催化信号放大系统的制备及其在免疫组化和原位杂？…

目的：应用本实验室制备的催化信号放大（ｃａｔａｌｙｚｅｄ ｓｉｇｎａｌ ａｍｐｌｉｆｉｃａｔｉｏｎ,ＣＳＡ）剂试,提高免疫组化（ＩＨＣ）和原位杂交（ＩＳＨ＿的敏感性。方法：ＣＡＳ法应用于ＩＨＣ检测５０例１０种不同类型的肿瘤切片中４０种不同的抗原和ＩＳＨ检测６种不同的ＲＮＡ成分,并与相应的常规方法进行敏感性比较。同时分析了存在的问题。结果：ＣＡＳ－ＩＨＣ法较传统的ＰＡＯＰ法、ＡＢＣ法和ＬＳＡＢ法敏感     

催化信号放大系统的制备及其在免疫组化和原位杂交中的应用

目的：应用本实验室制备的催化信号放大（ｃａｔａｌｙｚｅｄ　ｓｉｇｎａｌ　ａｍｐｌｉｆｉｃａｔｉｏｎ,ＣＳＡ）试剂,提高免疫组化（ＩＨＣ）和原位杂交（ＩＳＨ）的敏感性。方法：ＣＳＡ法应用于ＩＨＣ检测５０例１０种不同类型的肿瘤切片中４０种不同的抗原和ＩＳＨ检测６种不同的ＲＮＡ成分,并与相应的常规方法进行敏感性比较。同时分析了存在的问题。结果：ＣＳＡ－ＩＨＣ法较传统的ＰＡＰ法、ＡＢＣ法和 ＬＳＡＢ法敏感 ５００～１０００倍,较 ＥｎＶｉｓｉｏｎ法敏感 ２０～１００倍。 ＣＳＡ－ＩＳＨ法可使探针浓度从 ３ μｇ／ｍｌ降到 ０． ５～１μｐ／ｍｌ,杂交时间从 ２ ｄ缩短到 １ｄ,杂交阳性率明显提高。结论：本实验室制备的 ＣＳＡ系统与常规方法相比具有较高的敏感性、稳定性和低背景等优点,完全可取代进口试剂,非常适合于一抗昂贵、抗原性弱的ＩＨＣ检测,还可作为常规ＩＳＨ染色方法。     

p73基因在结肠癌组织中的表达及其临床意义

目的：研究p73基因在结肠癌组织中的表达及其与结肠癌发生，发展的关系。方法：采用逆转录聚合酶链反应和催化信号放大法检测48例结肠癌及癌旁组织中p73基因和P73蛋白的表达情况，分析其表达与临床分期及病理分级的关系。结果：结肠癌组织中p73mRNA的表达明显高于癌旁组织，免疫组织化学显示，48例结肠癌标本中21例呈阳性表达，癌旁组织无阳性表达。结肠癌不同临床分期之间以及不同分化程度之间P73阳性表达率有显著差异（分别为P＜0．005和P＜0．05）。结论：结肠癌组织中p73基因过主工表达，其蛋白表达水平与结肠癌临床分期，组织学分型有关。     

骨肉瘤组织中 Hedgehog 信号通路的表达及意义

目的：研究Hedgehog信号通路在骨肉瘤组织中的表达情况及临床意义。方法采用免疫组化法分别检测31例骨肉瘤、21例骨良性肿瘤（13例骨软骨瘤、8例骨样骨瘤）组织中IHH、PTCH、SMO和Gli1蛋白的表达情况,分析此4种蛋白相关关系以及其表达水平与骨肉瘤临床特征的关系。结果骨肉瘤中IHH、PTCH、SMO和Gli1蛋白在骨肉瘤组织中的阳性率分别为70．97％、72．73％、70．97％、70．97％,与骨良性肿瘤（19．05％、14．28％、14．28％、9．5％）比较,差异均有统计学意义（ P＜0．001）。 IHH、PTCH、SMO和Gli1蛋白表达与患者年龄、性别、临床分期、肿瘤部位、肿瘤大小及是否肺转移无关;相关分析显示IHH和PTCH、SMO、Gli1蛋白在骨肉瘤组织中的表达呈正相关。结论 Hedgehog信号通路中的IHH、PTCH、SMO和Gli1蛋白异常高表达,提示Hedgehog信号通路可能在骨肉瘤的发生和发展中起重要作用。     

