A microfluidic device containing membrane-immobilized antibodies for successively capturing cytosolic enzymes

A microfluidic device containing membrane-immobilized anti-esterase (ES) antibodies and anti-lactate dehydrogenase (LDH) antibodies was prepared. The membrane was prepared by transferring antibodies that had been separated by non-denaturing two-dimensional electrophoresis to a polyvinylidene difluoride membrane, which was then stained and cut into small pieces (16 mm2). In this microfluidic device, >0.014 Unit mL−1 of the purified porcine carboxylesterase was specifically captured by membrane-immobilized anti- ES antibodies and >147 Unit mL−1 of purified porcine LDH was specifically captured by membrane-immobilized anti-LDH antibodies. Furthermore, ES and LDH in micro-scale aliquots of porcine liver cytosol were successively captured by membrane-immobilized antibodies in the device, and the enzyme activities were quantitatively analyzed by spectrofluorometry. The results indicate that the microfluidic device containing membrane-immobilized antibodies can be used to investigate the activities of several types of intact enzymes.