Double diffusion in two dimensions

3.5 Double diffusion in two dimensions

In this technique the antigen and antibody are allowed to migrate towards each other in a gel and

a line of precipitation is formed where the two reactants meet. As this precipitate is soluble in excess antigen, a sharp line is produced at equivalence, its relative position being determined by the concentration of the antigen and antibody in the agar. The local concentration of each react- ant depends on: (a) absolute concentration in the well; (b) its molecular size; and (c) the rate at which it is able to diffuse through the gel. Multiple lines of precipitation will be present if the antigen and antibody contain several molecular species.

The particular advantage of the technique is that several antigens or antisera can be compared around a single well of antibody or antigen.

MATERIALS AND EQUIPMENT 2% agar in barbitone buffer

Antigen and antibody solutions (see procedure for details) Gel punch Pasteur pipettes Water vacuum pump

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

Antigen concentration: initially use 1 mg/ml, but vary the concentration to obtain optimal results Antiserum: use whole anti-IgG, non-absorbed. Specific anti-IgG sera available commercially are

often absorbed with light chains to render them class specific

METHOD

1 Melt the agar in a microwave oven.

2 Pour agar onto precoated slides; use a levelled surface.

3 Punch pattern required.

4 Suck out agar plugs with a Pasteur pipette connected to a water vacuum pump.

5 Fill the wells with antibody or antigen until the meniscus just disappears.

6 Place the slide in a humid chamber and incubate overnight at a constant temperature.

Suggested antibody and antigen patterns

A straightforward demonstration of identity and non-identity can be shown using the antigen mixtures as in Fig. 3.6. In addition, this technique may be used to show the relationships between IgG molecules and their enzymic digests prepared in Chapter 2. Arrange the wells as shown in Fig. 3.7.

IgG

IgG + HSA

Anti-IgG + anti-HSA

Fig. 3.6 Design of Ouchterlony plate to show reactions of identity and

IgG

HSA

non-identity IgG and albumin (HSA) with their respective antisera.

IgG Fc

Whole IgG

Anti-IgG

Fig. 3.7 Well arrangement to demonstrate spur formation, lines of identity and lines of non-identity.

IgG Fc prepared by papain digestion of IgG (Section 1.7). Light chains prepared

Light chain by reduction and alkylation of IgG

Light chain

(Section 1.6). 3.5DOUBLE DIFFUSION IN TWO DIMENSIONS

(a)

(b)

Fig. 3.8 The relationship of human IgG subclasses. In (a) and (b) wells 1–4 contain human IgG 1 , IgG 2 , IgG 3 and IgG 4 , respectively, and the central well contains rabbit anti-human IgG. (Antisera prepared by immunizing rabbits with IgG obtained from normal human serum and isolated by ion-exchange chromatography.) Antiserum (a) recognized subclass differences between IgG 1 and IgG 4 , hence the double spur, but failed to recognize IgG 3 . Antiserum (b) recognized subclass differences associated with IgG 1 alone and so produced a single spur. Both antisera were raised against the same pool of antigen, the variation is due to the rabbits used for immunization.

TECHNICAL NOTE The rate of diffusion is temperature dependent. Precipitin lines can often be seen within 3 h at 37°C.

Interpretation of results The basic patterns of precipitation as shown in Fig. 3.8.

(a) Reaction of identity. This occurs between identical antigenic determinants; the lines of precip- itation fuse to give one continuous arc. (b) Reaction of non-identity. Where two antigens do not contain any common antigenic deter- minants the two lines are formed independently and cross without any interaction. (c) Reaction of partial identity. This has two components: (i) those antigenic determinants which are common to both antigens give a continuous line of identity; (ii) the unique deter- minant(s) recognized on one of the antigens give(s), in addition, a line of non-identity so that

a spur is formed. Of course, the antiserum may recognize unique determinants in both anti- gens: this would give rise to two spurs. All these concepts of identity and non-identity are in terms of recognition by the antiserum.

An antiserum recognizing many determinants on the antigen molecules is necessary for the demonstration of all these features.