Determinations of Par j 1 by a competitive enzyme immunoassay using human specific IgE and IgG. Validation by skin prick testing.
Par j 1 is the major allergen of Parietaria judaica. The objectives of this study were the following: 1) to purify Par j 1; 2) to develop an enzyme immunoassay based on the bivalent properties of specific IgE and IgG to determine the Par j 1 content in several batches of P. judaica extracts; and, 3) to study the contribution of Par j 1 to the total allergenicity and antigenicity of P. judaica extracts. P. judaica pollen was extracted and subjected to hydrophobic interaction and gel filtration chromatography for the purification of Par j 1. Inhibition enzyme immunoassays, SDS-PAGE and immunoblotting were used to characterize the allergen content. The in vivo biological potency of the extracts was estimated by skin prick testing 26 P. judaica clinically sensitive patients. The new enzyme immunoassay showed a high degree of specificity and sensitivity, detecting from 2 to 100 ng Par j 1/ml. The range of Par j 1 content in nine batches ranged from 23% to 78% of the total protein in the extracts. The Par j 1 content showed a significant correlation with the allergenic potency of these extracts evaluated by specific IgE inhibition and skin prick testing; the correlation with the specific IgG inhibition capacity was not significant. Purified Par j 1 shows great specific IgE and IgG binding capacity; its content can be determined using this newly developed enzyme immunoassay. Par j 1 levels exhibit a significant correlation with the biological potency of the extracts. This method allows the detection of Par j 1 isoforms.
C.B.F. LETI, S.A. Research & Development Laboratories, Tres Cantos, Madrid, Spain.
L
L
Boluda
M
M
Casanovas
J L
JL
Prieto
E
E
Fernández-Caldas
D000485:Allergens / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000032:analysis / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012882:Skin Tests
D000485:Allergens; D007074:Immunoglobulin G; C083860:Par j I protein, Parietaria judaica; D010936:Plant Extracts; D010940:Plant Proteins; D007073:Immunoglobulin E