基于柯氏法血压信号放大装置的研制

为了使柯氏法测量的血压值结果进行放大,便于学生对血压值做出正确的判断。作者提取柯氏法血压时的振动信号,利用医学换能器把这种信号转换为电信号,再通过放大电路,把电信号进行放大。再利用换能器把这种电信号转换为声音信号,并把声音信号进行放大。     

免疫组织化学法检测非小细胞肺癌表皮生长因子受体突变

目的:采用免疫组织化学(immunohistochemistry,IHC)检测非小细胞肺癌(non-small cell lung cancer,NSCLC)常见的表皮生长因子受体(epidermal growth factor receptor,EGFR)突变,并对IHC检测的敏感性、特异性和成本-效益进行评估.方法...     

胶体金标法与酶免疫法检测乙肝表面抗原的比较

在临床免疫检测工作中，发现用酶免疫法(ELISA)和用胶体金标法(GICA)测乙肝表面抗原(HBsAg)有时会有不相符的现象，因此用这两法同时检测HBsAg，以比较ELISA和GICA的特异性和敏感性，现将比较结果报告如下：     

基于双重信号放大的电化学免疫法对前列腺特异性抗原的检测

以谷胱甘肽修饰的CdTe量子点(GSH-CdTe QDs)与金纳米粒(AuNPs)形成复合材料(AuNPs@GSH-CdTe)为信号标记物,并在还原氧化石墨烯(rGO)和AuNPs双重信号放大的作用下,建立了一种高灵敏检测前列腺特异性抗原(PSA)的三明治免疫夹心式电化学方法。在rGO/AuNPs表面固定二抗蛋白(Ab₂),捕获目标物PSA和标记有一抗蛋白(Ab₁)的AuNPs@GSH-CdTe信号物,形成“三明治”免疫夹心结构。经HNO₃溶解后,采用方波溶出伏安法(SWSV)测定酸解的Cd²⁺的峰电流用于定量分析PSA。其中AuNPs较大的比表面积以及较好的生物相容能力,达到了成功装载抗体以及放大信号的效果,同时具有较大表面积的rGO起到了协同放大的作用。所构建的免疫分析方法实现了对肿瘤标志物PSA的检测,其线性范围为0.5~200 ng/mL,检测限为5.0 pg/mL,并且该方法专属性、重复性以及稳定性好。此外,在实际样品的检测中,加标样回收率为98.20%~106.2%,结果准确度良好,为检测PSA提供了准确可靠且灵敏度高的新方法。     

端粒酶催化亚单位在细胞学诊断肺癌中的表达

目的:检测支气管镜涂片中端粒酶催化亚单位(hTERT)的表达,探讨其与肺癌发生的关系,探索细胞学诊断肺癌的新途径。方法:应用免疫组化和原位杂交的方法检测137例支气管镜涂片中hTERT和hTERTmRNA的表达,并与临床病理资料及细胞学诊断进行相关性分析研究。结果:137例病例中,hTERT和hTERTmRNA表达阳性率在细胞学诊断为癌细胞组分别为67.86%(57/84)和79.76%(67/84);疑癌或异型细胞组分别为46.67%(7/15)和60.00%(9/15);未见癌细胞组分别为5.26%(2/38)和39.47%(15/38)。二者的表达在癌细胞组明显高于其他2组(P<0.05)。其中有组织学对照的105例,hTERT和hTERTmRNA阳性表达率与细胞学诊断阳性率相一致。hTERT和hTERTmRNA的阳性表达率呈正相关性(r=0.994,P<0.05),但这二者的表达率与组织病理类型及其他临床特征差异均无显著性(P>0.05)。结论:端粒酶在支气管涂片中的阳性表达与肺癌的发生密切相关。hTERT和hTERTmRNA在癌组织中均存在高表达,且比传统形态学诊断更敏感。而hTERT蛋白的表达在细胞学涂片中更可靠(假阳性率低)。因此检测这二者的表达有助于提高肺癌的细胞学检出率,对早期发现、诊断肺癌有重要意义。     

