检测可溶型CD226双mAb夹心ELISA的建立及初步应用

目的 :建立一种检测可溶型CD2 2 6 (sCD2 2 6 )的双mAb夹心ELISA ,并用于临床标本的检测。方法 :确定包被mAbLeoA1和HRP mAbFMU3的最佳工作浓度及最佳稀释液后 ,建立双mAb夹心ELISA ,并对方法的特异性、敏感性和稳定性进行鉴定。再以建立的方法检测临床标本。结果 :包被mAbLeoA1的最佳工作浓度为 2 .5mg/L ,HRP mAbFMU3的最佳工作浓度 1∶4 0 0 ,标准品及HRP mAb的最佳稀释液分别为 1g/LBSA 1mL/LTween2 0 PBS和 30 g/LPEG PBS .建立的双mAb夹心ELISA ,其特异性 (与人IgG无交叉反应 ,阻断实验中阻断效应呈剂量依赖性 )、敏感性 (检出下限为 110ng/L标准品CD2 2 6 /Fc融合蛋白 )及稳定性 (CV <10 .2 % )均较良好。对部分临床标本的检测中 ,发现 6例类风湿关节炎(RA)患者血清sCD2 2 6的水平升高 ,与正常人血清sCD2 2 6水平相比差异显著 (P <0 .0 1)。结论 :建立一种特异性高、重复性好、较敏感的检测sCD2 2 6的双mAb夹心ELISA ,初步证明可用于临床标本的检测     

抗铜绿假单胞菌外膜蛋白F单克隆抗体制备及夹心ELISA检测方法的建立

目的:制备抗铜绿假单胞菌外膜蛋白F(OprF)的单克隆抗体(mAb),并建立双mAb夹心ELISA检测方法。方法:利用分子生物学方法克隆OprF基因,诱导表达并纯化OprF。用OprF免疫BALB/c小鼠后通过杂交瘤技术制备特异性的mAb,并用ELISA法测定mAb的免疫活性;建立检测铜绿假单胞菌的双mAb夹心ELISA方法,并对其敏感性、特异性进行初步评价。结果:成功地克隆OprF基因,经诱导表达获得铜绿假单胞菌的OprF。通过杂交瘤技术筛选出4株mAb:6D8F7、2H2B6、3C2F5和7B5D9,经鉴定mAb 6D8F7可作为捕获抗体,mAb 7B5D9被HRP标记后可作为检测抗体,以这2株mAb建立的双mAb夹心ELISA方法重复性好、特异性强,敏感性高达到1×103集落形成单位(CFU/mL)。检测的线性范围为1×103～108CFU/mL。与传统的细菌分离方法比较,检测临床标本的符合率高达94.7%。结论:通过杂交瘤技术制备抗铜绿假单胞菌OprF的mAb,并建立了高特异性、高敏感性检测铜绿假单胞菌抗原的双mAb夹心ELISA方法,可用于临床检测铜绿假单胞菌的感染。     

一种快捷、定量检测烟曲霉GM抗原双mAb夹心ELISA的方法

目的 :建立一种快速诊断烟曲霉病的双mAb夹心ELISA法。方法 :应用 4株抗烟曲霉GM单克隆抗体 (mAb) ,分别包被和制备HRP mAb ,用双mAb夹心ELISA法配对试验 ,选择捕获及检测mAb。结果 :经筛选得到捕获及检测mAb的最佳组合 ,并建立了双mAb夹心ELISA法。该法检测GM抗原的灵敏度为 0 .1μg/L ,测出范围在 0 .1～ 10 μg/L之间。连续 6d用ELISA法检测同一份样品 ,所获CV的平均值为 ( 7.2± 3.8) %。结论 :建立了一种可快速、定量检测烟曲霉GM抗原的双mAb夹心ELISA法 ,灵敏度高、重复性好 ,对研制试剂盒应用于烟曲霉病早期诊断和防治 ,具有重要的临床应用价值     

