人IFN-γ体外释放检测法的建立及其在结核病诊断中的应用

本研究旨在建立人IFN-γ体外释放检测法,用于人结核病的特异性诊断.克隆表达了人IFN-γ基因,利用纯化的重组IFN-γ免疫小鼠,获得两株高效价的单克隆抗体.用所获得的单克隆抗体及兔抗IFN-γ多克隆抗体建立了检测人IFN-γ的夹心ELISA,检测灵敏度达到31.25 pg/mL.采集111位结核病阳性病人与292位临床健康对照者肝素抗凝全血,利用结核菌特异性抗原ESAT-6/CFP-10融合蛋白体外刺激外周血淋巴细胞释放IFN-γ,用所建立的夹心ELISA及商品化试剂盒平行检测所有样本,结果表明两种方法的检测结果相符.结核患者的检测灵敏度为95.5%,健康对照的阳性检出率为16.7%,患者与健康对照的阳性检出率差异极显著(P<0.01),证实所建立的方法灵敏度与特异性均很高,具有良好的应用前景.     

双抗体夹心ELISA检测HIV-1 gp41抗原方法的建立

目的:建立检测HIV-1gp41抗原的双抗体夹心ELISA,并探讨其临床应用的可行性。方法:用饱和硫酸铵(SAS)纯化抗HIV-1gp41-5单克隆抗体(mAb),用HRP标记后建立双抗体夹心ELISA法,对其灵敏度及特异性进行检测,并用该方法对40份HIV-1阳性血清进行了检测。结果:用mAbE12(5μg/mL)为包被抗体,2H6为酶标记抗体(1∶900)建立了双抗体夹心ELISA法,检测gp41-5多肽的灵敏度是100pg/mL。对HIV-1阳性血清中gp41抗原的检出率为67.5%(27/40)。结论:建立了特异性强、灵敏度良好的检测HIV-1gp41抗原的双抗体夹心ELISA法。     

抗人甲状腺球蛋白单克隆抗体的制备

目的:制备分泌特异性抗人甲状腺球蛋白(thyroglobulin,Tg)单克隆抗体的杂交瘤细胞株,为建立高灵敏度的Tg检测方法做准备。方法:以天然人源甲状腺球蛋白为抗原经皮下免疫BALB/c小鼠,通过细胞融合制备分泌抗人甲状腺球蛋白单克隆抗体,并对其进行特异性鉴定,建立检测Tg的双抗体ELISA(enzyme-linked immunosorbent assay)夹心法。结果:获得7株可稳定分泌抗人甲状腺球蛋白单克隆抗体的杂交瘤细胞株,经ELISA鉴定,筛选抗体可与Tg抗原有良好的特异性反应。建立的双抗体夹心ELISA方法敏感性可达1 ng/mL。结论:成功制备了抗人Tg单克隆抗体并建立了检测人Tg双抗体夹心ELISA方法,为进一步研发Tg快速诊断试剂盒提供了原料。     

芜菁花叶病毒单克隆抗体的制备及检测应用

先以芜菁花叶病毒（TuMV）免疫BAL B/C小鼠,然后取其脾细胞使之与SP2/0鼠骨髓瘤细胞融合,经筛选、克隆,获得4株能稳定传代并分泌抗TuMV单克隆抗体（Mab）的杂交瘤细胞,并以之制备腹水单抗。4株单克隆抗体腹水ELISA效价在10-5～10-6之间,仅对TuMV起特异性反应。Western blot分析表明,4株单抗都能与TuMV 34kD的外壳蛋白亚基起特异反应。利用TuMV的多抗兔血清和单抗腹水建立了三抗体夹心ELISA检测TuMV的方法,检测病叶的灵敏度为1∶5120倍,检测提纯TuMV病毒绝对量为21.9 ng。利用三抗体夹心ELISA测定出7种作物上有TuMV侵染。      

