Direct binding assay

For ELISA, an enzyme is linked chemically to the antibody. The labeled antibody is allowed to bind to the unlabeled antigen, under conditions where nonspecific adsorption is blocked, and any unbound antibody and other proteins are washed away. Binding is detected by a reaction that converts a colorless substrate into a colored reaction product. The color change can be read directly in the reaction tray, making data collection very easy, and ELISA also avoids the hazards of radioactivity. This makes ELISA the preferred method for most direct-binding assays (Plested et al. 2003). 

The secondary bonds, which may be formed much more slowly than the primary bonds, actually contribute more to the overall affinity. For example, the primary (Coulombic) bond between bovine serum albumin (BSA) and anti-BSA IgG is 3.3kcalM 1 whereas the secondary bond (van der Waals) is 28kJ, for a total AH = 42 kJ. Because the formation of the secondary bond is much slower, it is easier to prevent formation of the strong complex rather than to try to dissociate it. This is one reason why the competitive immunoassays yield results that correlate with the equilibrium-binding constants, but any such direct-binding assays have to rely on the measurement of the initial rate of binding. 

FIGURE 16.5 A schematic representation of (a) SPR competitive and (b) direct binding assays of Con A-Man recognition, (c) The structure of clicked [G3]-Man glycodendrimer used for studying the glycoside cluster effect. 

Seeman P, Chau-Wong M, Tedesco J, Wong K (1975) Brain receptors for antipsychotic drugs and dopamine Direct binding assays. Proc Natl Acad Sci USA 72 4376 1380. 

Fig. 3. A direct binding assay between antigen and antibody. The antigen partitions into the top phase eft) and the antibody partitions into the bottom phase [middle]. Binding is observed as a displacement of antigen from the top phase to the bottom ph ise [right] (From Ling and Mattiasson (102).)...

It should be noted, however, that mixture-based hbraries are not limited to the solution state, but have also been screened while bound to a sohd support, such as plastic pins,P cellulose membranes,or resin beads, This assay format is favorable for direct-binding assays involving soluble acceptors. 

Determination of the receptor binding affinity of the radiolabelled conjugates under study by a direct binding assay using cell membranes, cells and antigen. 

The quality of an SPR based direct binding assay can be described in a similar manner to an HTS assay by measures that characterize the robustness and the reproducibility of the assay. In order to determine the reproducibility of a screen, a set of compounds is tested in replicate. It is important that all experimental steps of a given screen such as sample preparation, injection mode, washing procedures, data evaluation are included. The statistical data of the correlation (for instance slope and standard error) are indicative for the reproducibility of the data [39]. 

Figure 6.7-12. Left Panel Results for a direct binding assay for a In-DTPA-trastuzumab (Herceptin) IgG incubated with SK-BR-3 human breast cancer cells overexpressing HER2/ neu. The Ka and Bmax in this example were 1.2 x 10 L/mole and 1.0 x 10 receptors/cell, respectively. Right Panel Results for measurement of the immunoreactive fraction (IRF) of a Tn-DTPA-trastuzumab IgG incubated with SK-BR-3 cells using the Lindmo method [256]. In this example, the intercept on the ordinate (1/IRF) was 1.7 and the IRF was therefore 0.59. (Data generously provided by Kristin McLarty.)...

FLiK and FLIP Direct Binding Assays for the Identification of Stabilizers of Inactive Kinase and Phosphatase Conformations... 

Independent confirmation of primary hits is performed by a variety of reliable secondary assays. We use a number of direct binding assays such as biolayer interferometry (BLI) or (for strongly binding inhibitors) ITC as well as indirect binding assays such as thermal shift assays for hit validation. 

Direct Determination of Binding. With a of greater than 200 nM for Arfl GTP GGA interactions, conventional puU-down assays are not quantitative due to rapid dissociation during the washes. Nonetheless, these assays are useful for establishing that there is a specific interaction between GGA and Arfl GTP. We show an example of data from a direct binding assay in Fig. 1. In a typical experiment, 10 //g of GST or GST-VHSGATGGA3 are added to 450 y of a cell lysate, for example, bovine brain lysate, containing 20 mMTris-HCl, pH 8.0,100 mMNaCl, 0.1% (w/v)... 

Fig. 2. Results from a direct binding assay. The fraction of total Arfl GTP7S that was associated with GST-VHSGATggas immobilized on glutathione beads is plotted against the concentration of GST-VHSGATggas in the assay. The data were fit to Eq. (1).

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