Antibody affinity constants

The affinity and cross-reactivity of the whole serum and Fab fragments were determined using equilibrium dialysis for the affinity determination and RIA for the cross-reactivity studies. The average intrinsic affinity constant (Ko) of the antibody (Nisonoff and Pressman 1958) changed very little throughout the... 

Figure 17. Structure affinity relationship measurement using reflectometric interference spectroscopy of atrazine versus an antibody in water. A variety of derivatives of triazines are measured at a number of concentrations to obtain affinity constants.

Immunochemical techniques are based on the immunological reaction derived from the binding of the antibody to the corresponding antigen. This reaction is reversible and is stabilized by electrostatic forces, hydrogen bonds, and Van der Waals interactions. The formed complex has an affinity constant (k j that can achieve values around the order of 1010 M. This great affinity and specificity between the specific antibody and the antigen (or the analyte) have turned these techniques into powerful analytical tools to detect and quantify... 

Monoclonal antibody-binding affinity was determined by microchip-based capillary electrophoresis with LIF detection (33). The mixing was carried out off-chip, and the on-chip separations were performed in less than 60 s (Fig. 9). A Scatchard plot analysis resulted in an affinity constant for the monoclonal anti-BSA antibody to fluorescently labeled BSA (BSA ) of... 

Rey and McConnell40 have shown that the affinity constants k of specific IgG antibodies for simple nitroxide haptens such as... 

Many studies of the binding of monovalent haptens in solution to IgG antibodies have led to the conclusion that these bindings are essentially independent of one another. Thus the affinity constant for the second binding... 

Friguet, B., Chaffotte, A. F., Djavadi-Ohamance, L, and Goldberg, M. E (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent-assay. J Immunol Methods 77,305-319... 

Different techniques have been applied to study the protein-protein, protein-ligand and, in particular, MIP-ligand interactions. They may serve to estimate or determine the binding constant and the number of independent binding sites (N) of a ligand-to-receptor (MIP or antibody) interaction. The range of affinity constants that can be calculated depends on the sensitivity of the assay and, in those cases where the separation of the bound and free species is a step of the assay, perturbation of the equilibria in the separation step will also be important [22]. Direct nonseparation techniques such as spectroscopic techniques (e.g., SPR or fluorescence polarization) can be used as well as indirect separation techniques such as radiolabeling [22]. 

In addition, affinity CE separation was carried out to resolve monoclonal anti-BSA and BSA in dilute mouse ascites fluid. Then the affinity constant of this antigen-antibody interaction was measured [1004]. 

Interaction on the solid phase involves the hypervariable region of the antibody and sorption efficiency is related to the affinity constant. When this latter is very high and requires elution conditions that can have a deleterious effect on the integrity of the antibody, it may be necessary to chemically modify the structure of the antigen to decrease the affinity constant. 

Antigen affinity Bioaffinity Single antibody Availability of antigen affinity constant... 

Biotin has served this purpose well in both nucleic acid and antibody probe systems. As well as being easily detected with immunoglobulins specific for biotin, biotin may also be detected non-immunologically with avidin or streptavidin, two proteins which share a marked, highly specific affinity for biotin. The affinity constant for avidin-biotin interactions is approximately 10 - liters/mole, much higher than the range for antigen-antibody interactions which are commonly between 10 -10 liters/mole. Consequently, a vast number of detection complexes composed of avidin or streptavidin bound to a detection system are commercially available (e.g. streptavidin-alkaline phosphatase). 

As ACE can in principle differentiate between free and bound antigen (Ag) (or free and bound antibody, Ab), the method can be used for affinity-constant... 

Sensitivity The limit of detection for a certain compound is determined by its affinity constant or binding constant to the interaction partner immobilized to the surface. Therefore, the choice of the capture compound affects remarkably the assay performance. Because of the opportunity to create antibodies against different kind of compounds, e.g., peptides, proteins, enzymes, vitamins, environmental pollutants, herbicides, fungicides, etc., they are frequently used as a versatile tool for sensor assay developments. 

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