CFP-10/ESAT-6特异性IFN-g释放反应诊断活动性肺结核的研究

前期研究已经建立了基于结核分枝杆菌特异性抗原CFP-10/ESAT-6融合蛋白刺激外周血单核细胞IFN-γ释放反应,本研究旨在证实该方法在活动性肺结核及结核菌潜伏感染诊断中的意义。实验对象包括111例当地医院收治的活动性肺结核病人(病例组)及283例某大学入学新生(健康对照者)。采集肝素抗凝血,分别加入含结核菌抗原CFP-10/ESAT-6、植物血凝素及无刺激物(PBS)的细胞培养孔中,培养过夜,次日收集血清,进行IFN-γ检测。同时,对其中58位病人志愿者及46位健康对照志愿者进行了结核菌素(PPD)皮内变态反应。病例组与健康对照组的PPD皮内变态反应阳性率分别为79.3%(46/58)和76.1%(35/46),无显著差异(P>0.05),表明PPD皮内变态反应不能用于活动性肺结核的检测。病例组IFN-γ体外释放反应的阳性率为95.5%(106/111),而健康对照组阳性率为16.3%(46/283),两组间均存在极显著差异,表明IFN-γ体外释放反应诊断活动性肺结核具有很高的灵敏度(95.5%)与良好的特异性。在健康组中,IFN-γ体外释放反应与PPD皮内变态反应的总符合率为50.0%,且IFN-g体外...     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

抗HIV-p24单克隆抗体细胞株的建立及初步应用

拟建立抗HIV-P24蛋白的单克隆抗体细胞株及双抗体夹心法,用于检测艾滋病人血清中的P24抗原。用纯化的基因工程表达的HIV-P24蛋白免疫BALB/c小鼠。取免疫小鼠的脾细胞和小鼠骨髓瘤Sp2/0的细胞融合,有限稀释法克隆细胞,ELISA筛选特异性抗体。结果克隆筛选出12株细胞,初步建立了检测HIV-P24抗原的双抗体夹心酶联免疫吸附试验。本方法为监控艾滋病病毒感染的窗口期和艾滋病病人临床治疗效果提供了检测手段。     

抗埃博拉病毒核蛋白抗体的制备与双抗夹心ELISA检测方法的建立

目的:制备抗埃博拉病毒核蛋白(EBOV NP)单克隆抗体和多克隆抗体,建立针对EBOV NP的ELISA检测方法。方法:以重组EBOV NP免疫动物并制备多克隆抗体和单克隆抗体。在此基础上,通过优化抗体浓度、包被液等条件建立检测EBOV NP的双抗夹心ELISA方法。结果:制备出了兔多克隆抗体,筛选出2株可分泌单克隆抗体的鼠源杂交瘤细胞株。Western blot实验结果表明兔多抗与鼠单抗的结合区域均为N端1~35氨基酸。通过优化,建立了针对EBOV NP的双抗夹心ELISA检测方法。其线性范围是31.2~1 000 ng/ml,最低检测限为2.6 ng/ml。结论:制备出了抗EBOV核蛋白的高特异性多克隆抗体和单克隆抗体,建立了定量检测EBOV核蛋白的方法。     

泰泽氏病原体抗原表位相关肽的初步筛选与鉴定

目的获得泰泽氏病原体抗原表位相关肽,用于实验动物血清中该病原体感染相关抗体的检测。方法选用泰泽氏病原体的四种单克隆抗体(M2、M3、M4、M5)作为配基,从噬菌体表面展示的随机7肽文库中筛选单抗识别的抗原表位,获得特异性噬菌体克隆;并采用ELISA、Western blot方法对其进行分析鉴定,获得阳性噬菌体克隆。结果获得阳性噬菌体克隆5个,其展示的融合蛋白能被泰泽氏病原体的免疫血清识别,ELISA检测A值的P/N为8.0～17.1;Western blot分析显示单一特异性条带,相对分子质量约为38×103。结论本研究获得的5个阳性克隆所表达的融合蛋白,为泰泽氏病原体抗原表位相关肽,可作为该病原体隐性感染血清学检测的候选抗原。     

抗HLJ1单克隆抗体的制备及抗原检测方法的建立

为制备抗人肝脏DnaJ-like蛋白(Human liver DnaJ-like protein, HLJ1)的单克隆抗体, 并建立免疫组化和双抗体夹心ELISA检测HLJ1的方法。采用淋巴细胞杂交瘤技术, 获得两株能稳定分泌抗HLJ1单克隆抗体的杂交瘤细胞株A4C7和 C4C8。经鉴定, 两株单抗的亚类均为IgG1, 并且效价高、特异性好。以单抗A4C7和C4C8作为一抗, 对人胎肝组织石蜡切片进行免疫组化染色, 结果表明, 两株单抗均为阳性染色, 且HLJ1主要定位于胎肝细胞的胞浆。选取A4C7进行HRP酶标记, 并以HRP- A4C7作为酶标抗体, 以C4C8作为包被抗体, 建立双抗体夹心ELISA方法, 并进行棋盘滴定确定抗体的最佳工作浓度。该检测方法的线性范围是15~750 ng/mL, 灵敏度下限达15 ng/mL, 特异性良好。所建立的免疫组化和双抗体夹心ELISA 法可用于快速、灵敏地检测组织及血清中的HLJ1蛋白, 为HLJ1的肿瘤相关性研究提供了有力的工具。     

