Update on optimized purification and characterization of natural milk allergens.
Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.
Blanc
Fany
F
INRA Food allergy Laboratory, IBiTecS-SPI, CEA de Saclay, Gif sur Yvette, France.
Bernard
Hervé
H
Alessandri
Stefano
S
Bublin
Merima
M
Paty
Evelyne
E
Leung
Sandrine Ah
SA
Patient
Karine Adel
KA
Wal
Jean-Michel
JM
D000485:Allergens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002417:Cattle; D015550:Cell Degranulation; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006041:Goats; D006801:Humans; D007124:Immunoenzyme Techniques; D007768:Lactalbumin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D002364:Caseins; D007782:Lactoglobulins; D007768:Lactalbumin