Unexpected interaction of some anti-TNP hybridoma antibodies with Superose HPLC gel filtration resins.
Five different hybridoma antibodies (the isotypes IgG1, IgG2a and IgG2b), reactive with TNP, showed increased elution times when gel filtered on a Superose-12 HPLC column corresponding to apparent molecular weights ranging from 54 kDa to 120 kDa compared to the normal of 150 kDa. One of the antibodies (C1901-B4) was studied in detail showing that the unusual gel filtration behaviour was localized to the Fab part of the molecule. SDS-PAGE analysis showed that the intact antibodies had normal molecular weights. Gel filtration on a Toyosoda G-3000SW HPLC column and Sephadex G-150 as well as Sepharose 6B generally showed normal elution times. These results support the hypothesis that the retardation on the Superose gel is probably due to aromatic interactions between amino acid residues supposedly exposed in the hypervariable region (i.e., the antibody combining site) of the antibody, and the gel matrix which is rich in ether O-atoms created during the manufacturing process. If this hypothesis is correct one might expect such interactions between Superose resins and antibodies of many different specificities in which aromatic amino acids are exposed in the combining sites.
Michaelsen
T E
TE
Department of Immunology, National Institute of Public Health, Oslo, Norway.
Løfsgaard
M F
MF
Aase
A
A
Heyman
B
B
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D012685:Sepharose / Q000276:immunology*; D014301:Trinitrobenzenes / Q000276:immunology*
D007074:Immunoglobulin G; D014301:Trinitrobenzenes; D012685:Sepharose; C049225:Superose 12