通过双位点酶联免疫吸附测定法对螨虫和粉尘提取物中的Blo t 5进行定量分析。

Quantification of Blo t 5 in mite and dust extracts by two-site ELISA.

作者信息

Yi F C, Lee B W, Cheong N, Chua K Y

机构信息

Department of Paediatrics, National University of Singapore, Singapore.

出版信息

Allergy. 2005 Jan;60(1):108-12. doi: 10.1111/j.1398-9995.2004.00597.x.

DOI:10.1111/j.1398-9995.2004.00597.x

PMID:15575940

Abstract

BACKGROUND

Blomia tropicalis is an important mite species in the tropical and sub-tropical regions of the world. Blo t 5 is the major allergen with up to 70% sensitization rates in B. tropicalis allergic populations.

METHODS

Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 5 gene with in vivo electroporation. Blo t 5 monoclonal antibodies were generated using methylcellulose-based hybridoma kit. Monoclonal antibody (mAb) 4A7 was characterized by two-dimensional electrophoresis immunoblotting. A specific quantitative two-site enzyme-linked immunosorbent assay (ELISA) was developed with mAb 4A7 and guinea pigs Blo t 5 polyclonal antibody as capture and detection antibodies, respectively. This system was tested with Blo t 5 in crude extracts and dust samples.

RESULTS

A high-affinity mAb 4A7 recognizing several isoforms of Blo t 5 has been generated. Monoclonal antibody 4A7 is useful for immunoblotting and two-site ELISA. The two-site ELISA developed has a high sensitivity, with a detection limit of 10 pg/ml. The assay is species-specific and recognized the same epitopes on both native and recombinant Blo t 5. The assay developed is able to detect Blo t 5 in commercial diagnostic and therapeutic B. tropicalis extract. Blo t 5 quantification in dust samples showed that Blo t 5 is present in a high quantity in Singapore dust.

CONCLUSIONS

A highly sensitive and specific two-site ELISA has been developed. The assay system developed is useful for the quantification of Blo t 5 in mite and environmental dust extracts.

摘要

背景

热带无爪螨是世界热带和亚热带地区的一种重要螨类。Blo t 5是主要过敏原，在热带无爪螨过敏人群中的致敏率高达70%。

方法

用编码Blo t 5基因的裸质粒DNA通过体内电穿孔法对小鼠进行肌肉免疫。使用基于甲基纤维素的杂交瘤试剂盒制备Blo t 5单克隆抗体。通过二维电泳免疫印迹法对单克隆抗体（mAb）4A7进行表征。以mAb 4A7和豚鼠Blo t 5多克隆抗体分别作为捕获抗体和检测抗体，建立了一种特异性定量双位点酶联免疫吸附测定（ELISA）法。该系统用粗提物和粉尘样本中的Blo t 5进行测试。

结果

已产生一种识别Blo t 5多种异构体的高亲和力mAb 4A7。单克隆抗体4A7可用于免疫印迹和双位点ELISA。所建立的双位点ELISA具有高灵敏度，检测限为10 pg/ml。该测定具有物种特异性，能识别天然和重组Blo t 5上相同的表位。所建立的测定法能够检测商业诊断和治疗用热带无爪螨提取物中的Blo t 5。粉尘样本中Blo t 5的定量分析表明，新加坡粉尘中Blo t 5含量很高。