摘要
目的研制粉尘螨Ⅱ组变应原Der f 2单克隆抗体(Monoclonal Antibody,McAb),并建立双单抗夹心ELISA检测方法 ,测定标准化粉尘螨变应原脱敏疫苗中Der f 2的含量。方法应用基因工程方法获得粉尘螨变应原Der f2重组蛋白并通过亲和层析纯化,再免疫小鼠,利用间接ELISA筛选获得McAb杂交瘤细胞株并纯化、鉴定抗体的特异性;用一株单抗包被酶标板,同时用生物素标记另一株单抗,从而建立双单抗体夹心ELISA法;并用此法测定粉尘螨变应原脱敏疫苗中Der f2的含量。结果成功地获得了一株单抗3B12与屋尘螨抗原具有交叉反应,另外一株单抗3G7与屋尘螨抗原没有交叉反应,并建立了双单抗夹心ELISA方法测定Der f2的含量,其方法的检测限为0.5ng/mL,并在6.25ng/mL-300ng/mL范围内线性良好;标准化粉尘螨变应原脱敏疫苗中Der f2的含量为100ng/10000BAU。结论成功制备了高效价抗Der f 2单抗,并建立了双抗体夹心ELISA检测方法。该方法具有很高的灵敏度,可以测定标准化粉尘螨变应原脱敏疫苗中主要变应原Der f 2含量,为临床应用奠定基础。
The designed monoclonal antibodies（McAb）against rDer f 2 and sandwich ELISA were performed to detect the concentration of major allergen Der f 2 in Dermatophagoides farinae vaccine products.The rDer f 2 was expressed in E.coli and purified by affinity chromatograph.Balb/c mice were immunized with rDer f 2,and the indirect ELISA was used to screen the positive clones of hybridoma cell.With a McAb coated ELISA plate and a biotin labeled McAb,sandwich ELISA was performed to assay the concentration of major allergen Der f 2 in the vaccine products.Two monoclonal antibodies against Der f 2 were made and McAb 3G7 did not react with the extract from Dermatophagoides Pteronyssinus.In sandwich ELISA,the standard curve was linear and the rDer f 2 concentrations were from 6.25 ng/mL to 300 ng/mL with a low detection limit of about 0.5 ng/mL for rDer f 2.The concentration of major allergen Der f 2 in standard Dermatophagoides farinae vaccine products was about 100ng per 10000BAU.In conclusion,the two McAbs against Der f 2 were made successfully and the sandwich ELISA kit could detect the concentration of major allergen Der f 2 in the vaccine products.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2010年第7期663-667,共5页
Chinese Journal of Zoonoses
基金
国家"863"专题(No.2006AA02A231)
粤港关键领域重点突破项目(No.20054982207)联合资助
