Solid-Phase Binding Assay

Figure 6. Solid-phase binding assay for specific antigen.

Most one-bead-one-peptide libraries (see Section 4.3.7.3.1) are resin-bound and screened in direct solid-phase binding assays. In a typical binding assay, the library beads are incubated with the target protein labeled with a reporter molecule, such as an enzyme that catalyzes a reaction generating a colored compound, which in turn stains the beads to which the peptides that bind to the target protein are tethered. The positive (colored) beads are identified among the vast majority of noncolored beads, isolated, and submitted to structure determination of the peptides bound to them. 

This is a solid-phase binding assay that can easily be performed in a %-well plate (Fig. 9). ELISA measures antigen or antibody concentration, depending on the protocol used. The stages of a typical assay involve a series of addi-... 

A solid-phase binding assay similar to ELISA can be adapted to radioactive detection when a radioactively labeled antigen or antibody is used. The radioimmunoassay (RIA) is more sensitive and reliable than ELISA but is usually more time consuming and more expensive because of the cost of the radioactive label. 

The deprotected lactosides were evaluated as inhibitors against lectin binding in a solid-phase inhibition assay with immobilized ASF on the surface of microtiter plate wells, mimicking cell-surface presentation, while mammalian galectins-1, -3, and -5 were in solution. Strong multivalency effects and selectivity were observed for the... 

Many different radioactive tracer methods have been used for estimating the unsaturated thyroid hormone binding capacity. The simplest is the T3 uptake test. Traditionally, this test is performed by measuring the distribution of radiolabeled T3 between serum binding proteins and a solid-phase binding material. In a typical assay, a diluted sample of patient serum is allowed to equilibrate with a trace amount of I-Ts and the secondary binder. A portion of the radioac-... 

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). 

In 1990, a solid-phase immunobead assay (SPIA), with colored polystyrene particles coated with anti-CTX monoclonal antibody (MAb-CTX) began to be used for direct detection of ciguatoxins adsorbed on bamboo paddles coated with organic correction fluid. The membrane immunob-ead assay (MIA) presented by in 1998 Hokama et al. is based on the immunological principles used to develop the SPIA, using a MAb-CTX coated onto colored polystyrene beads. The polyether toxins extracted from a piece of fish tissue bind to the hydrophobic membrane on a plastic support (membrane stick), and can be detected with the MAb-CTX coated onto the colored polystyrene beads. The color intensity of the membrane is related to the concentration of the toxin bound to the membrane. 

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. 

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. 

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