title: 2-D Western blotting for evaluation of antibodies developed for detection of host cell protein.
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.
authors.affiliation: Life Science Group, Bio-Rad Laboratories, 6000 James Watson Dr., Hercules, CA, 94547, USA, Thomas_berkelman@bio-rad.com.
authors.forename: Tom
authors.initials: T
authors.lastname: Berkelman
authors.forename: Adriana
authors.initials: A
authors.lastname: Harbers
authors.forename: Sricharan
authors.initials: S
authors.lastname: Bandhakavi
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015180:Electrophoresis, Gel, Two-Dimensional; D011506:Proteins / Q000737:chemistry*
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D011506:Proteins