Two-dimensional or crossed immunoelectrophoresis
3.9 Two-dimensional or crossed immunoelectrophoresis
This technique combines the benefits of electrophoretic separation of antigens with their quantification by electrophoresis into an antibody-containing gel. It was originally introduced for the analysis of serum proteins but it has now been used in many systems. One application that is of particular interest is the analysis of C3 activation. The active and inactive forms of C3 share many antigenic determinants and so are detected simultaneously in simple immunodiffusion
assays. C3 in its inactive state has a β 1C electrophoretic mobility which changes to a β 1A mobility after activation. Thus it is possible to show the appearance of activated C3 and the disappearance of inactive C3. The two forms of C3 are first separated by electrophoresis in agarose. Rockets of immune precipitates are formed by a second electrophoretic step, at right angles to the first, into
a gel containing anti-C3. MATERIALS AND EQUIPMENT
Barbitone buffer containing 0.01 M ethylene diamine tetra-acetic acid (EDTA, disodium salt) Agarose Anti-C3 serum Serum samples for C3 quantification Glass microscope slide (not precoated)
8 × 8 cm glass plate (precoated with agar) Electrophoresis tank and power pack, e.g. Bio Rad, Shandon Scientific Ltd.
METHOD First dimension
1 Prepare a 2% agarose solution in the barbitone buffer containing EDTA.
2 Layer 3 ml of agarose solution onto the uncoated microscope slide and allow to set. Use a levelled surface.
3 Cut a 1-mm well in the centre of the slide, remove the agarose plug, and fill the well with the serum sample.
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3.9TWO-DIMENSIONAL OR CROSSED IMMUNOELECTROPHORESIS
(a) Fresh human serum (b) Serum treated with heat-aggregated IgG β
1A 1C
β 1C β
Fig. 3.13 Quantification of C3 activation. Although the inactive and active forms of C3 share many antigenic determinants, the activation of C3 is accompanied by a change in electrophoretic mobility from β 1C
to β 1A . It is therefore possible to quantify C3 activation by combining electrophoresis in one dimension with ‘rocket’ immunoelectrophoresis in the second dimension. Activation of C3: C1q, C1r, and C1s are linked, probably through calcium, to form a trimolecular complex. The binding of C1q to the Fc of the immune complex (in this case we have substituted heat-aggregated IgG) initiates the esterase activity of the C1s component which activates C4 and C2. (The complement components were numbered before their order in the activation sequence was known.) The resulting C4b2a complex has ‘C3 convertase’ activity and so splits
C3 (β 1C mobility) to C3a and C3b (β 1A mobility). The two rocket arcs of C3a and C3b are fused because of shared antigenic determinants.
4 Apply a potential difference of 150 V (constant voltage setting on power pack) for 2–3 h.
5 Cut and remove a 5-mm wide longitudinal strip of agarose from the centre portion of the slide, along its complete length. It must, of course, include the sample.
Second dimension
1 Prepare 12 ml of a 1 : 50–1 : 100 dilution of anti-C3 in 2% agarose solution at 56°C; the precise dilution of the antiserum to be used must be determined empirically.
2 Place the agarose strip at one end of the square glass plate (precoated) and cover the whole slide with 12 ml of agarose containing anti-C3.
3 Place the plate in the electrophoresis tank. The cathode must be at the end of the plate with the agarose strip, i.e. the electric field will cause the separated complement components to enter the antibody-containing gel at right angles to the first electrophoresis. Electrophorese at 40–50 V overnight (if cooling apparatus is available a higher voltage may be used for a shorter time).
4 Wash and stain the precipitin arcs (see Section 3.4.1).
Fresh serum should give a pattern similar to Fig. 3.13(a), while aged serum or serum with immune complexes should give a pattern more like Fig. 3.13(b).