Factors that affect the equilibrium constant include pH, ionic strength and temperature.
Most antibodies are not affected by changes in pH within the range 5.5-8.5. However, this is not true for all antibodies, and, in order to make one day's work similar to the next, routine serology should be carried out with saline buffered to pH 7.07.4. Below pH 4 and above pH 9, antigen-antibody complexes are largely dissociated and the antibody can be recovered in the supernatant. This is the basis of some elution techniques.
In saline of normal ionic strength, the ionized groups of both antigen and antibody are partially neutralized by oppositely charged ions in the medium. By lowering the ionic strength while maintaining tonicity, the ions become exposed and theoretically there should be an increase in attraction. Decreasing the ionic strength increases the rate of association (kx) of antigen with antibody but has little effect on their rate of dissociation (k2). For example, a 1000-fold increase in k1 with a threefold fall in k2 has been observed for anti-D by reducing the ionic strength to 0.03 mol/L. Low-ionic-strength solutions (LISS) containing 0.03 mol/L NaCl in a solution of sodium glycinate are used routinely in some blood banks to increase the speed and sensitivity of pretransfusion tests. Regrettably, LISS can lead to a failure in detection of some clinically important antibodies, in particular anti-K.
Low-ionic-strength solutions are also used to coat red cells with complement components via the alternative pathway. A few drops of fresh whole blood can be taken into 1-2 mL of 10% sucrose, incubated for 10 min at 37°C and washed, to provide control cells coated with C3 and C4 for the antiglobulin test. For this reason, the use of LISS under uncontrolled conditions can lead to unwanted positive direct and indirect antiglobulin tests.
The temperature of the reaction
The effect of temperature on antigen-antibody reactions includes:
(i) an alteration in the equilibrium constant of the antibody and
(ii) an alteration of the rate of encounter. With warm antibodies, the equilibrium constant is not changed by variations in temperature, but decreasing the temperature from 37°C to 4°C slows the rate of reaction 20-fold. With cold antibodies, there is an increase in the equilibrium constant with decreasing temperature and, even though the rate is reduced, stronger reactions and higher titres are found at lower temperatures.
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