Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing.
Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way.
Hagemann
Sascha
S
Department of Neurology, Philipps-University Marburg, Rudolf-Bultmann-Str. 8, 35037 Marburg, Germany.
Faude
Alexander
A
Rabenstein
Monika
M
Balzer-Geldsetzer
Monika
M
Nölker
Carmen
C
Bacher
Michael
M
Dodel
Richard
R
D001323:Autoantibodies / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016756:Immunoglobulins, Intravenous / Q000737:chemistry / Q000378:metabolism; D010956:Plasmapheresis; D015203:Reproducibility of Results
D001323:Autoantibodies; D000939:Epitopes; D055767:Immobilized Proteins; D007074:Immunoglobulin G; D016756:Immunoglobulins, Intravenous