liquid Chromatography Column Technology. Pharmaceutical Analysis Sample Preparation. 

The first quantitative immunoassays were based on the use of radioactive reporter molecules, mainly and " C. These radioimmunoassays (RIAs) have gradually been replaced by nomadioactive reporter molecules or labels, e.g., enzymes, fluorescent, and luminescent molecules and combinations of these. The nonradio-active labels facilitated development of automated immunoassays, and they also contributed to the design of assays with substantially reduced detection limits. However, nomadioactive assay have an advantage only in immunometric sandwich assays in inhibition assays, the use on nonradioactive labels has not provided any improvement in assay sensitivity as compared to RIA [1]. The development in immunoassay technology has been reviewed by Ekins [2]. 

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

Based on many of the advances described above in electrochemical approaches to immunoassay, it is tempting to conclude that commercialization of some of the approaches is imminent. This may be true, but the historical use of optical methods for many clinical chemistry tests coupled with their rapidly growing use in immunoassay is a difficult barrier for any radically different method to overcome, though electrochemical sensors have become more important in the clinical chemistry laboratory over the last decade. In any event, to be successful ECIA methods will have to demonstrate clear superiority over existing and emerging technologies in both cost and performance. Some of the more recently described approaches such as those using enzyme amplified amperometric detection and ecLIA appear... 

The first application of immunologically based technology to pesticides was not reported until 1970, when Centeno and Johnson developed antibodies that selectively bound malathion. A few years later, radioimmunoassays were developed for aldrin and dieldrin and for parathion. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked... 

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