Simultaneous production of immunoaffinity membranes.
We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3, haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids.
Shimazaki
Youji
Y
Graduate school of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Miyamoto
Masayuki
M
D000818:Animals; D055910:Antibodies, Immobilized / Q000378:metabolism*; D001391:Azo Compounds; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006242:Haptoglobins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008567:Membranes, Artificial; D010449:Peptide Mapping; D011145:Polyvinyls; D011228:Prealbumin / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000302:isolation & purification / Q000378:metabolism; D005465:Fluorocarbon Polymers
D055910:Antibodies, Immobilized; D001391:Azo Compounds; D001798:Blood Proteins; D006242:Haptoglobins; D008567:Membranes, Artificial; D011145:Polyvinyls; D011228:Prealbumin; D014168:Transferrin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C408744:Direct Blue 71