[Countercurrent immunoblotting].

Abstract

Urinary proteins, concentrated and separated on cellulose-acetate membranes by counterflow isotachophoresis (ITP), were transferred by direct contact onto a strip of nitrocellulose membrane (NCM). ITP in the system of electrolytes: tris-HCl, pH 6.7 (the leading one) and tris-beta-alanine, pH 8.6 (the terminal one) gives rise to a strong electroendo-osmotic flow (EEF) in NCM, directed to the cathode. The rate of the counterflow in the zone, occupied by the leading electrolyte, exceeds the migration rate of any protein possessing anode mobility and present in the zone. Under these circumstances EEF serves as a "conveyer belt" transferring immunological reagents (antibodies, immunoconjugates, peroxidase) through the protein bands, "printed" on NCM. The immunoblots were developed in a standard way with 4-ethyl-1-naphthol as a substrate for antibody-bound peroxidase. The counterflow immunoblotting makes it possible to reveal and characterize light chains of immunoglobulins when they are present in the urine in the range of 20 ng/ml.