Isolation of Der pI, the Dermatophagoides pteronyssinus major mite allergen, from a crude mite culture extract, purification by ion-chromatography, and comparison between the material obtained and a cDNA-coded Der pI.
A high degree of purity is a prerequisite for an allergen preparation to be suitable for clinical diagnosis and therapy. A pure allergen can easily be obtained from a crude mite culture extract by using an immunosorbent prepared with highly specific monoclonal antibodies or from a cDNA-coded material. However, up to now none of these methods has been performed on a process scale. Here large-scale purification is defined as a process in which a crude Dermatophagoides pteronyssinus mite culture extract is essentially fractionated by acetone and ammonium sulphate precipitations followed by anion-exchange high-performance liquid chromatography. A high yield of a very pure Der pI allergen is obtained during the first isocratic run, as shown by sodium dodecylsulphate-polyacrylamide gel electrophoresis, capillary electrophoresis, chromatofocusing and a two site monoclonal antibody enzyme-linked immunosorbent assay. Microsequencing revealed that the 25-residue sequence obtained is entirely in agreement with the sequence derived from the cDNA of Der pI.
Dandeu
J P
JP
Unité d'Immuno-Allergie, Institut Pasteur, Paris, France.
Rabillon
J
J
Lux
M
M
David
B
B
Guillaume
J L
JL
Camoin
L
L
D000485:Allergens / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides; D002853:Chromatography, Liquid; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D007477:Ions; D008925:Mites / Q000276:immunology*; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet
D000485:Allergens; D039741:Antigens, Dermatophagoides; D007477:Ions; D004247:DNA