Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biotinylation in vivo
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L. Huang [1] ; H. Bao [2] ; S. Li [4] ; J. Zhang [3] ; L. Li [1] ; B. Zhang [1] ; Y. Yu [1] ; Y. Liu [1] ; H. Li [1]
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[1] Tianjin Medical University
Tianjin Medical University
China
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[2] Tianjin Nankai Hospital
Tianjin Nankai Hospital
China
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[3] Tianjin Children's Hospital
Tianjin Children's Hospital
China
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[4] Academy of Traditional Chinese Medicine Affiliated Hospital, Tianjin, China
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- Localización: Allergologia et immunopathologia: International journal for clinical and investigate allergology and clinical immunology, ISSN-e 1578-1267, ISSN 0301-0546, Vol. 48, Nº. 4, 2020, págs. 348-354
- Idioma: inglés
- Texto completo no disponible (Saber más ...)
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Resumen
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.
