水生生物学报
水生生物學報
수생생물학보
ACTA HYDROBIOLOGICA SINICA
2010年
3期
509-516
,共8页
黄艺丹%汪开毓%郑建%黄锦炉
黃藝丹%汪開毓%鄭建%黃錦爐
황예단%왕개육%정건%황금로
豚鼠气单胞菌%单克隆抗体%生物特性%胶体金
豚鼠氣單胞菌%單剋隆抗體%生物特性%膠體金
돈서기단포균%단극륭항체%생물특성%효체금
Aeromonas caviae%Monoclonal antibody%Biological Characteristic%Colloidal gold
以福尔马林灭活的豚鼠气单胞菌按2.5×10~7个/只和5.0×10~7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IgG_1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.
以福爾馬林滅活的豚鼠氣單胞菌按2.5×10~7箇/隻和5.0×10~7箇/隻分成兩箇劑量組免疫BALB/c小鼠,通過雜交瘤技術製備針對豚鼠氣單胞菌的單剋隆抗體(McAb),用間接ELISA法對所需的雜交瘤細胞株進行特異性篩選,穫得瞭2株可分泌特異性McAb的雜交瘤細胞,併分彆命名為3F3和2C9C3.經過鑒定,這兩株McAb能夠特異性的針對豚鼠氣單胞菌,其抗體亞類分彆為IgG_1型和IgM型;腹水效價分彆為10~(-6)和10~(-5);相對親和力較高:3F3針對豚鼠氣單胞菌脂多糖錶位,而2C9C3針對非脂多糖抗原位點.利用實驗製備的McAb建立瞭以McAb為基礎的雙抗夾心法膜式超靈敏膠體金快速檢測方法,所研製的豚鼠氣單胞菌膠體金快速檢測卡靈敏度好,特異性高,重複性好,檢測時間快,操作簡便,為水產上豚鼠氣單胞菌的快速鑒定和診斷以及該菌流行的鑑測提供有力的工具.
이복이마림멸활적돈서기단포균안2.5×10~7개/지화5.0×10~7개/지분성량개제량조면역BALB/c소서,통과잡교류기술제비침대돈서기단포균적단극륭항체(McAb),용간접ELISA법대소수적잡교류세포주진행특이성사선,획득료2주가분비특이성McAb적잡교류세포,병분별명명위3F3화2C9C3.경과감정,저량주McAb능구특이성적침대돈서기단포균,기항체아류분별위IgG_1형화IgM형;복수효개분별위10~(-6)화10~(-5);상대친화력교고:3F3침대돈서기단포균지다당표위,이2C9C3침대비지다당항원위점.이용실험제비적McAb건립료이McAb위기출적쌍항협심법막식초령민효체금쾌속검측방법,소연제적돈서기단포균효체금쾌속검측잡령민도호,특이성고,중복성호,검측시간쾌,조작간편,위수산상돈서기단포균적쾌속감정화진단이급해균류행적감측제공유력적공구.
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.