Diagnostic Accuracy and Turnaround Time of the Xpert MTB/RIF Assay in Routine Clinical Practice

The Xpert MTB/RIF assay was introduced for timely and accurate detection of tuberculosis (TB). The aim of this study was to determine the diagnostic accuracy and turnaround time (TAT) of Xpert MTB/RIF assay in clinical practice in South Korea. We retrospectively reviewed the medical records of patients in whom Xpert MTB/RIF assay using sputum were requested. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of pulmonary tuberculosis (PTB) and detection of rifampicin resistance were calculated. In addition, TAT of Xpert MTB/RIF assay was compared with those of other tests. Total 681 patients in whom Xpert MTB/RIF assay was requested were included in the analysis. The sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay for diagnosis of PTB were 79.5% (124/156), 100.0% (505/505), 100.0% (124/124) and 94.0% (505/537), respectively. Those for the detection of rifampicin resistance were 57.1% (8/14), 100.0% (113/113), 100.0% (8/8) and 94.9% (113/119), respectively. The median TAT of Xpert MTB/RIF assay to the report of results and results confirmed by physicians in outpatient settings were 0 (0–1) and 6 (3–7) days, respectively. Median time to treatment after initial evaluation was 7 (4–9) days in patients with Xpert MTB/RIF assay, but was 21 (7–33.5) days in patients without Xpert MTB/RIF assay. Xpert MTB/RIF assay showed acceptable sensitivity and excellent specificity for the diagnosis of PTB and detection of rifampicin resistance in areas with intermediate TB burden. Additionally, the assay decreased time to the initiation of anti-TB drugs through shorter TAT.     

Xpert MTB/RIF在人类免疫缺陷病毒感染/艾滋病患者中诊断结核病的价值

为探讨利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术(Xpert Mycobacterium tuberculosis/rifampicin,Xpert MTB/RIF)在人类免疫缺陷病毒感染/艾滋病(human immunodeficiency virus infection/acquired immunodeficiency syndrome,HIV/AIDS)患者中诊断结核病的价值,本研究回顾性分析了2018年1月1日—2020年12月31日复旦大学附属公共卫生临床中心感染与免疫科收治的801例HIV/AIDS合并疑似结核病患者的临床资料。801例患者中,657例进行了Xpert MTB/RIF、外周血结核感染T细胞斑点试验(tuberculosis T cell spot test,T-SPOT.TB)、抗酸染色涂片镜检和BACTEC MGIT 960液体培养等检测。以液体培养及菌型鉴定结果作为结核病诊断的“金标准”,确诊结核病92例,Xpert MTB/RIF、T-SPOT.TB、抗酸染色涂片镜检在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)的灵敏度分别为72.8%、55.4%和69.6%,特异度分别为96.8%、90.3%和84.4%,与“金标准”行一致性检验,Kappa值分别为0.719 (P<0.01)、0.430(P<0.01)和0.424(P<0.01)。Xpert MTB/RIF检测502份呼吸道样本,结果显示其诊断肺结核的灵敏度和特异度分别为66.7%和96.0%;在痰涂片阳性和阴性的患者中,Xpert MTB/RIF诊断肺结核的灵敏度分别为77.4%和35.2%,特异度分别为87.7%和 97.8%。采用Xpert MTB/RIF检测343份肺外标本,结果显示其诊断肺外结核的灵敏度和特异度分别为63.3%和95.2%。以上结果提示,Xpert MTB/RIF在HIV/AIDS患者中诊断结核病(包括肺结核和肺外结核)具有较高的灵敏度和特异度,诊断肺结核的灵敏度高于肺外结核,因此推荐将其作为HIV/AIDS患者疑似结核病的首选检测方法。     