脑钠肽单克隆抗体的制备及临床应用研究

目的:制备抗脑钠肽(BNP32)单克隆抗体(mAb),并利用双抗体夹心ELISA法建立BNP抗原检测技术,应用于临床心脏患者脑钠肽水平的检测。方法:以基因工程原核重组表达BNP抗原免疫BALB/c小鼠,利用常规杂交瘤技术制备mAb,mAb经纯化和HRP标记后,利用双抗体夹心ELISA法筛选检测BNP32蛋白的最佳配对mAb,以其建立BNP32抗原检测技术,并与临床BNP检测的标准实验做平行比较。结果:成功筛选到16株稳定分泌抗BNP32mAb的杂交瘤细胞株,16株mAb的亚型分别为IgG1、IgG2a和IgM,并从中筛选出最佳mAb配对组合,该组合对BNP32蛋白的检测灵敏度为20ng/L。建立的双抗体夹心BNP检测ELISA法与临床BNP检测的标准实验平行比较具有很好的一致性(kappa值=0.828),两者没有统计学意义(P0.05)。结论:成功地建立了BNP32抗原的双抗体夹心ELISA法检测技术,并能够很好地运用于临床心衰患者BNP指标的检测。     

定量检测血和尿中Cystatin C含量在肾移植患者肾功能评价中的应用

目的:建立定量检测半胱氨酸蛋白酶抑制剂C(cys C)水平的夹心ELISA方法,对肾移植患者的肾功进行监测,以期作为术后急性排斥反应(AR)及肾功能延迟恢复(DGF)发生的一项新的预警指标.方法:建立分泌抗人cys C单克隆抗体(mAb)的杂交瘤细胞株,制备腹水纯化mAbs并进行辣根过氧化物酶标记,建立检测cys C的夹心ELISA方法;应用此方法检测正常人及23例肾移植患者移植手术前后的血清(scys C)及尿(IJcys C)标本中cys C的含量并分析其与血清肌酐(SCR)的相关性.结果:获得7株可稳定分泌抗cys CmAb的杂交瘤细胞.应用建立的ELISA方法检测cys C含量敏感度可达O.1ug/L.肾移植正常恢复(NC)组患者Scys C及Ucys C含量与SCR下降趋势一致.术后发生AR的患者Ucys C水平持续较高,直到术后21 d才出现下降.而发生DGF的患者同NC组患者相比术后4周内Scys C及Ucys C与SCB维持在较高水平,Ucys C的水平有逐渐升高的趋势,直到术后60 d才下降到与NC相近的水平.结论:建立了敏感稳定的检测cys C含量的ELISA方法,可用于检测Ucys C水平.而Scys C及Ucys C的持续升高可能提示肾移植患者术后AR或DGF的发生.     

检测结核分枝杆菌蛋白抗原b夹心ELISA的建立及其应用

目的 建立定量检测脑脊液中结核分枝杆菌蛋白抗原b的夹心ELISA,以便用于诊断结核性脑膜炎.方法 制备抗结核分枝杆菌蛋白抗原b鼠源性单克隆抗体(mAb)及兔多克隆抗体;以单克隆抗体为包被抗体,多克隆抗体为检测抗体,建立检测结核分枝杆菌蛋白抗原b的双抗体夹心ELISA;应用此方法检测正常人(n=6)、非结核性脑膜炎患者(n=26)及临床确诊结核性脑膜炎患者(n=42)的脑脊液标本中蛋白抗原b的含量,并分析其与结核性脑膜炎活动性感染的相关性.结果 成功建立了检测蛋白抗原b的夹心ELISA,敏感度可达0.4 μg/L.应用该方法在正常人及非结核性脑膜炎患者脑脊液中未检测到蛋白抗原b;在结核性脑膜炎患者脑脊液中蛋白抗原b含量显著升高.结论 建立了敏感、特异检测结核分枝杆菌蛋白抗原b含量的ELISA,为活动性结核性脑膜炎的诊断提供一种有效手段.     

