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Inicio Allergologia et Immunopathologia Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biot...
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Vol. 48. Issue 4.
Pages 348-354 (July - August 2020)
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Vol. 48. Issue 4.
Pages 348-354 (July - August 2020)
Original Article
DOI: 10.1016/j.aller.2020.02.002
Improvement of the sensitivity of the detection of Gal d 6-specific IgE via biotinylation in vivo
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L. Huanga,1, H. Baob,1, S. Lic, J. Zhangd, L. Lia,**, B. Zhanga, Y. Yua, Y. Liua,**, H. Lia,
a School of Medical Laboratory, Tianjin Medical University, Tianjin, China
b The Integrative Medical Diagnosis Laboratory, Tianjin Nankai Hospital, China
c Academy of Traditional Chinese Medicine Affiliated Hospital, Tianjin, China
d Department of Medical Laboratory, Tianjin Children's Hospital, Tianjin, China
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Table 1. Clinical characteristics of the study subjects.
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.

Keywords:
Biotinylation
Gal d 6
In vivo
In vitro
Specific IgE

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