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.

中文翻译：

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通过体内生物素化提高Gal d 6特异性IgE检测的灵敏度。

介绍和目标

这项研究旨在比较不同生物素化方法对过敏原特异性IgE检测性能特征的影响。

材料和方法

将Gal d 6基因克隆到pAN6 / pAC6载体中，得到rGal d 6-Bio / Bio-rGal d 6载体。融合蛋白在大肠杆菌AVB101中表达，并同时以位点特异性方式进行生物素化。通过PCR扩增Gal d 6基因，并将其克隆到pET-28a载体中，并转化到大肠杆菌BL21中，并通过Ni-NTA纯化，然后使用磺基-NHS-LC-生物素进行化学生物素化。检查了对蛋清过敏的28位患者对蛋黄的致敏性。开发了一种抗原捕获酶联免疫吸附测定法（AC-ELISA）以检测过敏原特异性IgE。

结果

使用不同的生物素结合模式制备rGal d 6，Bio-rGal d 6和rGal d 6以检测过敏原特异性IgE。rGal d 6-Bio（Kd = 0.6154）和Bio-rGal d 6（Kd = 0.6698）具有比rGal d 6（Kd = 28.93）明显更好的检测性能，而rGal d 6-Bio在以下条件下具有更好的检测性能。小剂量血清样品。

结论

rGal d 6-Bio提高了检测过敏原特异性IgE的灵敏度。