T. C. BøG‐HANSEN

Detection of Biospecific Interaction during the First Dimension Electrophoresis in Crossed Immunoelectrophoresis

Lectins were included in the first dimension gel in crossed immunoelectrophoresis of human serum proteins. Con A resulted in a retardation of most glycoproteins during the first electrophoresis and heterogeneous forms of individual glycoproteins could be detected. Ulex europeus lectin gave most of the proteins a higher migration velocity. Pokeweed mitogen did not interact with serum glycoproteins, but it did with serum lipoproteins. The method allows a close study of interacting components, determination of binding specificities, and detection of minor interacting components, including heterogeneous molecular forms.

An Artefact in Quantitative Immunoelectrophoresis of Spectrin Caused by Proteolytic Activity in Antibody Preparations

Many antibody preparations exhibit proteolytic activity due to the presence of plasmin. In crossed immunoelectrophoresis at pH 8.6 this enzyme can degrade certain proteins during electrophoresis in the antibody‐containing gel, resulting in artefacts in the form of extra precipitation arcs of congruent shape. The degradation behaviour of spectrin, a major protein of human erythrocyte membranes, was investigated. The artefact could be completely abolished by the addition of protease inhibitors, e.g. aprotinin and soya bean trypsin inhibitor, to the antibody preparations.

Identification of Glycoproteins with One and with Two or More Binding Sites to Con A by Crossed Immuno-Affinoelectrophoresis

Glycoproteins with one binding site and glycoproteins with two or more binding sites to concanavalin A may be identified by crossed immuno‐affinoelectrophoresis with free. and with immobilized concanavalin A. Human urinary acid phosphatase and human serum cholinesterase were demonstrated to be molecules with one and two or more binding sites to concanavalin A, respectively.

Enzyme Characterization in Quantitative Immunoelectrophoresis

Carboxylic ester hydrolases in human serum were identified, characterized, and quantified by means of crossed and rocket immunoelectrophoresis. Two types of esterase were found, arylesterase (EC 3.1.1.2) and cholinesterase (EC 3.1.1.8), exhibiting differences in specificity for several histochemical substrates. α‐naphthyl acetate and β‐naphthyl acetate were used to distinguish between the two enzymes. Arylesterase seemed to interact specifically with aJipoprotein. Quantitation of enzyme protein and correlation with enzyme activity is described. The experiments illustrate general methodological problems concerning immunochemical characterization of enzymes.

Detection of Concanavalin A — Binding Herpes Simplex Virus Type 1 and Type 2 Antigens by Crossed Immuno-Affinoelectrophoresis

The presence of four herpes simplex virus (HSV) glycoprotein antigens in cells infected with HSV type 1 and HSV type 2 was demonstrated by the use of crossed immuno‐affinoelectrophoresis with con A.

Affinity Electrophoresis with Lectins for the Study of Glycoproteins

Affinity chromatography and electroimmunoprecipitationcan be combined to obtain analytical procedures with the following aims: to identify ligand-binding proteins, to characterize the binding, and to predict the results of preparative affinity separations. The following methods differ in the order in which proteins interact with lectin. In Method A the sample is incubated with lectin before analysis by crossed immunoelectrophoresis. In Method B free (Bl) or immobilized (B2) lectin is incorporated in an intermediate gel in crossed immunoelectrophoresis. In Method C free lectin (Cl) or immobilized lectin (C2) is incorporated in the first-dimension gel of crossed immunoelectrophoresis. By comparing with control experiments it is possible to identify and characterize protein-lectin interactions by differences in electrophoretic and antigenic behavior of the protein under study. Protein-lectin interaction can then be identified and characterized by comparing of the electrophoretic and antigenic behaviour of the protein under study with the results of control experiments.

Immunoprecipitation in an Electric Field

200 240 280 320 360 FIG. 8.1. Development of human serum albumin immunoprecipitates. Human serum was submitted to rocket immunoelectrophoresis against rabbit anti-human albumin. The plate was photographed at various times after start of the electrophoresis, indicated on the figure in minutes. The electrical field strength was 3 V/crn. The buffer was Tris-barbital, pH 8.6, the ionic strength was 0.02.

Antisera against Purified Asialo‐Orosomucoid and Asialo‐Cholinesterase

Rabbits were immunized with highly purified desialylated human serum cholinesterase and orosomucoid. In immunoelectrophoresis and immunodiffusion the resulting antibodies had precipitation properties which were identical to those of antibodies against the native proteins. Spurring between native and asialo‐proteins was not seen. A linear correlation was observed between the rocket height obtained with the antiserum raised against the asialo‐protein and the rocket height with a normal antiserum. A monospecific antiserum against cholinesterase was obtained without absorption. The use of desialylated glycoproteins as immunogens for the production of monospecific antisera is discussed.

Enzyme Characterization in Quantitative Immunoelectrophoresis—Some Potentialities and Some Pitfalls

Enzyme characterization is carried out by histochemical methods after normal electroimmunoprecipitation, in which the enzymatic staining reaction serves to locate the immunoprecipitate containing the enzyme. The only modification of the standard routines is that the immunoplates may be dried in a stream of cold air (room temperature) or stained as wet plates after pressing and washing. Enzymes are usually active as immunoprecipitates in the pressed and dried agarose gel and can be kept for a long time before processing.