Rapid Ultrasensitive Gel-Free Immunoblotting with Magnetic Labels.

Immunoblotting is widely used for the detection of proteins using specific antibodies. We present here a new immunoblotting method, which is characterized by exceptional sensitivity, rapidness, and low consumption of antibodies. A thin conductive layer between touching hydrophilic cellulose membranes instead of polyacrylamide gel is used for the electrophoretic separation of proteins. Contrary to common Western blotting, the separation occurs in nondenaturing conditions. The membrane surface is smoothed by deposition of the cellulose layer and modified with azidophenyl groups, allowing for the photochemical in situ immobilization of proteins, which are carried out after the electrophoresis. Thus, the additional step of transferring the protein from the gel onto the membrane is eliminated. Specific protein bands are then visualized by decoration with magnetic beads. The limit of detection of interleukin IL-1β reaches 0.3 fg or ∼104 molecules, whereas the total blotting time is about 5 min. The application of the technique is demonstrated by the detection of IL-1β, total IgA, and IgA specific to Mycobacterium tuberculosis antigen in the exhaled breath samples, obtained from healthy subjects and tuberculosis patients.