Monolith peptide affinity chromatography for quantification of immunoglobulin M.
We have developed a method for quantification of a specific monoclonal IgM directed toward embryonic stem cells based on a peptide affinity monolith. A peptide affinity ligand with the sequence C-C-H-Q-R-L-S-Q-R-K was obtained by epitope mapping using peptide SPOT synthesis. The peptide ligand was covalently immobilized by coupling the N-terminal cysteine to a monolithic disk that was previously modified with iodated spacer molecules. The monolithic disc was used for quantification of purified IgM and for IgM present in mammalian cell culture supernatant. We observed 17% unspecific binding of IgM to the monolithic disk and additionally a product loss in the flow through of 20%. Nevertheless, calibration curves had high correlation coefficients and inter/intra-assay variability experiments proved sufficient precision of the method. A limit of quantification of 51.69 μg/mL for purified IgM and 48.40 μg/mL for IgM in cell culture supernatant could be calculated. The binding capacity was consistent within the period of the study which included more than 200 cycles. The analysis time of less than 2 min is an advantage over existing chromatographic methods that rely on pore diffusion.
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.
Silke
S
Neff
Alois
A
Jungbauer
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D002138:Calibration; D002417:Cattle; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D004058:Diffusion; D053595:Embryonic Stem Cells / Q000737:chemistry; D018604:Epitope Mapping; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism; D007075:Immunoglobulin M / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D055767:Immobilized Proteins; D007075:Immunoglobulin M; D010455:Peptides