The combination of both the electrophoresis and the immunofixation and of the thin layer gel filtration and the immunofixation followed by enzyme activity staining on antigen-antibody complexes were applied to detect and identify the enzyme-linked immunoglobulins which were observed as macromolecular enzyme complexes in human sera. For any given antigen-antibody system, the optimum concentration of antigen in the serum to be analysed must be given experimentally. Moreover, removing of unreacted proteins must be carefully performed with the solution which will protect the activity of the enzyme to be detected.
In the cases of LDH- and amylase-linked immunoglobulins the detection of minimum enzyme activities on Cellogel membrane were limited by staining sensitivity. Thus, two to ten times concentration of commercially available antibodies was required to give the optimum concentration of antigen-antibody complexes. In the cases of alkaline-phosphatase-linked immunoglobulins, the detection of the enzyme activity was easily amplified by the elongation of the incubation period. Thus, the optimum concentration of antigen was given without concentration of commercial antibodies.
In the studies of macromolecular enzyme complexes in human sera, the immunoprecipitation technique and enzymo-immunoelectrophoresis were conventionally carried out to identify the enzyme-linked immunoglobulins. Some cases, however, still ramain ambiguous because of unskilled and unstable techniques used for analysing these macromolecular enzyme complexes. Thus, these immunofixation techniques followed by enzyme activity staining (enzyme immunofixation), described in this paper can facilitate the detection and identification of such macromolecular complexes as enzyme-linked immunoglobulins.
