Affinity Determination

A comparison of affinity and kinetic data ensures consistency of the analysis. For a 1 1 interaction, affinity is equal to the quotient of dissociation and association rate constants ( d = dissAass)- However, some reactions do not follow a 1 1 interaction, and it must be noted that the apparent affinity of bi- or multivalent reactions can be directly affected by the density of immobilized interaction partners. Nevertheless, the chip surface resembles the situation on a cell surface and possibly reflects the in vivo environment better than an assay free in solution ]17]. 

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The method is based on the notion that both the concentration of the antagonist in the receptor compartment and its affinity determine the antagonism of agonist response. Since the antagonism can be observed and quantified and the concentration of the antagonist is known, the affinity of the antagonist (in the form of KB) can be calculated. 

A series of tricyclie pyrazoles has been synthesised and their CBi and CB2 affinity determined in mouse cerebellum membranes and mouse spleen homogenate, respectively [387]. Table 6.51 describes some of the pertinent data. 

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... 

Antibody affinity from the Latin, affinis = connected with, having things in common. In immunohistochemistry, antibody affinity determines the strength of binding of a monovalent antibody, such as Fab fragment, to one epitope, i.e., how tightly an antibody binds to its particular antigen. 

Doyle, M.L., G. Louie, P.R. Dal Monte, and T.D. Sokoloski. 1995. Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry. Methods Enzymol 259 183-194. 

The sensitivity of the microfluidic system was determined by measuring calibration curves of four cathepsin B inhibitors. The inhibitors caused negative peaks in the product mass chromatograms by inhibiting cathepsin B and thus the substrate turnover. The measured order of afEnities of the four inhibitors is in agreement with the affinities determined in microtiter plate assays and the macro-scale system. 

An additional problem for all direct methods is that they cannot use high ionic strength and nonvolatile buffers, which are needed to simulate physiological conditions, because ESI does not work under these conditions. Thus, nonspecific adducts may be produced, confusing the stoichiometry and affinity determinations. 

Furthermore, if the affinity is to be measured in water, then ESI must be done with solutions that have high contents of water, which is difficult or impossible. Another problem is that different source configurations (e.g. normal vs nano ESI), desolvation conditions, and instruments may give different results in affinity determinations [95]. 

Affinity determined by displacement of pig brain tubulin-bound [ H]vinblastine. Data are relative to vinblastine binding (1.00, K, = 106). 

NH Chiem, DJ Harrison. Monoclonal antibody binding affinity determined by microchip-based capillary electrophoresis. Electrophoresis 19 3040-3044, 1998. 

IgG molecules found outside the extravascular space can accumulate and penetrate into a tumor mass within a tissue by several mechanisms. The rate of IgG accumulation in tissue and subsequent penetration into the tumor mass depends on the affinity and avidity of the antibody for a tumor-associated antigen. According to one model, based on computer simulations, penetration of the tumor mass is influenced by diffusion and convection, while binding affinity determines residence time of the antibody molecule bound to antigen expressed on tumor cells [23-25]. 

Huff, M.E., L.J. Page, W.E. Balch, and J.W. Kelly. 2003. Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of finnish type. J Mol Biol. 334 119-27. 

Affinity Determination Between hIL8 and Its Antibody Fragments... 

Lehtio J., Sugiyama J., Gustavsson M., Fransson L., Linder M., and Teeri, T. T. 2003. The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules. Proc. Natl. Acad. Sci.,100,484-489. 

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