Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients.
A method for preparative isolation of human monoclonal antibody isoproteins is described in the present paper. A human monoclonal antibody directed against the transmembrane protein gp 41 from the human immunodeficiency virus (HIV-1) was used in this study. The antibody belongs to the IgG1 subtype and exhibits antibody dependent cellular cytotoxicity. The resolving power of conventional preparative protein separation techniques such as ion-exchange chromatography, chromatofocusing and lectin affinity chromatography is too poor for a complete separation of isoproteins. The more sophisticated technique of chromatofocusing on FPLC-based material (Mono P, Pharmacia) did not satisfy our expectation. With semipreparative IEF in immobilized pH gradients we were able to prepare the different isoproteins of a human monoclonal antibody in milligram amounts. No significant difference between the single isoproteins with respect to specificity and avidity to the recombinant antigen (rec gp 160) was detected. Therefore, we assume that the separation conditions did not influence the immunochemical nature of the antibody and significant denaturation and/or precipitation of the IgG did not occur. Furthermore the method affords preparative separation with resolution equivalent to analytical runs. Experiments for scale up and further characterization of isoproteins (carbohydrate composition, amino acid analysis, half life times etc.) are in progress.
Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria.
E
E
Wenisch
A
A
Jungbauer
C
C
Tauer
M
M
Reiter
G
G
Gruber
F
F
Steindl
H
H
Katinger
D000818:Animals; D000911:Antibodies, Monoclonal / Q000145:classification / Q000302:isolation & purification*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D015483:HIV Antibodies / Q000302:isolation & purification; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D051379:Mice
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies