A non-chromatographic method for the purification of a bivalently active monoclonal IgG antibody from biological fluids.
This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4-dinitrophenyl IgG: IgG(DNP); and mouse antidigoxin IgG: IgG(Dgn)) from ascites fluid. This procedure (for IgG(DNP)) has three steps: (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation of cyclic complexes of IgG(DNP) by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; (iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 microM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgG(DNP) and IgG(Dgn) from ascites fluid in yields of >80% and with >95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: (i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; (ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi- and/or trivalent analogues.
Bilgiçer
Başar
B
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Thomas
Samuel W
SW
Shaw
Bryan F
BF
Kaufman
George K
GK
Krishnamurthy
Vijay M
VM
Estroff
Lara A
LA
Yang
Jerry
J
Whitesides
George M
GM
D000645:Ammonium Sulfate; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D001201:Ascites / Q000276:immunology; D001666:Binding Sites, Antibody; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004077:Digoxin / Q000737:chemistry / Q000276:immunology*; D019281:Dimerization; D004136:Dinitrobenzenes / Q000737:chemistry / Q000276:immunology*; D006241:Haptens / Q000737:chemistry* / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008958:Models, Molecular; D051381:Rats
D000911:Antibodies, Monoclonal; D004136:Dinitrobenzenes; D006241:Haptens; D007074:Immunoglobulin G; D004077:Digoxin; D000645:Ammonium Sulfate