[Show abstract][Hide abstract] ABSTRACT: Electrophoretic separation together with immunoblotting as well as EAST-inhibition were used to investigate the importance of IgE cross reactivity between the four most important house dust mites, Dermatophagoides pteronyyssinus, D. farinae, D. microceras ans Euroglyphus maynei. Cross inhibition experiments achieved inhibition values in excess of 50%. In addition the allergen profiles in immunoblots were qualitatively comparable. The high cross-reactivity between the mite species as well as the good agreement between in vitro and in vivo diagnostic data suggest allergen specific immunotherapy with one of the Dermatophagoides mite species would be sufficient.
[Show abstract][Hide abstract] ABSTRACT: By EAST, SDS-PAGE, Westernblot and especially EAST inhibition we could show that there is a high degree of crossreactivity between short-, giant-, western and false ragweed. Nevertheless, we recommend that EAST measurements should be made with short ragweed allergen discs. This covers the most sensitivities in sera of ragweed-allergic patients. Short ragweed is the most common ragweed especially in Europe. Although we could not prove crossreactivity between ragweed and mugwort, we recommend that sera of mugwort-allergic patients should also be investigated using ragweed allergen disks. In 18.2% of mugwort-allergic patients we could also measure class >1 using ragweed allergen discs.
[Show abstract][Hide abstract] ABSTRACT: A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.
No preview · Article · Jul 2006 · Protein Expression and Purification