Serodiagnosis of extrapulmonary tuberculosis by enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycobacterium tuberculosis antigenic glycolipids were determined by enzyme-linked immunosorbent assay (ELISA). The 720 sera were collected from adult patients under investigation, suspected with extrapulmonary tuberculosis. The test performance was estimated according to definitive diagnosis in terms of specificity, sensitivity, positive predictive value and negative predictive value. These parameters calculated on 142 sera from patients with extrapulmonary tuberculosis and on 578 sera from patients with different nontuberculosis diseases were 92%, 81.6%, 70.9% and 95.1%, respectively. The specificity decreased to 85% when tuberculosis was associated with cancer or hepatic cirrhosis. In reactivated tuberculosis the sensitivity and the positive predictive value were 86.9% and 83.3%, respectively. Our results showed that ELISA was conclusive for patients with active tuberculosis, before the initiation of the treatment. The sensitivity decreased to 30% in inactive forms. It was demonstrated that ELISA was positive in cases with negative microscopy genitourinary tuberculosis. ELISA could be used as a supporting test in the laboratory diagnosis of active extrapulmonary tuberculosis in adults, disregarding the site involved.     

Rapid dot sputum and serum assay in pulmonary tuberculosis

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.     

Potential use of Amplicor PCR kit in diagnosing pulmonary tuberculosis from gastric aspirate.

Polymerase chain reaction (PCR) detection of mycobacteria from gastric aspirate for the diagnosis of tuberculosis is not fully evaluated up to now. A total of 116 gastric aspirate specimens were collected from patients with suspected pulmonary tuberculosis. The breakdown of diagnosis was 67 pulmonary tuberculosis, 16 nontuberculous mycobacterial infection, 5 extra pulmonary tuberculosis, and 28 other lung diseases. The conventional methods were shown to have a sensitivity of 47.8% and a specificity of 79.6%; on the other hand, Amplicor had 34.9% and 97.0%, respectively. The Amplicor provided a more rapid and specific method for diagnosing tuberculosis and was more useful than the conventional.     

三种免疫学方法在矽肺合并肺结核诊断中的价值研究

目的:探讨T淋巴细胞酶联斑点实验(T-SPOT)、结核菌素皮肤试验(tuberculin test,TST)以及结核抗体(tuberculosis antibody)在矽肺合并肺结核(pulmonary silicosis complicated with tuberculosis)诊断中的应用价值。方法:收集2015年8月7日至2016年5月20日确诊为矽肺结核感染患者41例、非矽肺结核患者90例、健康体检学生对照组40例,对三组人群经以上免疫学方法检测的结果进行统计分析。结果:1)矽肺结核、非矽肺结核及健康人群中T-SPOT阳性率分别为73.17%、93.33%、15%,三者两两比较差异有统计学意义;矽肺结核、非矽肺结核及健康人群中TST阳性率分别为58.54%、88.89%、32.5%,非矽肺结核患者与健康人群及矽肺结核患者之间,差异均有统计学意义,但矽肺结核患者与健康人群之间,差异无统计学意义;矽肺结核、非矽肺结核及健康人群中结核抗体阳性率分别为36.58%、42.22%、52.5%,三者两两比较差异均无统计学意义。2)三种检测方法在矽肺结核患者中的敏感性分别为73.17%、58.54%、36.58%,差异有统计学意义,其中T-SPOT及TST的敏感性均高于结核抗体方法(P0.05);特异性分别为85%、67.5%、47.5%,其中T-SPOT的特异性高于结核抗体方法(P0.05);阳性预测值分别为83.33%、64.86%、41.67%,阴性预测值分别为75.56%、61.36%、42.22%,三种方法的阳性及阴性预测值存在差异,均有统计学意义。3)T-SPOT在I、II、III期矽肺结核检测的阳性率分别为52.38%、94.12%、100%,I期与II期或III期的阳性率之间差异有统计学意义,II期与III期之间阳性率差异无统计学意义。结论:T-SPOT方法具有较高的敏感性及特异性,对矽肺结核辅助诊断及临床分期具有较好的应用价值。     

上海口岸2014-2015年入境人员肺结核筛查情况

为了解上海口岸入境人员肺结核的筛查情况及后续处理,防止结核病通过口岸跨境传播,本研究于2014年1月—2015年12月对所有在上海口岸办理入境体检的14岁以上人员进行结核病筛查,通过病史、体格检查和胸部X线摄影筛查疑似肺结核患者;对疑似肺结核患者进行痰细菌学检测、T‐SPOT ．TB和 Xpert MTB／RIF检测。结果显示,2014—2015年上海口岸入境人员共检出疑似肺结核患者215例,总检出率为229．76／10万;确诊肺结核患者33例,总检出率为35．27／10万,确诊率为15．3％。对210例疑似肺结核患者进行痰细菌学检测,结果显示结核分枝杆菌培阳率为14．3％,非结核分枝杆菌培阳率为17．1％。有95例和78例疑似肺结核患者分别接受 T‐SPOT ．TB和 Xpert MTB／RIF 检测,以痰细菌学检测为“金标准”,T‐SPOT ．TB的灵敏度为100％,特异度为49．4％;Xpert MTB／RIF的灵敏度为87．5％,特异度为96．8％。33例确诊肺结核患者中,25例（75．8％）离境,15例（45．5％）在离境前接受抗结核治疗,8例（24．2％）失访。本研究显示,上海口岸入境人员中肺结核确诊率仍有待提高。筛查与诊断中,T‐SPOT ．TB具备较高灵敏度, Xpert MTB／RIF具备较高特异度,两种方法均有较高应用价值,两者联用可提高检出率,缩短检出时间。对确诊病例或未确诊的可疑病例应加强后续监管。     

Evaluation of Rapid IgG4 Test for Diagnosis of Gnathostomiasis

Human gnathostomiasis is a parasitic disease caused by Gnathostoma nematode infection. A rapid, reliable, and practical immunoassay, named dot immuno-gold filtration assay (DIGFA), was developed to supporting clinical diagnosis of gnathostomiasis. The practical tool detected anti-Gnathostoma-specific IgG4 in human serum using crude extract of third-stage larvae as antigen. The result of the test was shown by anti-human IgG4 monoclonal antibody conjugated colloidal gold. The sensitivity and specificity of the test were both 100% for detection in human sera from patients with gnathostomiasis (13/13) and from healthy negative controls (50/50), respectively. Cross-reactivity with heterogonous serum samples from patients with other helminthiases ranged from 0 (trichinosis, paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis) to 25.0% (sparganosis), with an average of 6.3% (7/112). Moreover, specific IgG4 antibodies diminished at 6 months after treatment. This study showed that DIGFA for the detection of specific IgG4 in human sera could be a promising tool for the diagnosis of gnathostomiasis and useful for evaluating therapeutic effects.     

Differential expression of CD64 in patients with Mycobacterium tuberculosis infection: A potential biomarker for clinical diagnosis and prognosis

To evaluate the clinical utility of neutrophil (n)CD64 index to diagnose pulmonary tuberculosis (PTB) and extrapulmonary TB (ePTB) and to predict the outcome of Mycobacterium tuberculosis infection. We recruited 189 patients with active TB and 140 controls and measured the differential expression of nCD64 index using flow cytometry. The receiver operating characteristics (ROC) curve analysis was performed to estimate the diagnostic performance of the nCD64 index and T‐SPOT.TB assay for the diagnosis of TB. Furthermore, we analysed whether the nCD64 index in patients with TB was correlated with inflammatory indicators. Finally, we assessed the prognosis of patients by following the dynamic changes of the nCD64 index once a week. The nCD64 index was significantly higher in active TB group (PTB and ePTB), than in the anti‐TB and healthy controls (HC) groups. The sensitivity and specificity of nCD64 index for the differential diagnosis of PTB and pneumonia (PN) patients were 68.33% and 77.55%, respectively. The sensitivity and specificity of nCD64 index for the diagnosis of tuberculous meningitis (TBM) were 53.85% and 100%, respectively. Furthermore, there was a weak correlation between the nCD64 index and inflammatory indicators. More importantly, with the improvement in patient condition, the nCD64 index started to decline in the first week of anti‐TB therapy and significantly decreased at 4 weeks after treatment. Our study demonstrated that the CD64 assay is a rapid, non‐invasive and stable method for clinical application, and the nCD64 index can serve as a potential biomarker for the diagnosis and prognosis of TB.     

Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein ofMycobacterium tuberculosis H37Ra

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction(Mtb EST-6) of purifiedMycobacterium tuberculosis H₃₇Ra and affinity purified polyclonal antibodies againstMtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system usingMtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified antiMtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility ofMtb EST-6 as a diagnostic marker of pulmonary tuberculosis.