Crossedaffinity Immunoelectrophoresis CAIE

Affinity electrophoresis refers to any technique in which two or more components specifically interact during an electrophoretic run. Affinity electrophor-esis in agarose gels, combined with subsequent im-munochemical detection (CAIE), was introduced by Bog-Hansen in 1975 as a useful tool for the characterization of biospecific macromolecular interactions. This technique permits, among other things, the demonstration of ligand-binding proteins, enumeration of binding sites and estimation of binding constants, with the added advantage of being adequate for determination of very small quantities in impure materials.

An important field of application of CAIE has been the study of the interaction of glycoproteins and lec-tins. Lectins are animal and mostly plant-derived proteins that specifically interact with the carbohydrate components of glycoconjugates. There is a considerable amount of information in the literature about the biochemical properties of numerous lectins and on the type of glycan structures that they can recognize. Using different lectins in the first electrophoresis run (affinity electrophoresis step), CAIE permits the analysis of the heterogeneity of glycoproteins in complex mixtures. The serum protein glycoforms, after fractionation by lectin affinity electrophoresis, can be revealed with specific antibodies and finally quantified.

Figure 5A shows a schematic illustration of the CAIE technique. First, the serum proteins are subjected to electrophoresis (generally at pH 8.2-8.6)

2. Second-dimension electrophoresis

Antibody-containing agarose gel

First-dimension electrophoresis

Lectin-containing agarose gel i

2. Second-dimension electrophoresis

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Figure 5 (A) Schematic representation of the CAIE technique. O, origin, corresponding to the well where the sample is applied in the first-dimension electrophoresis. (B) CAIE patterns of human aracid glycoprotein from (right) healthy individuals and (left) patients with inflammatory processes. The numbered peaks correspond to microforms of aracid glycoprotein in decreasing order of mobility.

Figure 5 (A) Schematic representation of the CAIE technique. O, origin, corresponding to the well where the sample is applied in the first-dimension electrophoresis. (B) CAIE patterns of human aracid glycoprotein from (right) healthy individuals and (left) patients with inflammatory processes. The numbered peaks correspond to microforms of aracid glycoprotein in decreasing order of mobility.

in lectin-containing agarose gels (first-dimension gel). The lectin is added to the melted agarose (at around 55°C) before pouring the gel. Under these conditions most of the lectins used in CAIE do not have sufficient mobility. The proteins are fractionated in their different glycoforms, whose mobility depends on their corresponding affinity for the lectin. The first-dimension gel is then transferred into a second-dimension agarose gel containing specific antibodies against the protein to be analysed. The second-dimension elec-trophoresis produces for many serum glycoproteins fused precipitating peaks, which corresponds to the different microforms present in the sample. The CAIE patterns can be visualized by the methods described in the previous immunochemical techniques. The amount of each glycoform is related to the corresponding immunoprecipitating area that can be calculated by planimetry or by triangulation.

Figure 5B shows, as an example, the CAIE patterns of a1-acid glycoprotein from human serum. In the first dimension gel, 1 mg mL-1 of the lectin Concanavalin A was included. The second-dimension gel contained specific rabbit antiserum against human a1-acid gly-coprotein. The different peaks have been labelled in decreasing order of mobility. Peak 1 corresponds to glycoforms of the human a1-acid glycoprotein that did not react with the lectin and contains tri- and tetra-antenary glycans; peak 2 corresponds to weakly reactive glycoforms of the protein containing one bi-antennary glycan; finally, peak 3 corresponds to strong reactive glycoforms containing at least two bi-antennary glycans.

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