Definition and theoretical aspects

The antibody site to antigenic site association reaction at equilibrium can be written as:

antibody + antigen «-» complex with the concentrations of free antibody sites, free antigen sites and complex (saturated antibody or antigen sites) at equilibrium given as |Ab], [Ag] and [x] respectively.

The affinity, KA, is defined by the Law of Mass Action as:

The concentration of antibody sites [Ab] and the antigen sites [Ag] at equilibrium are related to the total antibody sites [AbJ and the total antigen sites [Agt] by:

If [Agt| is varied while [Abt] is kept constant:

with Kd defined as the equilibrium dissociation constant.

Consequently

Several linear plots have been proposed for the determination of Ku, the most commonly used of which are the Scatchard equation:

and the Klotz equation:

If Abt and Agt are known, the experimental determination of Kd (or Ka = VKd) requires precise measurement of only one of the three concentrations [Ab], [Ag] or [x] (Figure 1).

Ka depicts an equilibrium property and therefore does not reflect the speed at which equilibrium is reached. Yet, KA does depend on the association and dissociation rate constants. When binding is a simple one-step reaction, KA is equal to kuJkiM, where k„n and koff are the association and dissociation rate con-

(A)

Figure 1 (x)/(Abt) is usually referred to as v and corresponds to the fraction of bound antibody sites at equilibrium. (Ag) corresponds to the concentration of free antigenic site at equilibrium. (A) Scatchard plot; (B) Klotz plot.

Figure 1 (x)/(Abt) is usually referred to as v and corresponds to the fraction of bound antibody sites at equilibrium. (Ag) corresponds to the concentration of free antigenic site at equilibrium. (A) Scatchard plot; (B) Klotz plot.

stants. However, when a significant conformational change of either the monoclonal antibody or the antigen occurs upon binding, important deviations from that simple equation can be observed. For such cases, measuring only kon and ¿<>ff would provide an erroneous estimate of the affinity.

The true affinity cannot be determined for antibody-antigen interaction in heterogeneous phase systems. No straightforward thermodynamic theory describes the equilibrium in heterogeneous phase systems, such as the binding of a monoclonal antibody to an antigen present on a cell surface or immobilized on an ELISA plate or a biosensor chip. KA is defined in solution, with both the monoclonal antibody and the antigen diffusing and rotating freely in solution, while in the solid phase assay one partner is immobile. This can result in estimates of KA by solid-phase assay being orders of magnitude away from the real affinity in solution. In such cases, the strength of monoclonal antibody-antigen interaction is best described by an appropriate operational parameter that is dependent of defined experimental conditions. Moreover it is now well established that immobil ization of either antibody or antigen often results in a partial denaturation of the protein, thus modifying its binding properties.

How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment