TY - JOUR
T1 - Optimized techniques for the extraction of grape allergens appropriate for in vivo and in vitro testing and diagnosis
AU - Vassilopoulou, Emilia V.
AU - Zuidmeer, Laurian
AU - Akkerdaas, Jaap
AU - Rigby, Neil
AU - Javier Moreno, F.
AU - Papadopoulos, Nikolaos G.
AU - Saxoni-Papageorgiou, Photini
AU - Mills, Clare
AU - Van Ree, Ronald
PY - 2007/3
Y1 - 2007/3
N2 - Standardized allergen extracts are needed for diagnosis and therapy purposes. For grapes, standardization is hampered by low protein and high tannin and pectin concentrations. The aim of the current study was to develop an optimized method for the extraction of grape proteins and possibly extend this to other fruits. Several existing or modified extraction methods were compared by means of pro-tein concentration determination, SDS-PAGE, immunoblotting and radioallergosorbent test (RAST). An optimized extraction protocol was obtained in which we combined a high concentration of plant tissue, a concentrated, enriched and neutral buffer able to remove sugars and keep proteins soluble and a bivalent buffer for pectin removal. Both the quantitative (protein concentration) and qualitative parameters (SDS-PAGE protein patterns and IgE reactivity) were compared to standard protocols and commercial extracts used as diagnostic tools in the clinical practice. This method proved to be the most efficient mainly compared to the standard Björksten protocol in extracting the low molecular weight proteins, including the major grape allergen (lipid transfer protein, Vit v 1). It proved to be an easy, low cost and reproducible method proposed to prepare grape extracts that could replace the commercially available ones, used for diagnosis and possibly extend the method to other fruits especially in extracting LTPs.
AB - Standardized allergen extracts are needed for diagnosis and therapy purposes. For grapes, standardization is hampered by low protein and high tannin and pectin concentrations. The aim of the current study was to develop an optimized method for the extraction of grape proteins and possibly extend this to other fruits. Several existing or modified extraction methods were compared by means of pro-tein concentration determination, SDS-PAGE, immunoblotting and radioallergosorbent test (RAST). An optimized extraction protocol was obtained in which we combined a high concentration of plant tissue, a concentrated, enriched and neutral buffer able to remove sugars and keep proteins soluble and a bivalent buffer for pectin removal. Both the quantitative (protein concentration) and qualitative parameters (SDS-PAGE protein patterns and IgE reactivity) were compared to standard protocols and commercial extracts used as diagnostic tools in the clinical practice. This method proved to be the most efficient mainly compared to the standard Björksten protocol in extracting the low molecular weight proteins, including the major grape allergen (lipid transfer protein, Vit v 1). It proved to be an easy, low cost and reproducible method proposed to prepare grape extracts that could replace the commercially available ones, used for diagnosis and possibly extend the method to other fruits especially in extracting LTPs.
KW - Allergen
KW - Extract
KW - Grape
KW - Lipid transfer protein
KW - Pectins
UR - http://www.scopus.com/inward/record.url?scp=34250171245&partnerID=8YFLogxK
U2 - 10.1002/mnfr.200600194
DO - 10.1002/mnfr.200600194
M3 - Article
C2 - 17309118
AN - SCOPUS:34250171245
SN - 1613-4125
VL - 51
SP - 360
EP - 366
JO - Molecular Nutrition and Food Research
JF - Molecular Nutrition and Food Research
IS - 3
ER -