title: IgE antibodies to recombinant forms of Fel d I: dichotomy between fluid-phase and solid-phase binding studies.
abstract: BACKGROUND
  The major cat allergen Fel d I consists of two polypeptide chains linked by disulfide bonds, each of which has been expressed in bacteria. To investigate the antigenic structure of Fel d I, antibody binding to the native molecule and to each recombinant chain were compared.
  
  
  METHODS
  Polyclonal human IgE and IgG antibodies and monoclonal antibodies (mAbs) to Fel d I were compared for binding to Fel d I, chain 1, or chain 2 by fluid-phase inhibition radioimmunoassay, RAST, and immunoabsorption.
  
  
  RESULTS
  In the fluid-phase assay, neither recombinant chain significantly inhibited the binding of antibody to native Fel d I at concentrations of up to 10 micrograms/ml. Partial inhibition was observed when chain 1 was used, which inhibited the binding of two mAbs by 40% and 75%. In contrast, when the solid-phase RAST assay was used, IgE antibodies bound both chains with high specificity, and there was a good quantitative correlation between IgE antibody binding to Fel d I and both chain 1 (r = 0.58, p < 0.01) and chain 2 (r = 0.47, p < 0.01). Up to 70% of IgG or IgE anti-Fel d I antibodies could be absorbed by either chain 1 or chain 2, and both chains in combination produced similar absorption values in response to native Fel d I. Four mAbs were fully absorbed by chain 1, but not chain 2, and three mAbs were not absorbed by either chain.
  
  
  CONCLUSIONS
  The results demonstrate a dichotomy between antibody binding to recombinant Fel d I chains, which may be explained by confirmational differences between the chains in the fluid phase or on solid supports. The results also suggest that chain 1 is an important site for mAb-defined B-cell epitopes on Fel d I.
authors.affiliation: Department of Medicine, University of Virginia, Charlottesville 22908, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Slunt
authors.forename: B L
authors.initials: BL
authors.lastname: Rogers
authors.forename: M D
authors.initials: MD
authors.lastname: Chapman
mesh_terms: D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007163:Immunosorbent Techniques; D011852:Radioallergosorbent Test; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C059296:Fel d 1 protein, Felis domesticus