诺如病毒表达衣壳蛋白单克隆抗体的制备、鉴定和初步应用

目的:建立能稳定分泌抗诺如病毒(Norovirus)N蛋白的单克隆抗体(mAb)细胞株, 制备抗诺如病毒核衣壳蛋白的mAb, 为诺如病毒的早期快速检测及致病机制的研究提供实验材料.方法:用E.coli表达的GⅡ组广州株NVgz01(DQ369797)Norovirus-N蛋白免疫BALB/c小鼠, 通过PEG使小鼠脾细胞和Sp2/0细胞融合, 使用HAT选择性培养基培养融合细胞, 用间接ELISA和Western blot测定mAb的效价、免疫球蛋白亚型和mAb的特异性, 并将各mAb之间进行配对.结果:通过细胞融合和克隆化, 共筛选出4株分泌抗Norovirus-N蛋白抗体的杂交瘤细胞株N2C3、 N7C2、 N4B1、 N8A9.间接ELISA和Western blot检测结果表明, 这4株mAb都可以与E.coli表达的GⅡ组广州株Norovirus-N蛋白产生特异性反应, 并且能与天然粪便标本中的GⅡ组Norovirus产生特异性反应.配对结果显示N2C3和N7C2之间配对, 对表达蛋白和天然病毒都具有较强的检测灵敏度.结论:获得了诺如病毒GⅡ组特异性mAb, 为制备免疫诊断试剂盒及致病机制的研究奠定了基础.     

夹心ELISA检测N端利用物因子A(NusA)标签融合蛋白

目的创建基于夹心ELISA原理的定性和定量检测N端利用物因子A(NusA)标签融合蛋白的方法,为NusA标签融合蛋白的鉴定提供新的手段。方法以NusA标签蛋白作为免疫原免疫BALB/c小鼠,制备特异性识别NusA标签蛋白的单克隆抗体(mAb),常规标记辣根过氧化物酶,方阵法筛选夹心ELISA的配对抗体。结果获得14株NusA标签蛋白特异性mAb并最终确定一对可用于夹心ELISA检测的NusA特异性mAb。以NusA No.1作为捕获抗体, NusA No.10为检测抗体。结论成功建立双抗体夹心ELISA检测NusA标签融合蛋白的方法,检测敏感度可达1.5 ng/mL。     

检测可溶型TRAIL的ELISA的建立和初步应用

目的 :建立检测可溶型TRAIL的ELISA ,并评价其临床应用价值。方法 :采用本室制备的鼠抗TRAIL的单抗 (mAb)FMU1.1作为包被抗体 ,兔的抗TRAIL高效价多抗为夹心抗体 ,建立检测sTRAIL的ELISA ,并测定了 9例肾综合征出血热 (HFRS)患者和 4 0例银屑病患者血清sTRAIL的水平。结果 :建立了由单抗和多抗组成的检测人sTRAIL的夹心ELISA ,灵敏度为 0 .0 8μg/L。 9例肾综合征出血热 (HFRS)患者中有 3例急性期患者血清sTRAIL水平升高 ,4 0例银屑病患者中 8例升高。结论 :成功地建立了一种可用于检测血清sTRAIL的双夹心ELISA ,为判定某些相关疾病患者的病情、疗效及预后提供有用的工具     

抗幽门螺杆菌尿素酶B亚单位单克隆抗体制备和鉴定及其应用

目的制备抗幽门螺杆菌(Hp)的单克隆抗体(mAb),并对该Hp mAb进行分析鉴定,建立一种检测患者由于Hp感染而在血清中产生Hp抗体的竞争ELISA检测方法。方法用灭活的Hp免疫BALB/c小鼠,通过杂交瘤技术制备Hp mAb。我们采用Hp混合蛋白包括毒素相关蛋白A(CagA)、空泡毒素A(VacA)和尿素酶以及灭活的Hp菌体筛选阳性杂交瘤细胞株,用ELISA和Western blot等技术对所获得的Hp mAb进行鉴定。利用辣根过氧化物酶(HRP)标记所筛选的Hp mAb来建立一个可检测患者血清中Hp抗体的竞争ELISA。结果通过大规模的杂交瘤筛选,我们选择了1株将其命名为C3 Hp mAb,其抗体亚型为IgG2a,腹水效价可达1×107。Western blot法、ELISA和质谱检测结果显示,该C3 Hp mAb能特异地识别Hp的尿素酶B亚单位。用这个C3 Hp mAb,我们建立了一种可检测患者血清中Hp抗体的竞争ELISA。结论成功获得一种可以特异识别Hp尿素酶B亚单位的mAb,建立了一种可检测患者血清中Hp抗体的竞争ELISA。     

抗人Apo B100单克隆抗体的制备及夹心ELISA检测方法的建立

目的:制备抗人载脂蛋白B100(Apo B100)单克隆抗体(mAb),建立人Apo B100双抗体夹心ELISA检测方法。方法:将人Apo B100抗原免疫小鼠,通过细胞融合、筛选后得到杂交瘤细胞株。将细胞株用无血清培养基扩大培养并纯化上清获得抗体,测定抗体亲和力、亚型、特异性及表位,最后建立双抗体夹心ELISA方法。结果:获得4株抗人Apo B100的杂交瘤细胞株(4-1-2、4-2-2、4-3-2、4-6-3),其分泌的抗体不与其他相关蛋白交叉反应,亲和力达到1×109L/mol。用4-3-2和4-6-3建立的双抗体夹心法的检测范围为(1.3～80)ng/mL,灵敏度1.24 ng/mL,批内变异系数均小于10%,批间变异系数均小于15%,回收率在90%以上。结论:成功制备了抗人Apo B100mAb,建立了定量检测人Apo B100的双抗体夹心ELISA方法,为Apo B100检测及疾病的诊断奠定基础。     

抗Ig融合蛋白Fc段单克隆抗体的制备、鉴定与应用研究

目的：制备并鉴定抗Ig融合蛋白Fc段的单克隆抗体(mAb)，建立用于检测Ig融合蛋白的夹心ELISA法和纯化Fc融合蛋白的亲和层析法。方法：以hBCMA—Ig融合蛋白为抗原免疫BALB／c小鼠，通过细胞融合制备抗Fc段mAb，用ELISA等方法鉴定mAb的Ig亚类、表位以及种属特异性，建立用于检测Ig融合蛋白的夹心ELISA法;Western blot检测mAb对变性Ig融合蛋白的反应性。将mAb与Sepharose4B交联，制备亲和层析柱，对LAIR1-Ig融合蛋白进行纯化。结果：获得7株稳定分泌抗Fc段mAb的杂交瘤(FMUFcl-FMUFc7)。利用FMUFc4作为包被mAb，FMUFc5作为酶标记mAb，成功地建立了检测Ig融合蛋白的ELISA法，敏感度达到2μg／L；在7株mAb中，FMUFc6可用于Ig融合蛋白的western blot检测。用FMUFc 6mAb制备的亲和层析柱，可有效地纯化LAIRl—Ig融合蛋白。结论：成功地制备了抗Ig融合蛋白Fc段的mAb，建立了可用于Ig融合蛋白检测和纯化的方法，为Ig融合蛋白的应用提供了有力手段。     

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 10⁴ cfu/mL, and the linear range from 1 × 10⁵ to 1 × 10⁸ cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.     

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.     

Production and characterization of monoclonal antibodies against pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPP-A) was found to be a good first trimester maternal serum marker, together with free beta-human chorionic gonadotrophin (HCG) subunits, for the biochemical screening of fetal trisomy 21 (Down's syndrome). We have raised monoclonal antibodies (mAbs) against PAPP-A purified from human pregnancy serum. The different antibodies were characterized biochemically by Western blot analysis and in terms of specificity (reaction with non-pregnant and male serum). Their performance in Down's syndrome screening was assessed in comparison with an existing enzyme-linked immunosorbent assay method after labelling of the different mAbs with biotin or horseradish peroxidase. A pair of mAbs was eventually chosen for a double-antibody sandwich protocol. The selected combination was found to have a significantly increased specificity (P = 0.0116) over the method using (purified) polyclonal antibodies, together with slightly increased sensitivity. In our limited number of Down's syndrome pregnancy samples (n = 17) and controls (n = 18), the medians as well as the multiples of the median values (for the affected cases) were comparable between the two methods described.     

血清腱糖蛋白-C、hs-CRP与急性冠脉综合症的相关性分析

目的 通过观察血清腱糖蛋白-C(TN-C)和高敏C反应蛋白(hs-CRP)在不同类型急性冠状动脉综合征患者血中表达水平,探讨二者与不同类型冠脉综合征的相关性.方法 总共入组诊断为急性冠脉综合症的患者90例,进一步分为两组:急性心肌梗死(AMI)组(n=48),不稳定性心绞痛(UAP)组(n=42),以及另入组稳定性心绞痛60例稳定心绞痛(SAP)组,及健康体检者55例为对照组.采用酶联免疫吸附试验(ELISA法)、放射免疫法分别测定血清TN-C和hs-CRP水平,并在各组间进行比较.结果 AMI组和UAP组TN-C表达水平明显高于SAP组、对照组,差异有统计学意义(P＜0.05);SAP组TN-C水平高于对照组但差异无统计学意义(P＞0.05).AMI组、UAP组和SAP组hs-CRP水平明显高于对照组,差异有统计学意义(均P＜0.05),而AMI组和UAP组hs-CRP较SAP相比差异无统计学意义(均P＞0.05).结论 TN-C、hs-CRP在ACS患者血清中升高,TN-C可作为急性冠状动脉综合征斑块稳定性病变严重程度的预测因子.     

人可溶性B7-H3酶联试剂盒的研制及在肝病患者血清中水平的检测

目的:建立定量检测人的可溶性B7-H3(sB7-H3)酶联检测试剂盒,并分析肝病患者外周血中sB7-H3水平及其临床意义。方法:在已获得2株识别位点不同的小鼠抗人B7-H3单克隆抗体(4H7和2E6)基础上,采用单抗4H7为包被抗体,以经生物素标记的抗体2E6为检测抗体,以重组人可溶性B7-H3为标准品,建立可溶性B7-H3分子的酶标检测方法。在此基础上对健康献血者和不同肝病患者的血清样本进行了检测并分析其临床意义。结果:研制了sB7-H3酶联检测试剂盒,检测的线性范围是8.192～2 000 ng/L,批内、批间变异系数分别为3.35%和6.97%。酶标检测板在4℃放置1个月,变异系数(CV%)<±8.6,回收率为94%～117%,特异性试验显示无交叉反应,提示该检测试剂盒具有良好的灵敏度、稳定性和特异性。用该试剂盒分析不同肝病患者外周血sB7-H3水平后发现,(1)乙肝患者以及乙肝合并肝硬化或肝癌患者与健康对照组之间并不存在差异(P>0.05);(2)重症肝炎患者血清B7-H3水平低于健康对照组(P=0.0183);(3)血吸虫性肝硬化患者血清B7-H3水平显著高于健康对照组(P<0.01)。结论:建立了灵敏、特异的人sB7-H3酶联检测试剂盒,对肝病患者检测的结果表明,不同的肝病患者外周血sB7-H3表达水平不同,显示该试剂盒具有潜在的应用价值。     

Involvement of tenascin-C in proliferation and migration of laryngeal carcinoma cells

Tenascin-C (TN-C) is an extracellular matrix glycoprotein upregulated in various pathological processes. In this study, we investigated its distribution in dysplasia and carcinoma of the human larynx using immunohistochemistry and in situ hybridization (ISH) techniques. In all cancer tissues, TN-C immunostaining was markedly increased in the stroma, especially around the cancer cell nests. In addition, cytoplasmic staining of cancer cells was also observed in 62.5% of the invasive cases, the cells being distributed in the periphery of the nests adjacent to the stroma. TN-C mRNA signals in cancer cells were detected in all six cases examined by ISH. Furthermore, in vitro evaluation of the roles of TN-C demonstrated an increase in the proliferating cell fraction in a dose-dependent manner. In a wound closure assay, the addition of TN-C promoted migration. We conclude that TN-C secreted by cancer cells may be involved in their proliferation and migration in an autocrine fashion. Received: 5 March 1999 / Accepted: 7 June 1999     

Diagnostic utility of tenascin-C for evaluation of the activity of human acute myocarditis

Tenascin-C (TN-C) is an extracellular matrix protein that is expressed transiently in close association with tissue remodelling in various body sites. In the heart, TN-C is only present during early stages of development, is not expressed in the normal adult, but reappears in pathological states. The purpose of this study was to analyse the expression of TN-C in myocardial tissue from myocarditis patients, and to evaluate the diagnostic value of immunostaining for TN-C in the assessment of inflammatory activity in biopsy specimens. A total of 113 biopsy specimens obtained from 32 patients with a clinical diagnosis of acute myocarditis were examined by immunohistochemistry and in situ hybridization for TN-C. The immunostaining was semi-quantified and compared with histological diagnosis according to the Dallas criteria. Furthermore, serial biopsies from 22 patients were taken during convalescence, and sequential changes in TN-C levels were analysed. Expression of TN-C was specifically detected in endomyocardial biopsy specimens from patients with active-stage inflammation, and disappeared in healed stages. The degree of expression of TN-C correlated with the severity of histological lesions. These data suggest that TN-C reflects disease activity in cases of human myocarditis. Immunostaining for TN-C could enhance the sensitivity and accuracy of diagnosis using biopsy specimens.