Kondoh Masatoshi, Fukada Kazutake, Shimada Takashi, Kitamura Yoshihisa, Yasueda Hiroshi, Enomoto Tadao
Central Research Laboratories, Nichinichi Pharmaceutical Corporation Ltd.
Arerugi. 2010 Feb;59(2):109-16.
In order to specifically quantify the two major Dermatophagoides spp. allergens, Der p 1 and Der f 1, separately, we tried to establish a highly sensitive enzyme-linked immunosorbent assay (ELISA).
Ninety-six-well ELISA plates were coated with mouse monoclonal antibodies specific against Der p 1 or Der f 1. Allergen samples were incubated in the wells for 2 hours at 37 degrees C. After washing with PBS-T, biotinylated rabbit anti-Der 1 polyclonal antibody was added to the wells. The allergens were detected using horse radish peroxidase-conjugated streptavidin, an enzyme substrate (TMB/H2O2) and a microplate reader.
The working range of both ELISA systems for Der p 1 and Der f 1 was 40-2500 pg/ml. The intra- and inter-assay coefficients of variation for reproducibility were 0.99-4.38% and 0.68-3.02%, respectively, in Der p 1 ELISA and 1.54-3.65% and 0.39-4.77%, respectively, in Der f 1 ELISA. Moreover, these ELISA systems showed that there was no cross-reactivity between Der p 1 and Der f 1 allergens.
These ELISA systems may be useful for measuring less than 1 ng/ml of major mite allergens in house dust samples, various pharmaceutical studies such as evaluation of an allergen-inactivating agent, and standardizing recombinant/natural Dermatophagoides spp. allergens.
为了分别特异性定量两种主要的粉尘螨属过敏原，即Der p 1和Der f 1，我们试图建立一种高度灵敏的酶联免疫吸附测定（ELISA）方法。
用针对Der p 1或Der f 1的小鼠单克隆抗体包被96孔ELISA板。将过敏原样品在孔中于37℃孵育2小时。用PBS-T洗涤后，向孔中加入生物素化的兔抗Der 1多克隆抗体。使用辣根过氧化物酶偶联的链霉亲和素、酶底物（TMB/H2O2）和酶标仪检测过敏原。
Der p 1和Der f 1的两种ELISA系统的工作范围均为40 - 2500 pg/ml。Der p 1 ELISA中，用于重复性的批内和批间变异系数分别为0.99 - 4.38%和0.68 - 3.02%；Der f 1 ELISA中，批内和批间变异系数分别为1.54 - 3.65%和0.39 - 4.77%。此外，这些ELISA系统表明Der p 1和Der f 1过敏原之间没有交叉反应。
这些ELISA系统可用于测量室内灰尘样品中低于1 ng/ml的主要螨类过敏原，可用于各种药物研究，如评估过敏原灭活剂，以及对重组/天然粉尘螨属过敏原进行标准化。