TUNEL法原位检测凋亡细胞的某些改进

目的 :探索避免TUNEL法检测凋亡细胞出现的假阳性和消除非特异性反应的方法。方法 :脑和心肌组织石蜡切片 ,改进的TUNEL染色。选用多聚甲醛固定 ,蛋白酶K修复抗原 ,将H2 O2 封闭内源性过氧化物酶放在反应液标记DNA片段之后 ,先后用小牛血清或羊血清两次封闭非特异性反应。结果 :证实本实验方法避免了假阳性 ,背底十分清亮。结论 :改进后的TUNEL方法适用于凋亡细胞的检测     

应用"酶生物信号放大系统"放大DNA压电传感器检测信号的研究

目的 研究"酶生物信号放大系统"用于放大DNA压电传感器检测信号的可行性和有效性.方法 在压电石英基片金膜上固定标有biotin的金黄色葡萄球菌oligo探针,加入HRP和底物DAB生成附着于金膜的不可溶沉淀,观察频率对沉淀的响应.在金膜上固定金黄色葡萄球菌oligo探针后与标有biotin的靶序列杂交,加入HRP和底物DAB.比较直接检测杂交时频率变化值和酶系统放大后信号频率变化值.结果 该系统生成的沉淀可显著降低谐振频率.经酶系统放大后检测信号频率变化值显著高于直接检测信号频率变化值.结论 酶生物信号放大系统可有效地放大DNA传感器检测信号,降低非特异信号的产生.     

Preparation of CSA system and its application in IHC and ISH

CatalyZed st~ amplification(CSA) system was successfully ugh in ilnmunoaSSay by BobIDw et alll] and Adams et allzj reported that they aPPlied CSA system to immUnohistOChendstw (IHC ) and in situ hybridization(ISH), ~tively. The core reagent of CSA system is~de crass--linking a kind of ~er molecule such asbiotin, digokin, fluorescein or heaVy mend ion, etc. ThePrinciple of foe system is: twide is catalyZed by horseedsn pemtibe(ar) with HZOz to fonn covatent conjugation siteS in 15 …     

信号放大时间分辨荧光分析技术的研究进展及应用

信号放大时间分辨荧光分析技术是一种新型的超微量的标记分析技术。它是以生物信号放大为基础，以镧系元素螯合物为标记物，以时间分辨和波长分辨为测量方法，充分利用了酶标记分析和PCR扩增等分析技术优点的技术。它的独特优点是非同位素和超灵敏度。本综述介绍了TRFA的特点以及酶放大、二次酶放大、铕纳米颗粒、滚动循环放大、免疫PCR和荧光淬灭分析等信号放大时间分辨荧光分析技术的研究进展及应用。信号放大时间分辨荧光分析技术在生物分析中极其广泛的应用，显示出具有很好的发展前景。     

Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip

The identification of tumor-related microRNAs (miRNAs) exhibits excellent promise for the early diagnosis of cancer and other bioanalytical applications. Therefore, we developed a sensitive and efficient biosensor using polyadenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) for miRNA analysis based on strand displacement reactions on gold nanoparticle (AuNP) surfaces and electrokinetic signal amplification (ESA) on a microfluidic chip. In this FSNA, polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au. The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA, resulting in fluorescence quenching of FSNA probes induced by AuNP-based resonance energy transfer. Reporter DNA was replaced in the presence of target miRNA, leading to the recovery of reporter-DNA fluorescence. Subsequently, reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA. Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM. This method could also be used to detect miRNA-21 in human serum and urine samples, with recoveries of 104.0%–113.3% and 104.9%–108.0%, respectively. Furthermore, we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs (miRNA-21, miRNA-141, and miRNA-375), which increased the detection efficiency. Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.     

