title: The impact of structural integrity and route of administration on the antibody specificity against three cow's milk allergens - a study in Brown Norway rats.
abstract: BACKGROUND
  Characterisation of the specific antibody response, including the epitope binding pattern, is an essential task for understanding the molecular mechanisms of food allergy. Examination of antibody formation in a controlled environment requires animal models. The purpose of this study was to examine the amount and types of antibodies raised against three cow's milk allergens; β-lactoglobulin (BLG), α-lactalbumin (ALA) and β-casein upon oral or intraperitoneal (i.p.) administration. A special focus was given to the relative amount of antibodies raised against linear versus conformational epitopes.
  
  
  METHODS
  Specific antibodies were raised in Brown Norway (BN) rats. BN rats were dosed either (1) i.p. with the purified native cow's milk allergens or (2) orally with skimmed milk powder (SMP) alone or together with gluten, without the use of adjuvants. The allergens were denatured by reduction and alkylation, resulting in unfolding of the primary structure and a consequential loss of conformational epitopes. The specific IgG1 and IgE responses were analysed against both the native and denatured form of the three cow's milk allergens, thus allowing examination of the relative amount of linear versus conformational epitopes.
  
  
  RESULTS
  The inherent capacity to induce specific IgG1 and IgE antibodies were rather similar upon i.p. administration for the three cow's milk allergens, with BLG = ALA > β-casein. Larger differences were found between the allergens upon oral administration, with BLG > ALA > β-casein. Co-administration of SMP and gluten had a great impact on the specific antibody response, resulting in a significant reduced amount of antibodies. Together results indicated that most antibodies were raised against conformational epitopes irrespectively of the administration route, though the relative proportions between linear and conformational epitopes differed remarkably between the allergens.
  
  
  CONCLUSIONS
  This study showed that the three-dimensional (3D) structure has a significant impact on the antibodies raised for both systemic and orally administered allergens. A remarkable difference in the antibody binding patterns against linear and conformational epitope was seen between the allergens, indicating that the structural characteristics of proteins may heavily affect the induced antibody response.
authors.affiliation: Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
authors.forename: Jeanette Lund
authors.initials: JL
authors.lastname: Madsen
authors.affiliation: Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
authors.forename: Stine
authors.initials: S
authors.lastname: Kroghsbo
authors.affiliation: Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
authors.forename: Charlotte Bernhard
authors.initials: CB
authors.lastname: Madsen
authors.affiliation: NNF Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Irina
authors.initials: I
authors.lastname: Pozdnyakova
authors.affiliation: Department of Systems Biology, Enzyme and Protein Chemistry, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Vibeke
authors.initials: V
authors.lastname: Barkholt
authors.affiliation: Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark.
authors.forename: Katrine Lindholm
authors.initials: KL
authors.lastname: Bøgh
keywords: Animal model; Conformational epitopes; Cow’s milk allergens; Food allergy; IgE; Linear epitopes; Oral administration; i.p. immunisation