Immunofixation electrophoretic techniques applied to identification of proteins in serum and cerebrospinal fluid

We describe the application of immunofixation staining of agarose-gel electrophoretograms in areas where its use in the clinical laboratory is appropriate. Immunofixation electrophoresis consists of an electrophoretic phase followed by a fixation phase in which antiserum is used to precipitate the protein. As long as the antibody is in slight excess or near equivalency, the antigen/antibody complex remains insoluble. The reaction can be detected by visual inspection in indirect light, by protein staining, or by use of antibodies labeled with fluorescein, enzyme, or isotope. In the method described here we primarily have used protein staining (Coomassie Blue) to accentuate the proteins fixed by antisera. All unreacted proteins are removed by pressing with filter paper and saline washing. In the clinical laboratory, this method expedites immunochemical evaluation of samples and may also supplement immunoelectrophoresis. It has been applied successfully in identifying small obscure monoclonal proteins in the serum and cerebrospinal fluid of patients with multiple sclerosis, subacute sclerosing panencephalitis, biclonal gammopathies, serum monoclonal light chains, and mobility shifts of certain proteins, particularly of the complement series. Immunofixation demonstrates that the protein bands present in spinal fluid from multiple sclerosis and subacute sclerosing panencephalitis patients are of the IgG class of immunoglobulins; and non-IgG protein, such as beta and gamma trace proteins, are not detected. We also comment on reverse immunofixation with labeled antigen as a branch of the procedure that allows detection of function of the immunoglobulins separated by electrophoresis.