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 mm(2)). 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.
Department of Chemistry and Biology, Graduate School of Science and Engineering, Ehime University, Matsuyama, Japan. Electronic address: shimazaki.yoji.my@ehime-u.ac.jp.
Youji
Y
Shimazaki
Faculty of Science, Ehime University, Matsuyama, Japan.
Ai
A
Hashimoto
D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D003600:Cytosol / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004867:Equipment Design; D004950:Esterases / Q000737:chemistry; D005470:Fluorometry; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D008567:Membranes, Artificial; D046210:Microfluidic Analytical Techniques; D044085:Microfluidics; D011145:Polyvinyls / Q000737:chemistry; D015203:Reproducibility of Results; D013552:Swine; D005465:Fluorocarbon Polymers
D055910:Antibodies, Immobilized; D004950:Esterases; D007770:L-Lactate Dehydrogenase; D008567:Membranes, Artificial; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers
Enzyme activity; Esterase; Immunoaffinity membrane; Lactate dehydrogenase; Spectrofluorometry