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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Clinical and Translational Allergy 1/2014

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

Clinical and Translational Allergy > Ausgabe 1/2014
Jeanette Lund Madsen, Stine Kroghsbo, Charlotte Bernhard Madsen, Irina Pozdnyakova, Vibeke Barkholt, Katrine Lindholm Bøgh
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​2045-7022-4-25) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JLM participated in the denaturation of proteins, participated in the conduction of the native-gel and performed all ELISAs. JLM performed statistical analyses, contributed to the discussion of the results, wrote a student report on the presented results and reviewed the manuscript. SK contributed to the design and carried out the oral animal study as well as the β-casein i.p. animal study. SK reviewed the manuscript. CBM contributed to the design of all animal studies, discussed the results and reviewed the manuscript. IP performed CD spectra, the analyses and graphs of these. IP participated in writing of the manuscript and reviewed the paper. VB designed the denaturation protocol and reviewed the manuscript. KLB drew up the study design. KLB participated in the denaturation of proteins and conduction of native-gel. KLB contributed to the design and carried out the BLG and ALA i.p. animal study. KLB participated in the discussion of the results and converted the student report to a paper manuscript. All authors read and approved the final manuscript.



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.


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.


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.


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.
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