鱼类致病性豚鼠气单胞菌单克隆抗体-胶体金检测方法的建立

以福尔马林灭活的豚鼠气单胞菌按2.5×107个/只和5.0×107个/只分成两个剂量组免疫BALB/c小鼠,通过杂交瘤技术制备针对豚鼠气单胞菌的单克隆抗体(McAb),用间接ELISA法对所需的杂交瘤细胞株进行特异性筛选,获得了2株可分泌特异性McAb的杂交瘤细胞,并分别命名为3F3和2C9C3。经过鉴定,这两株McAb能够特异性的针对豚鼠气单胞菌,其抗体亚类分别为IgG1型和IgM型;腹水效价分别为10-6和10-5;相对亲和力较高;3F3针对豚鼠气单胞菌脂多糖表位,而2C9C3针对非脂多糖抗原位点。利用实验制备的McAb建立了以McAb为基础的双抗夹心法膜式超灵敏胶体金快速检测方法,所研制的豚鼠气单胞菌胶体金快速检测卡灵敏度好,特异性高,重复性好,检测时间快,操作简便,为水产上豚鼠气单胞菌的快速鉴定和诊断以及该菌流行的监测提供有力的工具。  

DEVELOPMENT OF A RAPID DETECTION METHOD ON MONOCLONAL ANTIBODIES CONJUGATING TO COLLOIDAL GOLD AGAINST PATHOGENIC AEROMONAS CA VIAE IN FISH

The purpose of this study was to prepare Aeromonas caviae hybridoma cell lines, and to establish the rapid, super sensitive detection method of 'double-antibody-sandwich' membrane colloidal gold basing on monoclonal anti-bodies that was used for the rapid diagnosis of Aeromonas caviae. In this study, BALB/c mice were immunized with Aeromonas caviae killed by formalin as the dosage of 2.5×10~7 cell per mouse and 5.0×10~7 cell per mouse respectively. The hybridoma cell lines which consistently secreted monoclonal antibody (McAb) against Aeromonas caviae were obtained through cell fusion. The specificity of the McAb was analysed by indirect ELISA. Then the subtypes were identified, the titer and affinity constant were measured, and its specificity was analyzed. Tests were made to identify the antigen epitope of the two McAbs by combined experimental site and indirect ELISA with LPS of Aeromonas caviae. The McAb conjugating to colloidal gold against Aeromonas caviae was established, and its specificity, sensitivity, re-peatability was estimated respectively. From over hundreds of positive hybridomas which secreted anti-Aeromonas caviae McAbs, two strains of hybridomas were screened out, and designated with 3F3 and 2C9C3. The subtypes of the McAb were IgG_1 and IgM. The titer of 3F3 and 2C9C3 McAb produced by ascites fluid were 1 : 10~6 and 1 : 10~5. The McAbs had high relative affinity. The two strains of monoclonal antibodies were targeted to different antigen epitope: 3F3 was targeted to the LPS ofAeromonas caviae, while 2C9C3 was targeted to the non-LPS sites of Aeromonas caviae. Basing on the McAbs, the rapid detection McAbs conjugating to colloidal gold against Aeromonas caviae was devel-oped to detect the thalli antigen of Aeromonas caviae specifically. Therefore, the results demonstrated that this method had high specificity, sensitivity and repeatability. It could serve as an effective detection measure for clinic, and as the method of the rapid identification for Aeromonas caviae in aquaculture and monitoring the epidemic of Aeromonas caviae.