中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
2期
180-184
,共5页
蔡江丽%秦莲花%刘忠华%王洁%胡忠义
蔡江麗%秦蓮花%劉忠華%王潔%鬍忠義
채강려%진연화%류충화%왕길%호충의
MPT64抗体%SELEX%适体%ssDNA%血清学检测
MPT64抗體%SELEX%適體%ssDNA%血清學檢測
MPT64항체%SELEX%괄체%ssDNA%혈청학검측
MPT64 antibodies%SELEX%Aptamer%ssDNA%Serological detection
目的 利用SELEX技术筛选MPT64抗体的适体建立混合夹心ELISA检测体系,应用于临床血清标本的检测,探讨该方法 潜在的实验室诊断价值.方法 利用竞争ELISA检测方法 ,测定不同浓度的MPT64抗原对最后一轮ssDNA文库与靶物质亲和力的抑制率,选取优势序列且亲和性值较高的适体用于检测,优化混合夹心ELISA检测方法 ,确定MPT64抗体的检测下限和线性范围,同时检测230例临床血清标本.结果 在竞争ELISA结果 中,总反应体系为100μl,MPT64抗原浓度由2μg/ml增加到256 μg/ml时,对ssDNA文库的抑制率也由0.25%增加到80%.优化的混合夹心ELISA的检测体系:ssDNA包被浓度为0.1μg/孔、血清稀释度为1/200、辣根过氧化物酶标记的羊抗人IgG抗体浓度为1/40 000.MPT64抗体的检测下限为3 mg/L,线性范围为10～1000 mg/L.利用该体系检测100例结核病患者、100例健康体检者和30例非结核病患者血清,检测结果 用GraphpadPrism软件进行分析,结核病组与健康体检组、结核病组与非结核疾病对照组差异均具有统计学意义(P<0.001).统计学分析得到检测的特异性与敏感性分别为96.1%和31.0%.结论 利用适体建立的混合夹心ELISA用于结核病的血清学诊断具有一定的诊断价值.
目的 利用SELEX技術篩選MPT64抗體的適體建立混閤夾心ELISA檢測體繫,應用于臨床血清標本的檢測,探討該方法 潛在的實驗室診斷價值.方法 利用競爭ELISA檢測方法 ,測定不同濃度的MPT64抗原對最後一輪ssDNA文庫與靶物質親和力的抑製率,選取優勢序列且親和性值較高的適體用于檢測,優化混閤夾心ELISA檢測方法 ,確定MPT64抗體的檢測下限和線性範圍,同時檢測230例臨床血清標本.結果 在競爭ELISA結果 中,總反應體繫為100μl,MPT64抗原濃度由2μg/ml增加到256 μg/ml時,對ssDNA文庫的抑製率也由0.25%增加到80%.優化的混閤夾心ELISA的檢測體繫:ssDNA包被濃度為0.1μg/孔、血清稀釋度為1/200、辣根過氧化物酶標記的羊抗人IgG抗體濃度為1/40 000.MPT64抗體的檢測下限為3 mg/L,線性範圍為10～1000 mg/L.利用該體繫檢測100例結覈病患者、100例健康體檢者和30例非結覈病患者血清,檢測結果 用GraphpadPrism軟件進行分析,結覈病組與健康體檢組、結覈病組與非結覈疾病對照組差異均具有統計學意義(P<0.001).統計學分析得到檢測的特異性與敏感性分彆為96.1%和31.0%.結論 利用適體建立的混閤夾心ELISA用于結覈病的血清學診斷具有一定的診斷價值.
목적 이용SELEX기술사선MPT64항체적괄체건립혼합협심ELISA검측체계,응용우림상혈청표본적검측,탐토해방법 잠재적실험실진단개치.방법 이용경쟁ELISA검측방법 ,측정불동농도적MPT64항원대최후일륜ssDNA문고여파물질친화력적억제솔,선취우세서렬차친화성치교고적괄체용우검측,우화혼합협심ELISA검측방법 ,학정MPT64항체적검측하한화선성범위,동시검측230례림상혈청표본.결과 재경쟁ELISA결과 중,총반응체계위100μl,MPT64항원농도유2μg/ml증가도256 μg/ml시,대ssDNA문고적억제솔야유0.25%증가도80%.우화적혼합협심ELISA적검측체계:ssDNA포피농도위0.1μg/공、혈청희석도위1/200、랄근과양화물매표기적양항인IgG항체농도위1/40 000.MPT64항체적검측하한위3 mg/L,선성범위위10～1000 mg/L.이용해체계검측100례결핵병환자、100례건강체검자화30례비결핵병환자혈청,검측결과 용GraphpadPrism연건진행분석,결핵병조여건강체검조、결핵병조여비결핵질병대조조차이균구유통계학의의(P<0.001).통계학분석득도검측적특이성여민감성분별위96.1%화31.0%.결론 이용괄체건립적혼합협심ELISA용우결핵병적혈청학진단구유일정적진단개치.
Objective To establish mixed-sandwich ELISA detection system by screening aptam-ers of MPT64 antibodies with SELEX to detect clinical serum samples, and explore the potential laboratory diagnosis value of this method. Methods To detect the affinity of the final round ssDNA library to MPT64 antibodies inhibited by MPT64 antigen with the competitive ELISA method, optimize the mixed-sandwich ELISA detection method that was aptamer-serum-horseradish peroxidase labeled goat anti-human IgG anti-body detection system to detect 230 cases of clinical serum samples as well as the lowest concentration of MPT64 antibodies and the linear range. Results In competitive ELISA test results, the percentage of inhi-bition effect of MPT64 antigen to final round ssDNA library is from 0.25% to 80% when the MPT64 antigen concentration rised from 2 μg/ml to 256 μg/ml. The Optimized detection system of mixed-sandwich ELISA was constitute of the concentration of ssDNA coated with 0.1μg/hole, serum dilution of 1/200, horseradish peroxidase labeled goat anti-human IgG antibody concentration of 1/40 000. The lowest concentration of MPT64 antibody is 3 mg/L and the linear range is between 10 mg/L and 1000 mg/L. The serum samples of 100 cases of tuberculosis patients, 100 healthy individuals and 30 cases of non-tuberculesis were tested in this system and the test result was analyzed with Graphpad Prism, the difference of tuberculosis group and healthy group was statistically significant (P<0.001 ), the difference of TB group and non-TB control group was also statistically significant (P<0.001). The specificity and the sensitivity was 96.1% and 31.0% re-spectively. Conclusion The aptamer mixed-sandwich ELISA method will play an important role in the sero-logical diagnosis of tuberculosis.
