Development of a carbohydrate binding assay for the B-oligomer of pertussis toxin and toxoid.
Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and, in its detoxified form PTd, is an important component of pertussis vaccines. The in vivo histamine sensitization test (HIST) is currently used for the safety testing of these vaccines. However, an alternative test is needed because of large assay variability and ethical concerns with regard to animal usage. PTx has two functionally distinct domains: the enzymatic A-protomer and the B-oligomer that facilitates host-cell binding and entry of PTx into the cell. The development of a quantitative PTx binding assay using glycoproteins or defined oligosaccharides is reported. PTx was found to bind preferentially to multiantennary N-glycans, with the highest binding toward the fully sialylated structures. In contrast, PTd lost the ability of PTx to bind to sialylated multiantennary structures but retained some capacity to bind to neutral multiantennary structures. The developed assay was shown to be specific, sensitive, and robust and could be used for investigating the mechanisms of PTx detoxification and for monitoring PTx binding activity in vaccine formulations. This assay could also be used to complement a PTx-enzymatic assay, developed recently, and together they may form the basis of a potential alternative in vitro assay to replace the in vivo HIST.
Gomez
Sheena R
SR
Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, UK.
Xing
Dorothy K-L
DK
Corbel
Michael J
MJ
Coote
John
J
Parton
Roger
R
Yuen
Chun-Ting
CT
D001667:Binding, Competitive; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D037342:Pertussis Toxin / Q000737:chemistry* / Q000378:metabolism; D010567:Pertussis Vaccine / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D014121:Toxoids / Q000737:chemistry* / Q000378:metabolism
D006023:Glycoproteins; D009844:Oligosaccharides; D010567:Pertussis Vaccine; D011134:Polysaccharides; D014121:Toxoids; D037342:Pertussis Toxin