Immunoassays competitive binding

Competitive binding immunoassays require a pure, labelled sample of the antigen... 

The presence of a cross-reacting antigen in a sample will result in falsely increased test values when using competitive binding immunoassays... 

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. 

Competitive binding immunoassay (e.g., RIA) Enzyme-linked immunosorbent assay (ELISA) Immunoradiometric assay (IRMA) — dual monoclonal antibody assay Receptor binding assay Cell binding assay... 

Hage, D.S., Thomas, D.H., Chowdhuri, A.R., and Clarke, W. 1999. Development of a theoretical model for chromatographic-based competitive binding immunoassays with simultaneous injection of sample and label. Anal Chem 71 (15) 2965—2975. 

On the basis of the split-peak equations, Hage et al. I8) developed a model to interpret competitive binding immunoassays and applied the method to the determination of low levels of albumin in urine 47). An equation was derived to predict the assay response as a function of flow rate, amounts of analyte and... 

Fig. 1 Typical schemes for (a) the on-off mode of immunoaffinity chromatography and (b) a chromatography-based competitive binding immunoassay. The solid circles represent the analyte, the open circles represent a labeled analog of the analyte, and the open squares represent nonretained sample components.

Another important technique in lAC is the use of immobilized antibody columns to perform chromatographic (or flow-injection) immunoassays. One way this can be done is in a competitive binding format. The simplest approach to a competitive binding scheme is to mix the sample and a labeled analyte analog (the label) and apply these simultaneously to the lAC column this is a method known as a simultaneous injection competitive binding immunoassay. If the sample is applied to the lAC column and followed later by a separate injection of the label, then the technique is called a sequential injection competitive binding immunoassay. In both formats, an indirect measure of the sample analyte is obtained by examining the amount of label that elutes in either the nonretained or retained lAC fractions. 

The early development of reliable immunoassays for PRL was hampered by extensive cross-reactivity of the antibodies used and the lack of purified human PRL calibrators. To minimize such problems, heterologous systems in which the hormones used for immunization and labeling were derived from different species were used. As purified human PRL became more readily available, homologous competitive-binding immunoassays were developed for the specific determination of PRL in microhter volumes of serum or plasma specimens." Initially, RIA was the predominant method for... 

Lubbers and Optlz (9) and Andrade et al (10 have employed fiber optic sensors for the continuous measurement of chemical reactions in biological systems. High sensitivity may be achieved using these sensors to measure fluorescent-tagged antigens or antibodies in competitive binding immunoassay reactions In solution. 

Hage, D.S. Thomas, D.S. Beck, M.S. Theory of a sequential addition competitive binding immunoassay based on high-performance immunoaffinity chromatography. Anal. Chem. 1993, 65, 1622-1630. 

An alternative format for a chromatographic immunoassay is the displacement competitive binding immunoassay. In this format, the lAC column is first saturated with the labeled analogue, followed by application of sample to the column. As the analyte travels through the column, it is able to bind to any antibody sites that are momentarily unoccupied by the label as the analyte and antibodies undergo local dissociation and reassociation. This situation results in competition between the analyte and the label... 

Fig. 3 Typical scheme for a chromatographic competitive binding immunoassay. This particular example is for the simultaneous format. Source From Chromatographic Immunoassays, in Handbook of Affinity Chromatography...

Figure 4A outiines the basis of a simple competitive binding immunoassay where the antigen has been labelled. Antibody (preferably afifinity purified, or an IgG fraction) is coated onto a solid support (e.g. a microtitre plate well), and a constant amount of labelled antigen is allowed to compete for the antibody binding sites with vaiying amounts of unlabelled standard or sample antigen. 

Figure 5 Competitive binding immunoassay—labelled antibody.

Discuss the similarities of cation-anion exchange methods and competitive binding immunoassay techniques. 

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