Immunoassay competitive binding analysis

The utilization of lAC in analytical methods has received increasing retention in recent years [23,24], Of particular interest is the use of immobilized antibody columns in performing immunoassays, a technique known as a chromatographic immunoassay or flow-injection immunoassay. This approach has already been reported in a number of formats such as those involving simple analyte adsorption/desorption, sandwich immunoassays, competitive binding immunoassays, and multianalyte methods (see Figure 13,9) [23,24,73,74], Typical advantages of these methods include decreased analysis times and improved precision versus manual immunoassays. 

Triazines such as atrazine, propazine or simazine, are widely used herbicides. Currently, the determination of atrazine in water and soil samples is mainly done by GLC (1,2) or HPLC (3-5). However, these procedures require cumbersome cleanup steps which could be avoided by using immunoassays as an alternative approach to residue analysis. Such immunochemical determination based on competitive binding of herbicides or pesticides to an antibody (6,7), has been described recently for s-... 

The MIPs have also been utilized as the recognition elements in pseudoimmunoassays. " In this approach, MIPs are substituted for antibodies to quantify the amount of analyte in a biological sample, such as blood plasma. Most MIP immunoassays are competitive binding studies in which a radio- or fluorescent-labeled analyte is added to a mixture of the MIP and imlabeled analyte. After equilibrium is reached, some fraction of the labeled species is bound to the polymer surface and thus can be separated from the supernatant. The supernatant is then analyzed via scintillation or fluorescence techniques to determine the concentration of the original unlabeled analyte. Mosbach et al. have demonstrated that MIP-based immunoassays can rival the selectivity of antibody-based assays. Imprinted polymers for the opioid receptor ligands enkephalin and morphine were prepared and showed submicromolar (pM) level selectivity in a radioligand competition assay in aqueous buffers. The analysis... 

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). 

In CE, analysis by immunoassays depends on the principles that the antigen and antibody migrate differently when they are bound compared to when they are free. One of these two compounds (mostly the antigen) is labeled with a fluorescent tag. The unknown sample is mixed with labeled antigen for competitive binding assay and the mixture is separated by CE. The label in the bound fraction (or... 

In many instrumental analysis methods the instrument response is proportional to the analyte concentration over substantial concentration ranges. The simplified calculations that result encourage analysts to take significant experimental precautions to achieve such linearity. Examples of such precautions include the control of the emission line width of a hollow-cathode lamp in atomic absorption spectrometry, and the size and positioning of the sample cell to minimize inner filter artefacts in molecular fluorescence spectrometry. However, many analytical methods (e.g. immunoassays and similar competitive binding assays) produce calibration plots that are intrinsically curved. Particularly common is the situation where the calibration plot is linear (or approximately so) at low analyte concentrations, but becomes curved at higher analyte levels. When curved calibration plots are obtained we still need answers to the questions listed in Section 5.2, but those questions will pose rather more formidable statistical problems than occur in linear calibration experiments. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

The most common, but by no means the only or even the most promising, immunochemical assay for small molecules is radioimmunoassay (R1A). As an overview, an immunoassay involves chemically attaching the small molecule of interest (or a derivative of it) to a carrier protein and raising specific antibody titers to it in the serum of an animal. Very dilute antibody solutions are then used to bind the small molecule which has been radiolabeled. The competition of varying known concentrations of unlabeled material is measured and the resulting standard curve used to determine unknown concentrations (Table 1). The steps leading to the development of an R1A are outlined below followed by a description of other immunochemical procedures and an analysis of the attributes and limitations of immunoassay. 

Odell, W.D. Daughaday, W.H. Principles of Competitive Protein Binding Assays Lippincott Philadelphia 1971. Christian, G.D. Clinical chemistry. Analytical Chemistry John Wiley and Sons Inc. New York, 1994 611-628. Ullman, E.E., Langen, J., Clapp, J.J., Eds. Liquid Assay Analysis of International Development on Isotopic and Non-Isotopic Immunoassay Masson New York, 1981 113. Sharma, A. Schulman, S.G. Fluorescence analytical methods and their applications. Introduction to Fluorescence Spectroscopy, WHey-lntexsdeace.l ew York, 1999 123-158. 

The reaction is not as simple as it appears though, because a labelled substance (tracer) is needed to facilitate the detection of the Ab-Ag binding event. These labels can be radioisotopes (radio immunoassay (RIA), not used in environmental analysis), fluorophores (fluoro immunoassay (FIA)) or enzymes (enzyme immunoassay (EIA)). Therefore a competition of the Ag with the label (L) is involved, and is defined by the association and dissociation rate constants ka2 and kd2 (Equation (3.3.2)) ... 

Immunoassays are a class of analytical methods based on the selective affinity of an antibody for an analyte [1-3]. Biological antibodies are highly useful analytical reagents, which may be produced by innoculation of laboratory animals with the analyte or with a derivative. The archetypal immunoassay, the radioimmunoassay or RIA, involves competition for a limited number of antibody binding sites between the analyte, present at unknown concentration in the sample, and a radiolabelled probe. Immunoassays are widely employed in clinical analysis in particular, but also in other fields including environmental analysis and research. 

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