Equilibrium dialysis

3.1.2 Equilibrium dialysis

This technique was devised in 1932 as a direct method for studying the primary interactions between antibody and hapten, and generally regarded as the standard method for affinity deter- mination against which the other methods are judged. However, it is rather cumbersome to perform and uses up large amounts of antibody and antigen. Since many haptens (but not oligosaccharides) bind to albumin, it is necessary to work with antibody isolated by ammonium sulphate precipitation, though further purification is not usually required.

3.1DETERMINATION OF ANTIBODY AFFINITY

Before incubation

After incubation

Fig. 3.2 Equilibrium dialysis showing relative distribution of the antibody and hapten molecules at time zero and after the equilibrium

is established. Cell to left shows distribution at the beginning of the experiment, cell to right shows distribution at equilibrium. There is

a greater concentration of hapten molecules in the inner cell of the diagram on the right because of

Antibody molecule

Hapten molecule

the hapten molecules bound to the antibody.

Constant amounts of antibody (approximately the reciprocal of the expected equilibrium constant) are placed in dialysis bags and allowed to equilibrate with various concentrations of hapten over an antigen excess of 1 to 40. Free hapten will enter the dialysis bag along its concen- tration gradient (Fig. 3.2). Some of the hapten will complex with antibody and so will not con- tribute to the free hapten concentration. At equilibrium, the free hapten concentration will

be the same on either side of the dialysis membrane, but the total hapten concentration will be relatively greater inside the dialysis bag. For each experimental point on the binding curve the following samples, each in triplicate, are required: (a) antibody immunoglobulin; (b) irrelevant immunoglobulin of the same isotype for the determination of non-specific bind-

ing; and (c) buffer alone, to check that equilibrium has in fact been established.

Outline of the technique

Typically each sample of protein or buffer solution (about 0.5 ml) is placed in a small dialysis bag. For each concentration of free hapten on the binding curve, nine sample bags can be equilibrated against a single pool of radioactive hapten (or hapten with alternative label), usually in a 50-ml bottle. Bottles are left in a water bath with mixing for 24–48 h and then triplicate volumetric samples are taken from each free hapten solution and each dialysis bag. The free hapten con- centration at equilibrium may be determined directly from the counts per minute (c.p.m.) of the samples taken from the bottles. The bound hapten concentration may be calculated as follows:

Antibody-bound hapten, Hb = (H Ab ) – (H f + [H N –H f ])

where H Ab is c.p.m. of sample from antibody bag, H f is c.p.m. of free hapten in bottle and H N is c.p.m. in bag containing irrelevant immunoglobulin.

76 C H A P T E R 3: Antibody interactions with antigens

Then allow for the specific activity of the hapten. The association constant may then be calculated using the Langmuir plot as detailed in the

following section. TECHNICAL NOTE

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