Comparison of polymerase chain reaction and catalyzed signal amplification in situ hybridization methods for human papillomavirus detection in paraffin-embedded cervical preneoplastic and neoplastic lesions

BACKGROUND: Two molecular methods for HPV genotyping in formalin-fixed, paraffin-embedded tissue were evaluated: in house polymerase chain reaction assay (PCR) with consensus and type-specific primers and a novel procedure of in situ hybridization-a catalyzed signal amplification system (CSA-ISH, Genpoint, DAKO, Glostrup, Denmark). The number of HPV positive cases and detected viral types were compared in cervical biopsies and cone specimens according to histopathological diagnosis. Primer efficiency in detecting various types of HPV by PCR method was evaluated. METHODS: DNA samples (101) were used as a template to amplify with three pairs of consensus (MY09/11, GP5+/6 +, CPI/IIG) and four type-specific HPV primers (HPV-6/11, 18, 16 and 33). The according histological tissue sections were analyzed with CSA-ISH method, using commercial HPV biotinylated probes HPV-6/11, 16/18 and 31/33/51. RESULTS: The degree of concordance for PCR and CSA-ISH was 64.4%. In 63 of 101 samples (62.4%), HPV was detected by PCR, while only 35 (34.7%) were positive using CSA-ISH. CSA-ISH found lower percentages for all HPV types, except HPV-6/11. A lower percentage of positive results in all high-grade lesions was detected by CSA-ISH. Multiple infections were detected by PCR in only one sample and in three samples by CSA-ISH. Detection with My09/11 primers followed by Gp5+/6+ primers, in nested reaction, gave the highest number of positive results: 58 of 63 (92%). None of the samples diagnosed as condylomata planum or CIN I was positive for HPV-6/11 (low risk type), which was detected exclusively in condylomata acuminatum group. CONCLUSIONS: A significantly higher number of positive samples was detected with PCR than with CSA-ISH method. CSA-ISH method should be improved, especially in detecting HPV in high-grade lesions. CSA-ISH may be more accurate in detection of multiple infections. GP5+/6+ in nested reaction after MY09/11 detected the highest number of positive results. Samples diagnosed as benign lesions positive on HPV-X must be monitored as possible candidates for progression. CIN I lesions, which were HPV negative, probably will not progress. This finding may be important in planning therapy and avoiding unnecessary treatment.     

荧光定量聚合酶链反应与分支链DNA信号扩增法检测血清HBV DNA的比较

目的了解荧光定量聚合酶链反应(PCR)法与分支链DNA信号扩增(bDNA)法在检测血清中HBV DNA含量上的优缺点.方法分别用这两种方法平行检测44份乙型肝炎患者的血清标本,并与定性PCR检测结果比较.同时用荧光定量PCR法观察15例乙肝患者干扰素治疗期间HBV DNA含量变化.结果①荧光定量PCR法和bDNA法比较,a前者测出的HBV DNA含量均值为(3.50×105±33.6)copies/μl;后者为(3.07×105±12.9)copies/μl,两者比较差异无显著性(P＞0.05).b前者的HBV DNA阳性率79.5%;后者及常规PCR定性法均为59.1%.荧光定量PCR法的阳性检出率显著高于bDNA法及常规定性PCR法(P＜0.05).②15例中5例获完全应答的乙型肝炎患者其血清HBV DNA含量在治疗16周内均逐渐下降至完全转阴.结论荧光定量PCR法与bDNA法在检测血清HBV DNA含量上,结果大多一致,且敏感性强,检验也较简便、价廉,并能较好评价和检测乙肝患者抗病毒疗效,宜于临床推广.     

酪氨信号放大间接免疫荧光组织化学方法

0　引言　免疫组织化学方法是对细胞及细胞内的某种抗原物质进行定性、定位或定量检测的方法 .随着标记技术的不断改进 ,使得免疫组织化学方法检测的敏感性大大增强 ;随着显微镜技术的发展 ,且与计算机技术的不断结合 ,可以对抗原物质作更加精确和全面的定位 .我们应用酪氨信号放大间接免疫荧光组织化学方法 ,并结合激光共聚焦显微镜术对脑动脉 Y1 受体免疫反应物质分布进行了观察 .1　材料和方法1.1　酪氨信号放大间接免疫荧光组织化学方法　 11只 SD大鼠 (2 0 0～ 2 5 0 g) ,戊巴比妥钠 (5 0 mg· kg- 1 ,ip)麻醉 ,经心脏灌注 37℃生理…     

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease IV-assisted exponential signal amplification

Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3′-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3′-PO₄ end of the substrate and generates 3′-OH, TdT can effectively elongate the 3′-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3′-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10⁻³ U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection.