Has immunoblotting replaced electroimmunoprecipitation? Examples from the analysis of autoantigens and transglutaminase-induced polymers of the human erythrocyte membrane.
The virtues and drawbacks of immunoblotting and electroimmunoprecipitation in the characterization of macromolecules in crude mixtures are presented. Interactions between autoantibodies and human erythrocyte membrane proteins were studied by means of crossed-affinoimmunoelectrophoresis with autologous immunoglobulins incorporated into the first dimension gel and by immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis separated erythrocyte membrane proteins with autologous immunoglobulins as primary antibodies. Substrates for transglutaminase in calcium-activated human erythrocyte membranes were examined by immunoelectrophoretic and immunoblotting methods. The experiments concerning autoantibodies complemented each other and showed that epitopes on Band 3 protein, spectrin and ankyrin are recognized by circulating immunoglobulin autoantibodies in normal individuals. The polymer experiments showed the presence of spectrin, ankyrin, Band 3, Band 4.1, glucose transporter, actin and haemoglobin epitopes in the polymer (Mr 3.10(6)-5.10(6]. It is concluded that the two techniques complement each other. The most evident advantage of immunoblotting is its sensitivity and applicability while electroimmunoprecipitation in some instances allows an easier identification of distinct protein species and still has a role for quantification and certain monitoring purposes.
Bjerrum
O J
OJ
Immuno Technology, Novo Industri A/S, DK-2880 Bagsvaerd, Denmark.
Heegaard
N H
NH
D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000032:analysis*; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D004910:Erythrocyte Membrane / Q000276:immunology / Q000378:metabolism*; D005972:Glutaminase / Q000378:metabolism*; D015151:Immunoblotting; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G / Q000276:immunology; D011108:Polymers; D011233:Precipitin Tests
D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D007074:Immunoglobulin G; D011108:Polymers; D005972:Glutaminase; D002118:Calcium