Antigen binding kinetics

Sapsford etal. (2001) examined microarray-based antibody-antigen binding kinetics in real time to determine the effect of spot size. Capture antibodies were immobilized in an array pattern onto silver-clad microscope slides. Antimouse IgG was directly attached to the surface or attached via neutravidin capture of the biotinylated antibody. Cy5-labeled mouse IgG capture was monitored based upon the signal generated from the excitation of an evanescent wave guide (slide) with a 635-nm laser source detection was achieved by a charge-coupled device (CCD) camera system. Both static and flow-through conditions were employed. 

The binding kinetics were characterized in terms of the apparent time constant (K pp = kf C + k ) where C = analyte concentration kf = association rate constant and k = dissociation rate constant. In closed loop experiments, a plateau value for K pp of 0.0024/s was reached at a linear flow rate of 2.67 mL/min. K ppWas foxmd to decrease with decreasing antigen concentration (C), with equilibrium achieved only at the highest level (1 pg/mL). The association rate constant Kf was calculated at 3.6 x 10 M/s for IgG binding. 

Sapsford, K.E., Liron, Z., Shubin, Y.S., and Ligler, RS., Kinetics of antigen binding to arrays of antibodies in different sized spots. Anal. Chem., 73,5518-5524,2001. 

Incorporation of (111) into a kinetically stable complex suitable for attachment to an antibody calls for an octadentate ligand, and so pendant-arm cyclen derivatives are appropriate. In particular, two or three antigen-binding fragments of an antibody (Fabs) have been attached to the di- or trimaleimide ligands 80-82 (Scheme 8) (90, 91)... 

Sadana, A. A single- and a dual-fractal analysis of antigen-antibody binding kinetics for different biosensor applications. Biosens. Bioelectron. 1999, 14, 515-531. 

In future work, the methods illustrated in this paper will be applied to a variety of problems in macromolecular kinetics. More detailed studies of substrate binding to superoxide dismutase and antigen binding to antibody molecules are in progress. Other studies that are planned or in progress include the examination of Coulombic contributions to polymer growth and to DNA-ligand interactions. 

Determine dissociation rate constant of clone(s) of interest exactly as outlined previously in Subheading 3.3.3. Keep in mind that mutant clones should hopefully have slower dissociation kinetics and time points will likely have to be extended to observed complete or near-complete decay of antigen-binding fluorescence. It is also good practice to include the parental WT scFv clone for comparison and to confirm reproducibility with previous results. 

Hu G, Gao Y, Li D (2007) Modeling micropattemed antigen-antibody binding kinetics in a microfluidic chip. Biosens Bioelectron 22 1403-1409... 

A conventional microtiter plate, ELISA requires equilibrium of the antibody-antigen reaction that would require an incubation time of approximately 1-2 h. Currently, most of the commercially available ELISA test kits for aflatoxins are working in the kinetics phase of antibody-antigen binding, which reduces the incubation time to minutes. Although reduction of incubation time may lead to some loss of assay sensitivity, the test kit can provide accurate and reproducible results [75]. 

Borrebaeck, C.A., Malmborg, A.C., Furebring, C. et al (1992) Kinetic analysis of recombinant antibody-antigen interactions relation between structural domains and antigen binding Biotechnology, 10, 697-698. 

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