Identification of the major Ascaris allergen and its purification to homogeneity by high-performance liquid chromatography.
The major allergen from the body fluid of adult Ascaris suum (ABF) has been identified and purified to homogeneity using gel permeation high-performance liquid chromatography (HPLC). The purity of the Ascaris body fluid allergen (ABA-1) was confirmed by HPLC and SDS-PAGE. ABA-1 appears to be a 25-kDa dimer in its native form, which runs as a 10-kDa monomer on SDS-PAGE under reducing conditions. The allergenicity of the HPLC-purified protein was confirmed using isolated in vivo-sensitised mast cells from infected rats in an in vitro histamine release assay. ABA-1 is responsible for less than 80% of the allergenicity of ABF in this sensitive and specific system. Amino acid composition analysis and N-terminal amino acid sequencing were performed on ABA-1. Comparisons are made between ABA-1 and some of the heterogeneous Ascaris allergen preparations previously published. It is suggested that Asc-1 and allergen A both contain ABA-1 in large quantities and that discrepancies in the literature result from contaminating proteins in these preparations and technical differences in characterization of the predominant molecules present in the preparations. Compositional data suggests that the ABA-1 monomer is a molecule of 94 amino acids (based on a molecular weight estimate of 10 kDa) with a composition resembling that previously published for allergen A. The first 10 amino acids are identical to those of a protein affinity purified from the body fluid of Ascaris suum at the Wellcome Laboratories for Experimental Parasitology (WLEP-14K). The similarity between ABA-1 and WLEP-14k is also apparent on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular Biology of Infectious Diseases Group, Faculty of Medicine, University of Calgary, Alberta, Canada.
A M
AM
McGibbon
J F
JF
Christie
M W
MW
Kennedy
T D
TD
Lee
D000485:Allergens / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001200:Ascaris / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006636:Histamine Release; D008407:Mast Cells / Q000378:metabolism; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
D000485:Allergens; D000596:Amino Acids