Chapter title |
2-D Western Blotting for Evaluation of Antibodies Developed for Detection of Host Cell Protein
|
---|---|
Chapter number | 28 |
Book title |
Proteomic Profiling
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2550-6_28 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2549-0, 978-1-4939-2550-6
|
Authors |
Tom Berkelman, Adriana Harbers, Sricharan Bandhakavi |
Abstract |
Recombinant proteins generated for therapeutic use must be substantially free of residual host cell protein (HCP). The presence of host cell protein (HCP) is usually assayed by ELISA using a polyclonal antibody mixture raised against a population of proteins derived from the host cell background. This antibody should recognize as high a proportion as possible of the potential HCPs in a given sample. A recommended method for evaluating the assay involves two-dimensional electrophoretic separation followed by Western blotting.We present here a method using commercial anti-HCP antibody and samples derived from Chinese Hamster Ovary (CHO) cells. The 2-D electrophoresis procedure gives highly reproducible spot patterns and entire procedure can be completed in less than 2 days. Software analysis enables the straightforward generation of percent coverage values for the antibody when used to probe HCP-containing samples. |
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