title: Validation of an enzyme-linked immunosorbent assay for the quantification of human IgG directed against the repeat region of the circumsporozoite protein of the parasite Plasmodium falciparum.
abstract: BACKGROUND
  Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines.
  
  
  METHODS
  The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum.
  
  
  RESULTS
  The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time.
  
  
  CONCLUSIONS
  This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine.
authors.affiliation: Center for Vaccinology, Ghent University, Ghent, Belgium.
authors.forename: Frederic
authors.initials: F
authors.lastname: Clement
authors.forename: Vincent
authors.initials: V
authors.lastname: Dewar
authors.forename: Eva
authors.initials: E
authors.lastname: Van Braeckel
authors.forename: Isabelle
authors.initials: I
authors.lastname: Desombere
authors.forename: Marianne
authors.initials: M
authors.lastname: Dewerchin
authors.forename: Christine
authors.initials: C
authors.lastname: Swysen
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Demoitié
authors.forename: Erik
authors.initials: E
authors.lastname: Jongert
authors.forename: Joe
authors.initials: J
authors.lastname: Cohen
authors.forename: Geert
authors.initials: G
authors.lastname: Leroux-Roels
authors.forename: Pierre
authors.initials: P
authors.lastname: Cambron
mesh_terms: D000328:Adult; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D057230:Limit of Detection; D017780:Malaria Vaccines / Q000276:immunology*; D016778:Malaria, Falciparum / Q000276:immunology / Q000517:prevention & control; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D018985:Epitopes, B-Lymphocyte; D007074:Immunoglobulin G; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041595:circumsporozoite protein, Protozoan