Single radial immunodiffusion as a method for the assay of the acellular pertussis vaccine components, pertussis toxoid, filamentous haemagglutinin and pertactin.
The development of acellular pertussis vaccines has raised a number of issues relevant to the control of these products. Of particular importance is the need for robust and accurate in vitro assays for the antigen content of the vaccines which might contain up to five different antigen components, each of which needs to be independently assayed. This paper describes a simple method for the quantification of three component antigens. Because relatively high doses of purified antigens are used in those preparations, the elimination of residual toxicity is a major concern. This is achieved by genetic modification of chemical treatment. The latter results in modification of the immunological reactivity of the antigens making direct assay by such methods as ELISA ineffective. A single radial diffusion technique using polyclonal antisera for the assay of pertussis toxoid (PTxd), chemically treated filamentous haemagglutinin (FHA) and pertactin (69 kDa) has been developed. The method uses low concentrations of antisera, allowing accurate and reproducible quantification of antigen content as low as 25 microg/ml of protein for pertussis toxoid and filamentous haemagglutinin and 5 microg/ml for pertactin. Since by the addition of detergent, diffusible subunits are produced irrespective of the original physical state of the antigens, the assay is suitable for assay of these antigens after detoxification/or stabilization by chemical treatment and is able to determine the differences between preparations which have the same protein concentration but different antigenic contents. This provides a means for assuring the consistency of the antigens after detoxification/or chemical stabilization which could be used as an in-process control method for acellular pertussis vaccines.
Xing
D K
DK
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, U.K.
Canthaboo
C
C
Corbel
M J
MJ
Schild
G C
GC
D018829:Adhesins, Bacterial / Q000032:analysis; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001886:Bordetella pertussis / Q000276:immunology; D005260:Female; D006388:Hemagglutinins / Q000032:analysis; D006801:Humans; D005779:Immunodiffusion / Q000379:methods*; D051379:Mice; D010567:Pertussis Vaccine / Q000032:analysis* / Q000592:standards*; D011786:Quality Control; D012756:Sheep; D014121:Toxoids / Q000032:analysis; D010566:Virulence Factors, Bordetella
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D006388:Hemagglutinins; D010567:Pertussis Vaccine; D014121:Toxoids; D010566:Virulence Factors, Bordetella; C065273:filamentous hemagglutinin adhesin, Bordetella pertussis; C051093:pertussis toxoid; C068929:pertactin