Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biotinylation in vivo

ABSTRACT

INTRODUCTION AND OBJECTIVES: This study aimed to compare the effects of different biotinylation methods on the performance characteristics of allergen-specific IgE detection. MATERIALS AND METHODS: The Gal d 6 gene was cloned into the pAN6/pAC6 vector, resulting in rGal d 6-Bio/Bio-rGal d 6 vector. The fusion protein was expressed in Escherichia coli AVB101 and simultaneously biotinylated in a site-specific manner. The Gal d 6 gene was amplified via PCR and cloned into the pET-28a vector and transformed into E. coli BL21 and purified via Ni-NTA, followed by chemical biotinylation using Sulfo-NHS-LC-Biotin. Twenty-eight patients allergic to hen's egg white were examined for sensitization against egg yolk. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed to detect allergen-specific IgE. RESULTS: rGal d 6, Bio-rGal d 6, and rGal d 6 were prepared using different biotin binding modes to detect allergen-specific IgE. rGal d 6-Bio (Kd = 0.6154) and Bio-rGal d 6 (Kd = 0.6698) had a markedly better detection performance than rGal d 6 (Kd = 28.93), and the rGal d 6-Bio had a better detection performance in small-volume serum samples. CONCLUSIONS: rGal d 6-Bio improved the sensitivity for the detection of allergen-specific IgE

RESUMEN

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