Solid phase competitive assays

Most of the assays now carried out with isoluminol derivatives are solid-phase competitive assays (Fig. 2), in which the analyte to be determined competes with the labeled analyte for the available binding sites on antibodies that are immobilized on the solid phase. Thus, plastic microspheres have been used to immobilize antibodies for estriol (K12) or thyroxine (W9). Estradiol antibodies have been immobilized onto plastic tubes (K7) or beads (K16). In all of the above cases, the amount of immunoconjugated hormone label is inversely proportional to the amount of free hormone in the analytical sample. Unconjugated labels are removed by decantation or aspiration from the solid phase, and the specific, immunoconjugated labels are measured in a luminometer. This approach has been successfully applied to progesterone analyses in either serum (D6) or saliva (D5). A review of several separation-based assays with isoluminol analogs was presented a few years ago by Kohen et al. (K18). 

Yamaguchi and Ruoslahti (71) first noted that overexpression of decorin in Chinese hamster ovary cells reduced their rate of proliferation and density at saturation. This effect was subsequently attributed to the direct binding and inactivation by decorin of the transforming growth factor-(3 (TGF-p) that was considered to be an autocrine stimulator of cell proliferation in these cultures (72). Biglycan was also reported to bind to TGF-p in the solid-phase competition assay system used in this study. 

After Zemplen treatment under usual conditions, the sparingly water-soluble hexamers 49 and 51 were obtained in quantitative yields. Their modest solubility in water has however not jeopardized their biological evaluations in solid-phase competitive inhibition assays as well as in their cross-linking abilities with a model plant lectin, Concanavalin A, known to form cross-linked lattices in the presence of multiantennary glycans (39). 

Employing variations of the ELISA technique, the fluorogenic ELISA assays are based on the capture of antigens or antibodies from a solution by an insoluble solid phase. These assays are used to determine concentrations of high molecular weight analytes, to indicate the presence of specific antibodies, or for competitive analysis of low-concentration haptens. A linear capillary... 

Fig. 7.4 Examples of solid-phase ECL assay formats, a DNA hyinidization assay based on an immobilized ssDNA hybridizes with a labeled target ssDNA. b Sandwieh-type DNA biosensor, c Assay used for integrase aetivity test with immobilized and Iree-labeled dsDNA. d Sandwich-type immunoassay, e Direct immunoassay, f Competitive assay in which analyte competes with labeled analyte for antibody-binding sites on immobilized antibody, g Protease activity assay in which cleavage of the immobilized peptide results in the decrease in ECL emission due to the removal of the ECL label, h Kinase activity assay using a labeled antibody to recognize the phosphorylated product. Adapted from Ref. [23]. Copyright 2008 American Chemical Society...

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

Direct competition. The solid phase (a microtiter plate) is coated with an antibody specific for the antigen being assayed. The sample and enzyme-labelled antigen (antibiotic) are added. There is a competition for the antibody between the labelled and unlabelled antigen (antibiotic). Substrate is added and the color produced by the enzymatic hydrolyse is inversely proportioned to the concentration of antigen in the sample... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the solid phase. The analyte-indicator on the solid phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Competitive immunoassays can be based on different formats [3, 22, 23] (1) an anti-analyte antibody is immobilized on a solid support and the analyte in the sample competes with an added labeled analyte for a limited number of antibody binding sites (2) a solid phase is coated with an antibody that binds to the antianalyte antibody and the assay is carried out with the analyte and the labeled derivative mixture (3) the immobilized antigen approach consists of the competition of the analyte in the sample with the immobilized antigen for binding to labeled antibody molecules. 

In literature, a direct competitive assay performed using magnetic beads protein G coated as solid phase and carbon screen-printed electrodes as transducers is reported [4], The main steps of the assay are shown in Fig. 25.4. 

The demonstration that MTs from a wide variety of fish species are recognized by an antiserum raised against one piscine MT has enabled the development of immunotechniques based on ELISA143 and radioimmunoassay (RIA) procedures144 for the quantification of these compounds. A competitive solid-phase assay based on dissociation-enhanced lanthanide fluoroimmuno-detection (DELFI A) of anti-MT monoclonal antibody bound to a solid phase has been reported.145 An electrochemical determination of MTs by square wave cathodic stripping voltammetry has also been developed and optimized.146... 

The most popular form of this technique is solid-phase heterogeneous ELISA, and the inherent use of passive adsorption facilitates flexibility in assay design. ELISA is generally classified as direct, indirect, sandwich (double-antibody) or competitive. Principles of each of these formats are briefly discussed below. 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

A brief description of techniques used in EIA follows. The various assays have been classified as either competitive or noncompetitive assays, depending on whether or not the technique involves a reaction step in which unlabeled and labeled antigen compete for a limited number of antibody sites (competitive assay) or whether the antigen (or antibody) to be measured is first allowed to react with antibody (antigen) on a solid phase followed by measurement of the binding of enzyme-labeled immune reactant (noncompetitive assay)... 

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