Counterflow immunoisotachophoresis on cellulose acetate membranes

Discontinuous electrophoresis on cellulose acetate membranes with the use of 0.06 M Tris-HCl (pH 6.7) as the leading electrolyte and 0.012 M Tris-beta-alanine (pH 8.6) as the terminating one results in concentration of the proteins present in the system on the Cl-/beta-alanine- boundary. If the antigen solution is placed in a "pocket" ahead of the moving boundary, a counterflow to the cathode arises due to electroendosmosis. At constant voltage the migration rate of the boundary drops and that of electroendosmosis does not change until they become equal. In such a stationary position, the antigen-containing solution is passing through the Cl-/beta-alanine- boundary to the cathode, while all the proteins are completely "absorbed" on the boundary as highly concentrated bands. Addition of ampholytes to the antigen solution contributes to the isotachophoretic separation of a protein mixture on the strip. The concentrated and separated antigens can be revealed by immunofixation, immunodiffusion, or crossed immunoelectrophoresis in gel. The technique is approximately 100 times more sensitive compared to the usual immunodiffusion and immunoelectrophoresis methods on cellulose acetate membranes, and is applicable to the detection of trace amounts of antigens in the urine, liquor, amniotic fluid, tears, and other biological fluids with low protein contents.