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

L. Huang, H. Bao, S. Li, J. Zhang, L. Li, B. Zhang, Y. Yu, Y. Liu, H. Li

• 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.