Immunoassay competitive equilibria

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Many immunoassays require a separation step prior to quantitation, in order to separate the bound and free fractions of the labeled species. Consider an immunoassay in which a labeled ligand, Ag, competes with unlabeled analyte, Ag, for a limited quantity of antibody binding sites. This competitive equilibrium is represented by Eqs. 6.5 and 6.6,... 

A homogeneous enzyme immunoassay based on antibody inhibition of enzyme conversion from the apo- to the holo- form has been developed for DNP-aminocaproic acid, DNP-ACA (Ngo et al., 1985). A competitive equilibrium is established between the analyte DNP-ACA and DNP-conjugate apoglucose oxidase, DNP-CAGO, which is the labeled hapten. Flavin adenine dinucleotide, FAD, added to the mixture binds to free DNP-CAGO to give DNP-CAGO FAD, which is enzymatically active and... 

In a so-called competitive immunoassay format the antigen competes with a labeled antigen for a limited number of antibody-binding sites. It can be shown that in this case the ultimate sensitivity of the assay (when the [Ab] approaches zero) is dependent on the equilibrium constant K and the reliability of the signal measurement of the bound fraction at zero dose [15],... 

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. 

Competitive immunoassays are based on the measurement of antibodybinding sites unoccupied by the analyte. An increase in the concentration of analyte causes a reduction of unoccupied sites. The measurement is performed indirectly by means of competition between the analyte and a competitor/tracer molecule (usually a labelled analyte derivative) for a limited number of antibody-binding sites. In principle, such assays can be described by Eqs. (9.4) and (9.5), together with their corresponding equilibrium constants K and K ... 

The first immunoassay developed was based on the principle described above, with an insulin competitor, i.e., an analyte derivative labelled with a radioactive isotope ( I) and the anal3d e (insulin) competing for a limited amoimt of immobilized anti-insiilin antibodies. After the equilibrium was reached and the unbmmd competitor was removed, the residual radioactivity was correlated to the concentration of insulin [2], Since then, numerous other variants of competitive immimoassay formats have been described, either in a... 

Fig. 9.6. Direct competitive immunoassay with immobilized antibodies-. The analyte competes with a labelled analyte derivative ( tracer ) for a limited amount of antibodybinding sites (1) until equilibrium (2) is established. Then, the excess of analyte and tracer (3) are removed by washing of the solid support, and the fraction bound to the antibody is detected (4).

An important aspect in the development of competitive immunoassays is to decide how long to allow the incubation of reactants to progress in order to attain equilibrium. Motulsky [84] demonstrated that the incubation time until attaining equilibrium depends on the relative values of the dissociation rate constants of the competitor and analsde from their corresponding antibody complexes, as well as on their concentration. When the dissociation of the antibody-competitor complex is much faster than that of the antibody-analyte complex, the predicted equilibration time is strongly dependent on the analyte concentration, whereas the kinetic constants and the concentration of the competitor do not matter. The time necessary to reach the equilibrium at IC50 is equal to 1.75/ 2j whereas full equilibrium at maximum analsde binding is reached at (i.e., the lower the analyte concentration, the faster the... 

Fig. 9.15. Evolution of the signal at different moments (a-e) before equilibrium at different levels of the calibration curve in a competitive immunoassay. (Adapted from Ref [84].)...

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... 

Immunoassays for small organic compounds usually are formatted as competition assays (13.141. There are many assay formats and choices of label for the eventual quantification of binding. An equilibrium is approached for formatirxi of complexes between the antibody, labeled or immobilized hapten (hapten ), and fiee analyte in the sample, according to the following equation. 

Homogeneous enzyme immunoassays use a competitive assay format that relies on a reduction in the rate of enzyme catalysis of Ag as it binds to the Ab. The general equilibrium scheme for homogeneous immunoassays, which also holds true for the nonenzymatic ones discussed later, is shown in Fig. 4. The... 

From simple solvent extraction to RIA, sample preparation and screening tests share many common elements. The pivotal characteristics are (1) the existence of a difference in properties between the materials to be separated and (2) an exploitable equilibrium. An understanding of these ideas facilitates an understanding of partitioning and competitive equilibria. All of the sample preparation uid screening procedures and protocols described in this chapter can be characterized by invoking some combination of these foimdational principles. Furthermore, the theory behind TLC and immunoassay forms a bridge that leads naturally into a discussion of instrumentation, the subject of the next chapter. 

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