中华临床感染病杂志
中華臨床感染病雜誌
중화림상감염병잡지
CHINESE JOURNAL OF CLINICAL INFECTIOUS DISEASES
2013年
1期
23-26
,共4页
冷建杭%寿成珉%吴志刚%卓广超%王克义%沈俊娅%黄晟
冷建杭%壽成珉%吳誌剛%卓廣超%王剋義%瀋俊婭%黃晟
랭건항%수성민%오지강%탁엄초%왕극의%침준아%황성
分枝杆菌,结核%培养滤液蛋白10%抗原肽%抗体,单克隆%免疫学技术
分枝桿菌,結覈%培養濾液蛋白10%抗原肽%抗體,單剋隆%免疫學技術
분지간균,결핵%배양려액단백10%항원태%항체,단극륭%면역학기술
Mycobacterium tuberculosis%Culture filtrate protein 10%Antigenic peptide%Antibodies,monoclonal%Immunologic techniques
目的 制备抗结核分枝杆菌培养滤液蛋白10(CFP-10)特定抗原肽的单克隆抗体,为建立结核病临床早期诊断的免疫学方法奠定物质基础.方法 设计并合成结核分枝杆菌CFP-10第53 ～ 66位氨基酸(14肽AAVVRFQEAANKQK)作为特定的抗原肽序列,经免疫BALB/C小鼠,取其脾细胞和小鼠骨髓瘤细胞SP2/0融合,经酶联免疫吸附试验(ELISA)检测、多次有限稀释亚克隆建立能稳定分泌抗结核分枝杆菌特异抗原肽单克隆抗体的杂交瘤细胞系,纯化该单克隆抗体,进行免疫球蛋白亚型分析,并鉴定分析该单克隆抗体在蛋白质免疫印迹法(Western blotting)、免疫沉淀法和ELISA检测中的应用效果.共检测结核分枝杆菌培养上清样本38份,非结核分枝杆菌培养上清样本20份,结核性胸水样本32份,非结核性胸水样本24份以及健康对照血清样本20份.结果 单克隆抗体免疫球蛋白亚型分析显示该杂交瘤细胞系产生的抗体类型为IgG1和κ型.该抗结核分枝杆菌CFP-10特异性单克隆抗体可成功用于Western blotting和免疫沉淀法分析.ELISA定量检测显示,该特异性抗体检测结核分枝杆菌的敏感性为78.6％ (55/70),特异性为92.2％ (59/64).结论 CFP-10特定抗原肽单克隆抗体用于结核分枝杆菌检测有较高的敏感性和特异性,可用于结核病的早期诊断.
目的 製備抗結覈分枝桿菌培養濾液蛋白10(CFP-10)特定抗原肽的單剋隆抗體,為建立結覈病臨床早期診斷的免疫學方法奠定物質基礎.方法 設計併閤成結覈分枝桿菌CFP-10第53 ～ 66位氨基痠(14肽AAVVRFQEAANKQK)作為特定的抗原肽序列,經免疫BALB/C小鼠,取其脾細胞和小鼠骨髓瘤細胞SP2/0融閤,經酶聯免疫吸附試驗(ELISA)檢測、多次有限稀釋亞剋隆建立能穩定分泌抗結覈分枝桿菌特異抗原肽單剋隆抗體的雜交瘤細胞繫,純化該單剋隆抗體,進行免疫毬蛋白亞型分析,併鑒定分析該單剋隆抗體在蛋白質免疫印跡法(Western blotting)、免疫沉澱法和ELISA檢測中的應用效果.共檢測結覈分枝桿菌培養上清樣本38份,非結覈分枝桿菌培養上清樣本20份,結覈性胸水樣本32份,非結覈性胸水樣本24份以及健康對照血清樣本20份.結果 單剋隆抗體免疫毬蛋白亞型分析顯示該雜交瘤細胞繫產生的抗體類型為IgG1和κ型.該抗結覈分枝桿菌CFP-10特異性單剋隆抗體可成功用于Western blotting和免疫沉澱法分析.ELISA定量檢測顯示,該特異性抗體檢測結覈分枝桿菌的敏感性為78.6％ (55/70),特異性為92.2％ (59/64).結論 CFP-10特定抗原肽單剋隆抗體用于結覈分枝桿菌檢測有較高的敏感性和特異性,可用于結覈病的早期診斷.
목적 제비항결핵분지간균배양려액단백10(CFP-10)특정항원태적단극륭항체,위건립결핵병림상조기진단적면역학방법전정물질기출.방법 설계병합성결핵분지간균CFP-10제53 ～ 66위안기산(14태AAVVRFQEAANKQK)작위특정적항원태서렬,경면역BALB/C소서,취기비세포화소서골수류세포SP2/0융합,경매련면역흡부시험(ELISA)검측、다차유한희석아극륭건립능은정분비항결핵분지간균특이항원태단극륭항체적잡교류세포계,순화해단극륭항체,진행면역구단백아형분석,병감정분석해단극륭항체재단백질면역인적법(Western blotting)、면역침정법화ELISA검측중적응용효과.공검측결핵분지간균배양상청양본38빈,비결핵분지간균배양상청양본20빈,결핵성흉수양본32빈,비결핵성흉수양본24빈이급건강대조혈청양본20빈.결과 단극륭항체면역구단백아형분석현시해잡교류세포계산생적항체류형위IgG1화κ형.해항결핵분지간균CFP-10특이성단극륭항체가성공용우Western blotting화면역침정법분석.ELISA정량검측현시,해특이성항체검측결핵분지간균적민감성위78.6％ (55/70),특이성위92.2％ (59/64).결론 CFP-10특정항원태단극륭항체용우결핵분지간균검측유교고적민감성화특이성,가용우결핵병적조기진단.
Objective To prepare a novel monoclonal antibody (mAb) that specifically against Mycobacterium tuberculosis culture filtrate protein 10 (CFP-10).Methods The BALB/c mice were immunized by a peptide with 14 amino acids (aa residues 53 to 66) of CFP-10,and then the splenocytes of mice were fused with myeloma cell line SP2/0.The resultant fused cells were subjected to screening culture,enzyme linked immunosorbent assay (ELISA) assay and subcloning by limited dilution to establish hybridoma cell lines of stable secreting anti-the peptide of CFP-10 antibody.The antibody was purified,and its isotypes were analyzed.Then,the antibody was further evaluated by Western blotting,immunoprecipitation and ELISA in 38 culture supernatant samples of Mycobacterium tuberculosis,20 culture supernatant samples of non-Mycobacterium tuberculosis,32 samples of tuberculous pleural effusion,24 samples of non-tuberculous pleural effusion,and 20 serum samples from healthy controls.Results The isotype of the mAb against the specific peptide of CFP-10 was an IgG1 with κ chain,and it was applicable for Western blotting and immunoprecipitation analysis.ELISA quantitative test showed that the sensitivity and specificity for diagnosis of Mycobacterium tuberculosis were 78.6％ (55/70) and 92.2％ (59/64),respectively.Conclusion The mAb generated against the specific peptide of CFP-10 is high in sensitivity and specificity,and it might be used in the early diagnosis of tuberculosis.
