Affinity precipitation of monoclonal antibodies by nonstoichiometric polyelectrolyte complexes.
The nonstoichiometric polyelectrolyte complex (PEC) formed by poly(methacrylic acid) (degree of polymerization 1830) (PMAA) and poly(N-ethyl-4-vinyl-pyridinium bromide) (degree of polymerization 530) (PEVP) undergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5-6.5 depending on the ionic strength and PEVP/PMAA ratio in the complex. The antigen, inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit was covalently coupled to PEVP. The resulting GAPDH-PEVP/PMAA complex was used for the purification of antibodies from a 6G7 clone specific towards inactivated GAPDH. The crude extract was incubated with GAPDH-containing PEC and the precipitation of the PEC was carried out at 0.01 M NaCl and pH 4.5, 5.3, 6.0 and 6.5 using PEC with PEVP/PMAA ratios of 0.45, 0.3, 0.2 and 0.15, respectively. Purified antibodies were eluted at pH 4.0 where PECs of all compositions used were insoluble. PEC precipitation is accompanied only by small nonspecific coprecipitation of proteins. Precipitated PEC could be dissolved at pH 7.3 and used repeatedly.
Dainiak
M B
MB
Department of Biotechnology, Lund University, Sweden.
Izumrudov
V A
VA
Muronetz
V I
VI
Galaev IYu
Mattiasson
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011108:Polymers; D011817:Rabbits
D000911:Antibodies, Monoclonal; D011108:Polymers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases