Immunoassay principles

Salmain, M., Vessieres, A., Brossier, P., Butler, I.S., and Jaouen, G. (1992) Carbonylmetallo-immunoassay (CMIA) a new type of nonradioisotopic immunoassay. Principles and application to phenobarbital assay.J. Immunol. Meth. 148, 65-75. 

The commercially available Enzygnost HBe monoclonal kit (distributed by Dade Behring) can be considered as an example for an immunoassay which combines many favorable features, e.g. nonisotopic label, sandwich immunoassay principle, use of monoclonal antibodies, determination of antigen and anti-antigen antibodies in a single test, microtiter plate format, possibility for semi- and fully automation. 

Since the immunoassay principle was introduced by Yalow and Berson [2] for the determination of insulin and by Ekins [3] for the determination of thyroxine, there has been an exponential growth in immunoassay development, not only in the range of applications, but also in the number of novel and ingenious designs. This enormous scientific interest is clearly reflected by the number of scientific publications that have appeared in the last four decades in the immunoassay field (Fig. 9.1). 

K Helgert, et al. Enzyme immunoassay—Principle and application. Pharmazie 44 ... 

Moser, A.C. Hage, D.S. Capillary electrophoresis-based immunoassays Principles and quantitative applications. Electrophoresis 2008, 29, 3279-3295. 

The ELISPOT (enzyme-linked immunospot) assay is based on classical immunoassay principles and the detection procedures employed in ELISA assays. Its sensitivity, ease of use, employment of highly reactive, standardized monoclonal antibodies, conunercial availability, and ready application to studies on individual cell types makes it a good choice for research and diagnostic... 

Figure 12.9 Microfluidic chip layout and immunoassay principle (IRC, immunological reaction chamber and ERC, enzymatic reaction chamber). (Adapted with permission from Ref [64]. Copyright The Royal Society of Chemistry)...

Apoptosis and necrosis were detected using the Cell Death Detection EL1SA kit, Roche (Version 11.0), a photometric enzyme-immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone associated DNA fragments after induced cell death. Assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones that allows specific determination of mono- and oligonucleosomes in the cytoplasmatic fraction of the cell. 

Turbidimetry and Nephelometry. In contrast to classical absorbance methods, immunoassay reactions frequently involve agglutination in which the optical scatter signal of the agglutinated particles is measured by turbidimetric or nephelometric means. The principles of light scattering as it relates to analytical methods is discussed in reference 6. 

The use of immunoassay methodology for residue trial analysis is in principle just as acceptable as for enforcement methods, provided that the method has been adequately validated. Because the validation of such methods requires a different approach, as opposed to chromatographic and spectrometric methods, some important points to be aware of in the use are explained in SANCO/3029/99. The authors do not go into detail on this subject here, since on the one hand very few methods have been submitted up to the present, and on the other it would go beyond the scope of this article. 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

In the following discussion, the detection of pesticides and veterinary dmgs in food animals by immunoassay will be described. Discussion will be organized by compound class, the specific analyte, and, finally, the tissues examined. The general principles described in the first part of this review provide the rationale in the applications described in the following pages. 

Assay principle Radioimmunoassay (RIA) Microparticle enzyme immunoassay (MEIA)... 

According to the practical equipment there are useful tools, so-called test kits, which are units that contain all the reagents and a simple instrumentation in form of plates, tubes and wells. The test kits work rapidly, are easily to handle and field-portable. Frequently, biochemical principles are applied, especially immunoassay techniques which use body-antibody reactions. 

Figure 11 The basic principle of ECL immunoassay using streptavidin-coated magnetic beads, and labels based on Ru(bpy)32+.

Using the same PAbs an optical biosensor system has been developed for 2,4,6-TCP [224]. The principle is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d=58.4 mm) produced by a piezoelectric generator system. A continuous Ar ion laser (488 nm) excites the fluorescent tracer and its fluorescence is detected by a spectrometer attached to a cooled, charge-coupled device (CCD) camera... 

Fig. 31 (A) Principle of a sandwich immunoassay using FDA particulate labels. The analyte is first immobilized by the capture antibody preadsorbed on the solid phase (a) and then exposed to antibody-coated microparticle labels (b). Every microparticle contains 108 FDA molecules. High signal amplification is achieved after solubilisation, release, and conversion of the precursor FDA into fluorescein molecules by the addition of DMSO and NaOH (c). (B) Calibration curves of IgG-FDA microcrystal labels with increasing surface coverage of detector antibody (a-d) compared with direct FITC-labeled detector antibody (e). The fluorescence signals increase with increasing IgG concentration. FDA microcrystals with a high IgG surface coverage (c,d) perform better than those with lower surface coverage (a,b). (Reprinted with permission from [189]. Copyright 2002 American Chemical Society)...

Despite many novel developments in immunoassay design the principles are confined to two broad approaches those that rely on the competition between antigens labelled with a molecule which may be readily observed (for example, a radioisotope) and unlabelled antigens for a limited number of antibody binding sites and those in which the antibody is available in excess and for which there is no competition for binding sites. 

Other successful homogeneous assays have been developed such as the fluorescence polarization technique made available by Abbott Laboratories of North Chicago, USA. This competitive immunoassay technique relies upon the principle... 

E. P. Diamindis, Immunoassays with time-resolved fluorescence spectroscopy Principles and applications, Clin. Biochem. 21, 139-150(1988). 

The aim of this chapter is to discuss fluorescence concepts that are used in selected immunoassay applications. The primary focus is on fluorescence topics of recent interest that provide insight into the characteristic properties of antibodies and antigens in immunoassays, or that describe enhancements in immunoassay technologies. The basic reagents and instrumentation required for immunoassay purposes are discussed first, followed by a brief description of immunoassay formats. The principles that are utilized in various fluorescence immunoassay technologies are outlined with specific examples and their significance. Since it is beyond the scope of this chapter to review all of the applications of fluorescence immunoassays, apologies are extended to authors that this chapter fails to cite. A number of comprehensive treatments of fluorescence immunoassay (FIA) applications and related topics are available. 18 ... 

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