Investigation of allergen proteins in five tomato cultivars

Investigation of putative allergens from tomato berries is challenging as differences between human serum IgE specificity and reactivity as well as the non-specific binding of the primary and secondary antibodies often cause difficulties. In this study five tomato cultivars were investigated to evaluate their potential allergenicity in Hungarian tomato sensitive patients. The major allergens proved to be low molecular weight proteins, but several previously described allergens could be identified as well using IgE-Western blotting. IgE binding to cross-reactive carbohydrate determinants (CCDs) was ruled out through the use of CCD inhibitor, but non-specific binding of the secondary antibody remained an issue. IgE binding activity of a purified, immunoblot positive protein (band at 40 kDa), and non-specific binding of the secondary antibody to the same protein, was demonstrated with an Optical Waveguide Lightmode Spectroscopy (OWLS) based immunosensor. LC-ESI-MS/MS analysis showed this protein is an as-yet undescribed vicilin-type putative allergen

Study of the effect of instant controlled pressure drop (DIC) treatment on IgE-reactive legume-protein patterns by electrophoresis and immunoblot

Détente Instantanée Contrôlée (DIC) technology was used to reduce immunoglobulin E (IgE) reactivity of legume proteins. Soybean, roasted peanut, chickpea and lentil seeds were treated at three or six bars for 60 or 180s. The effect of this treatment on the IgE-binding pattern of the legume proteins - separated by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis - was monitored by five individual paediatric legume allergic and - two individual negative control human sera. A highly cross-reactive legume positive serum was selected for the two-dimensional electrophoreses immunoblots to compare the IgE reactive protein patterns, before and after the DIC treatment. The number of the identified IgE reactive spots was highly reduced for soybean (0/7) and chickpea (2/7), and moderately reduced for lentil (4/7) when the seeds were treated at a higher pressure (6 bar) and for a longer time (180 s), but this treatment was not effective for roasted peanut (6/8) where the intensity of the IgE reactive resistant spots were even stronger. © 2013 Taylor & Francis

Application of zinc hydroxide in the purification of bean alpha-amylase inhibitor

A novel step in bean (Phaseolus vulgaris) α-amylase inhibitor (AAI) purification, based on the application of an inorganic adsorbent, zinc hydroxide, was developed. The new method was substantially faster than existing protocols. Up to 98% of bean seed proteins were bound to the white precipitate in the range of 1–4% (w/v) zinc hydroxide, while the amount of bound bean AAI was far less in the range of 1–2%. The AAI-enriched fraction, unbound by zinc hydroxide, was further purified by DEAE-(diethylaminoethyl)chromatography and gel filtration. It was found that zinc hydroxide binds the majority of soluble proteins of bean, while it leaves α-amylase inhibitor in solution. The binding of proteins to zinc-hydroxide occurs in a short time and the change caused in the buffer composition is insignificant, thus it may open new approaches in purification of other proteins, too