Immunoelectrophoresis

These methods are based on immune-precipitate (precipitin) formation at antigen/antibody equivalence, analogous to Ouchterlony immunodiffusional analyses in which antibody and antigen placed in separate wells in agar(ose) form precipitin arcs in the gel at position(s) depending on levels of antigen and antibody in the wells. In the method of Grabar and Williams the proteins under analysis are separated on agar gels, then lateral slots are cut approximately 8 mm to each side of the gel to accommodate antibody. After a day lateral diffusion of the protein bands towards the counter-diffusing antibody one or more precipitin arcs will form in the gel depending on the antibody and antigen heterogeneity. Quantitation of antigen/antibody by this method requires considerable effort and multiple runs. A simpler approach to quantitation was devised by Laurell, using a method known as 'rocket immunoelectrophoresis'.

Figure 3 (See Colour Plate 39). Direct dual immunostaining of a polyacrylamide/glyoxyl-agarose composite gel to profile fibrinogen y-chain (grey), a-chain (amber) cross-linking and hybrid a/y-chain (umber) cross-linking by plasma transglutaminase (right lane), and the chain composition of plasma fibrinogen. The illustration depicts sieving equivalent to a regular polyacrylamide gel, and subsequent rendering of the gel for antibody permeation by removing the polyacrylamide. (Reproduced with permission from Shainoff etal., 1991, JournalofBiological Chemistry 266: 6429.)

Figure 3 (See Colour Plate 39). Direct dual immunostaining of a polyacrylamide/glyoxyl-agarose composite gel to profile fibrinogen y-chain (grey), a-chain (amber) cross-linking and hybrid a/y-chain (umber) cross-linking by plasma transglutaminase (right lane), and the chain composition of plasma fibrinogen. The illustration depicts sieving equivalent to a regular polyacrylamide gel, and subsequent rendering of the gel for antibody permeation by removing the polyacrylamide. (Reproduced with permission from Shainoff etal., 1991, JournalofBiological Chemistry 266: 6429.)

In the method of Laurell, the gel is poured with antibody added to it at 56°C, and wells are punched in the solidified gel to accommodate the antigen solution. As the antigen moves by electrophoresis out of the well it sweeps soluble immune complexes along with it in a comet-shaped profile until the antigen/ antibody levels become equivalent, whereupon the comet-shaped precipitin arc comes to a virtual stop because the antibodies themselves do not migrate at around pH 8.6. The area contained within the arc is usually directly proportional to the antigen level. Because of the ease with which agarose gels can be cut and filled with interposed gels open-faced, this technique is amenable to a myriad of variations, as described by Axelsen and associates.

In one variation of the Laurell method, called crossed immunoelectrophoresis, the proteins are subjected to pre-electrophoresis, and antibody-containing agarose is cast around a strip of the gel. Then, in a crossed electrophoresis the antigens are transferred out of the primary gel into the antibody where they form rocket(s) in line with their initial position. The technique is 'found' useful for quantifying multiple antigens and variant forms of an antigen. If the initial gel contains SDS, it should be quenched by adding nonionic detergent such as Lubrol® to the antibody-containing gel. In the event that the protein becomes insoluble when stripped of SDS, initially it can be fixed, then probed with charge-enhanced car-bamylated primary antibody. Retained antibody is measured by electrophoresis into secondary antibody, as illustrated in Figure 4. Use of secondary antibodies is also essential when using monoclonal IgG antibodies as primary probes, because these monoclonal antibodies do not form immunoprecipitates on their own unless the epitope is multiply expressed in the antigen.

Direct Immunoprobing

Because of the permeability of agarose gels to antibody it is possible to probe electropherograms directly. Direct immunoprecipitation within the gel is seldom used because of uncertainties of levels or antibody required for substantial precipitation of un-

Figure 4 Crossed immunoelectrophoresisto profile plasma fibrinogen derivatives in plasma by: (i) probing the electropherogram with primary antifibrinogen antibodies; and (ii) measuring the retained antibody by displacing to form rockets in secondary gel containing anti-IgG antibodies, with standards for the IgG forming the left- and right-most peaks. This approach was made necessary because insolubility of fibrinogen and its high molecular mass derivatives, once denatured by SDS, cannot be transferred out of the primary electropherogram to form rockets directly. (Reproduced from Dardik etal, 1989, Cleveland Clinical Journal ofMedicine 56: 451.)

known levels of antigen. The proteins are usually fixed or immobilized, and probed by imbibing labelled antibody into and out of the gel. If secondary antibodies are to be used to report retention of the primary antibody, the primary antibody should in turn be fixed before the secondary probing to avoid dissociative losses of the primary during the secondary probing which takes periods of the order of an hour. Glyoxyl agarose was developed to enable these fixations by chemical immobilization.

Blotting

This widely used procedure involves transfer of components out of the gel on to a blotting membrane to immobilize them for immunostaining or compositional analysis on an open-faced surface. It is an essential means for probing reactivities of components separated on polyacrylamide because of the low permeability of the gel. Much uncertainty attends this 'blotting', because the transfers are incomplete, and frequently nil with high molecular mass proteins. These proteins not only transfer slowly, but often precipitate within the gel as SDS transfers away from them. Also, very low molecular mass peptides fail to be retained by the blotting membrane.

While blotting from polyacrylamide gels is usually effected by crossed electrophoresis, the transfers out of agarose gels are more simply effected by either compressing them against the membrane supported on filter paper stacks (a method that yields only partial transfer), or by light vacuum suctioning of buffer through the gel on to the membrane supported on a 'gel drier'. However, when SDS is present it must either be quenched by a prior 15 min immersion in buffer containing 1-2% nonionic detergent, or precipitated by immersing the gel in 0.1 M KCl.

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