engleski

HIGH THROUGHPUT ISOLATION OF IgG FROM HUMAN PLASMA SAMPLES BY USE OF CIM PROTEIN G MONOLITHIC DISKS

Immunoglobulin G is the second most abundant protein in human plasma (10-15 mg/ml). All IgG molecules carry N-glycans at the highly conserved N-glycosylation site (Asn-297) of the fragment crystalizable (Fc) part of their heavy chains. Fc N-glycosylation modulates the biological activity of IgG and furthermore changes in N-glycosylation have been found to be associated with various diseases. Using recently developed high-throughput HPLC method we want to perform a large scale study of IgG N-glycans in normal human population and use these glycans as quantitative traits for GWAS (genome wide association study) analysis to identify genes that regulate glycosylation in plasma cells. We used newly developed CIM® Protein G 96-Monolithic technology for optimization and later isolation of immunoglobulin G from 1984 human plasma samples. This affinity system consists of a unique monolithic stationary phase that is installed inside each well of an ELISA plate. Results obtained with the use of Protein G 96-Monolithic plate were comparable to those of Protein G and A bulk monolithic columns. With the proper sample preparation, optimized isolation conditions and regeneration at the end of each chromatographic run, we were able to isolate IgG from about 300 plasma samples per day. CIM® Protein G 96-Monolithic plates enable high throughput and highly efficient purification of immunoglobulin G. After this optimization step, a robotic system will be used for high throughput isolation of IgG and other less abundant proteins in order to perform further analysis of human glycoproteome

protein G; IgG; monoliths

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