Quantification of the thaumatin-like kiwi allergen by a monoclonal antibody-based ELISA.
Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.
Gavrović-Jankulović
Marija
M
Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia. mgavrov@chem.bg.ac.yu
Spasić
Milena
M
Cirković Velicković
Tanja
T
Stojanović
Marijana
M
Inić-Kanada
Aleksandra
A
Dimitrijević
Ljiljana
L
Lindner
Buko
B
Petersen
Arnd
A
Becker
Wolf-Meinhard
WM
Jankov
Ratko M
RM
D029042:Actinidia / Q000737:chemistry*; D000485:Allergens / Q000032:analysis* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D005638:Fruit / Q000737:chemistry*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology
D000485:Allergens; D000911:Antibodies, Monoclonal; D010936:Plant Extracts; D010940:Plant Proteins; C003427:thaumatin protein, plant