Kinetics of antigen binding

In the ideal case, the binding of a univalent antigen (hapten) to a single antibody-binding site can be represented by the law of mass action, such that at equilibrium 

From this relationship it can be seen that if 50% of the available antibodybinding sites were associated with ligand then [5]free = [5 L] and Ka = 

Let the number of binding sites per antibody be n, so that the total site concentration is x [Ab] 

It follows from (2) and (3) that the free site concentration [5]free= [Ab] [LJbound 

If A// is 0, then K2 = K and the binding constant is independent of temperature. In this case the driving force for binding is entropy or A5. 

---

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. 

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. 

Using a concentration jump as the perturbation, Sutherland et a/.(113) measured the kinetics of binding of fluorescein-labeled human IgG (present as an antigen in solution) to surface-immobilized sheep anti-human IgG. Two TIRF surfaces were used a planar slide and a fiber-optic cylinder. Also using a TIRF recovery after a concentration jump, Kalb et a/,(114) measured the slow ( 10 4 s 1) unbinding kinetics of anti-trinitrophenol (TNP) antibodies in solution and a TNP-derivatized lipid in a planar bilayer. 

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. 

Specific, rapid, and high-affinity recognition of antigens by antibodies is essential for the host s immune system to respond to invasion by foreign cells and pathogens. Pecht and Lancet have presented a cogent analysis of antibody interactions with haptens, and the interested reader will find their examination of the kinetics and thermodynamics to be particularly lucid. Briefly, for the simple one-step binding scheme. 

Antibodies interact noncovalently with their target epitope, and the strength of this interaction is characterized by the kinetics of association and dissociation of the antibody. Antibody-antigen interactions are in principle reversible, and appropriate conditions must therefore be selected for a given antibody to bind with reproducible stoichiometry to its target antigen. Linkage of the antibody to an appropriate fluorochrome will mean that the number of antibody molecules bound will be reflected by the fluorescence intensity/cell. 

Roe, R., Robins, R. A., Laxton, R R., and Baldwin, R W (1985) Kinetics of divalent monoclonal antibody binding to tumor cell surface antigens using flow cytometry—standardization and mathematical analysis Mol Immunol. 22, 11—21... 

---