Ramos J D A, Teo A S M, Lee B W, Cheong N, Chua K Y
Department of Paediatrics, National University of Singapore, Singapore.
Allergy. 2004 May;59(5):539-47. doi: 10.1046/j.1398-9995.2003.00409.x.
Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production.
To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs).
The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA).
Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization - time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11.
A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.
Blo t 11是来自热带血厉螨的一种高分子量变应原，具有显著的免疫球蛋白（Ig）E结合频率。天然和重组的Blo t 11易降解，变应原的分离和表达存在问题，因此获得足够量的纯化Blo t 11用于抗体生产受到限制。基于DNA的免疫是一种有吸引力的替代策略，可绕过抗原纯化来生产抗体。
使用基于DNA的免疫方案生产和鉴定Blo t 11单克隆抗体（mAb）。
将编码Blo t 11的2625 bp cDNA克隆到哺乳动物表达载体中，通过电穿孔法进行肌肉注射免疫小鼠。使用基于甲基纤维素的杂交瘤克隆试剂盒产生针对Blo t 11的单克隆抗体。这些单克隆抗体用于天然Blo t 11的分离和夹心酶联免疫吸附测定（ELISA）的开发。
产生并鉴定了六种识别天然和重组Blo t 11的单克隆抗体。从Bt提取物中亲和纯化天然Blo t 11，并通过基质辅助激光解吸/电离-飞行时间质谱法确认其身份。天然Blo t 11与67%的螨过敏血清显示出IgE反应性。开发的双位点ELISA显示对Blo t 11的检测限为100 pg/ml。
基于DNA的免疫方案成功用于产生具有位于整个分子的一系列不同表位的Blo t 11单克隆抗体，它们可用于免疫亲和纯化和免疫测定。
