CAJ | 학술논문

以小麦主要过敏原Glb-1蛋白为免疫原免疫BALB/c小鼠，取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用细胞融合和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞株，用杂交瘤细胞株诱生小鼠腹水并用蛋白A亲和层析法纯化抗体后检测。采用间接ELISA法鉴定该单克隆抗体的IgG亚型；通过间接ELISA鉴定该单克隆抗体的特性和交叉性。利用双单抗夹心ELISA法检测单抗的抗原表位特异性。结果表明：共获得4株可稳定分泌小鼠抗小麦主要过敏原 Glb -1蛋白的单克隆抗体，分别命名为1 C4、4H5、1A9、4F5，经检测其Ig亚型均为IgG1，且4株单抗效价均在10-9以上。ELISA结果分析表明该4株单抗均能特异性识别小麦主要过敏原Glb-1蛋白且和其他常见食物无交叉反应性。将4株单抗两两配对进行ELISA实验，结果发现1 C4与4H5可能有不同的抗原表位，以此建立的双抗夹心ELISA系统可以检测小麦Glb1-G3蛋白。实验成功制备了鼠抗小麦主要过敏原Glb-1蛋白抗原的单克隆抗体，并且建立了双单抗夹心ELISA检测系统，为小麦主要过敏原蛋白的检测奠定了基础。
이소맥주요과민원Glb-1단백위면역원면역BALB/c소서，취면역소서비세포여소서골수류NS-1세포융합。채용세포융합화유한희석법상결합적방법쾌속사선획득은정분비적특이성잡교류세포주，용잡교류세포주유생소서복수병용단백A친화층석법순화항체후검측。채용간접ELISA법감정해단극륭항체적IgG아형；통과간접ELISA감정해단극륭항체적특성화교차성。이용쌍단항협심ELISA법검측단항적항원표위특이성。결과표명：공획득4주가은정분비소서항소맥주요과민원 Glb -1단백적단극륭항체，분별명명위1 C4、4H5、1A9、4F5，경검측기Ig아형균위IgG1，차4주단항효개균재10-9이상。ELISA결과분석표명해4주단항균능특이성식별소맥주요과민원Glb-1단백차화기타상견식물무교차반응성。장4주단항량량배대진행ELISA실험，결과발현1 C4여4H5가능유불동적항원표위，이차건립적쌍항협심ELISA계통가이검측소맥Glb1-G3단백。실험성공제비료서항소맥주요과민원Glb-1단백항원적단극륭항체，병차건립료쌍단항협심ELISA검측계통，위소맥주요과민원단백적검측전정료기출。
BALB/c mice were immunized with G1 b -1 protein,the main allergen from wheat.The splenocytes of the immunized mice were fused with NS-1 myeloma cells.Cell fusion and limited dilution assay were used to screen hybridoma with stable secretion character and mice ascites were induced by the selected bybridoma.Monoclonal antibodies (McABs)were purified using affinity chromatography and further tested by their specificity,subtype,titers and cross-reactivity.The antigen epitope specificity of the McABs was examined by ELISA method.Four cell lines secreting McABs against wheat G1 b-1 pro-tein were obtained,named 1C4,4H5,1A9 and 4F5,respectively.The four McABs belong to IgG1subtype and their titer was above 9 -10 .Moreover,all the four McABs specially recognized wheat G1 b-1 protein and had no cross-reaction with other familiar foods.In order to establish a double-antibody sandwich ELISA system to detect wheat GIB1 proteins,we paired off the four monoclonal antibodies for ELISA assay and found 1 C4 and 4h5 might have different epitopes.Four mice anti-wheat G1 b-1 protein McABs were prepared successfully and a double-antibody sandwich ELISA method was set up in this work,which may facilitate other wheat allergen protein detection.