利用生物素标记法和 Western blotting技术检测血小板膜糖蛋白

为了建立一种简便的血小板膜糖蛋白检测方法,用生物素标记血小板膜糖蛋白,然后用westernblotting法进行检测。结果显示生物素可标记十几种血小板膜糖蛋白。用以标记的生物素浓度以5mmol/L较为适宜。在Western blotting图谱上,血小板无力症患者的膜糖蛋白Ⅱb、Ⅲa与正常对照相比明显减少。结果提示该方法可用于血小板膜糖蛋白减少性疾病的检测。

用 Western Blot 法鉴定感染鸡外周血淋巴细胞中马立克氏病毒抗原

马立克氏病毒是鸡的一种高度致肿瘤性疱疹病毒，主要以淋巴瘤为特征，有关该病毒的致病机理还不清楚。本研究利用澳大利亚分离的ＭＤＶ致病毒株ＭＰＦ５７感染１日龄ＳＰＦ雏鸡，并在感染后４周对其外周血淋巴细胞中的ＭＤＶ特异性抗原进行了检测。结果表明，临床上呈急性感染并伴有明显症状的鸡的外周血淋巴细胞中的ＭＤＶ抗原分子量主要为２８、７３、６０和４９ｋＤａ；而无明显症状的鸡的外周血淋巴细胞中的ＭＤＶ抗原分子量主要为７３ｋＤａ。试验证明，Ｗｅｓｔｅｒｎｂｌｏｔ是一种较好的检测病毒抗原的方法，可以检测到荧光抗体方法检测不到的淋巴细胞中的ＭＤＶ特异抗原。

Interpretation Criteria for Standardized Western Blot for the Predominant Species of Borrelia Burgdorferi Sensu Lato in China

Objective Western blotting （WB;immunoblotting） is a widely used tool for the serodiagnosis of Lyme borreliosis （LB）,but so far,no generally accepted criteria for its performance and interpretation have been established in China.The present study was designed to determine the criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China,in which WB was produced with strain PD91 as the representative strain attributed to predominant genospecies Borrelia garinii of Borrelia burgdorferi sensu lato.Methods Approximately 13 bands between 14 and 100 kD were differentiated for strain PD91 by using Gel-Pro analysis software.In a study with 631 serum samples （taken from 127 patients with Lyme borreliosis and 504 controls）,all observed bands were documented.To establish criteria for a positive WB result for strain PD91,receiver operating characteristic （ROC） curves were used.Results The following interpretation criteria were recommended：for IgG,at least one band of P83/100,P58,P39,P30,OspC,P17,P66,and OspA;for IgM,at least one band of P83/100,P58,OspA,P30,OspC,P17 or P41.In addition,syphilis,leptospirosis and other related diseases should be excluded when the positive band is P41 in IgM.For IgG criteria,the sensitivity is 73.2%,the specificity is 99.4% and Youden index is 0.726;for IgM criteria,the sensitivity is 50.6%,the specificity is 93.1% and Youden index is 0.437.Conclusion Standardization of WB assays is necessary for comparison of results from different laboratories.Moreover,the criteria of other genospecies of Borrelia burgdorferi sensu lato should be determined in the future to complete the criteria of WB for the diagnosis of the Lyme disease in China.

A Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China

Objective To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. Methods FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and irnmunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples （159 patients with Lyme disease and 292 controls）, all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

Analysis of Protoscoleces-specific Antigens from Echinococcus Granulosus with Proteomics Combined with Western Blot

Objective To establish and optimize the proteomic analysis of protoscoleces-specific antigens from Echinococcus granulosus. To provide a foundation for identifying specific antigens in the soluble proteins of E. granulosus protoscoleces for further research. Methods Brood capsules were collected aseptically from fertile E. granulosus cysts from the livers of an infected patient. The fertile E. granulosus cysts were fractured, and protoscoleces were collected by centrifugation. The soluble proteins of protoscoleces were acquired using the 2D Quant kit according to the manufacturer＇s instructions. We employed two-dimensional electrophoresis （2-DE） combined with immunoblot assay （Western blot） to analyze the soluble components of E. granulosus protoscoleces antigens. The 2-DE and immunoblot maps obtained were analyzed with PDQuest 8.0 image analysis software. Results About 233 soluble protein spots were identified with Coomassie-stained gels. Most of the proteins had a molecular weight of 16 000 Da to 117 000 Da, and an isoelectric point value of 3.0 to 10.0. 2-DE immunoblot was conducted and 57 specific antigen spots were observed, among which 23 spots were identified. Conclusion 2-DE combined with Western blot is the key to successful proteomic analysis and presents a new possibility for searching the specific E. granulosus protoscoleces antigens.

Study of penetration mechanism of labrasol on rabbit cornea by Ussing chamber, RT-PCR assay, Western blot and immunohistochemistry

Labrasol, as a non-ionic surfactant, can enhance the permeation and absorption of drugs, and is extensively used in topical, transdermal, and oral pharmaceutical preparations as an emulsifier and absorption enhancer. Recent studies in our laboratory have indicated that labrasol has a strong absorption enhancing effect on different types of drugs in vitro and in vivo. This study was performed to further elucidate the action mechanism of labrasol on the corneal penetration. In this research, the fluorescein sodium, a marker of passive paracellular transport of tight junction, was selected as the model drug to assess the effect of labrasol on in vitro corneal permeability. To investigate the continuous and real-time influence of labrasol on the membrane permeability and integrity, the Ussing chamber system was applied to monitor the electrophysiological parameters. And, furthermore, we elucidated the effect of labrasol on excised cornea at the molecular level by application of RT-PCR, Western blot, and immunohistochemical staining. The results indicated that labrasol obviously enhance the transcorneal permeability of fluorescein sodium, and the enhancement was realized by interacting with and down-regulating the associated proteins, such as Factin, claudin-1 and β-catenin, which were contributed to cell-cell connections, respectively.

Detection of H pylori infection by ELISA and Western blot techniques and evaluation of anti CagA seropositivity in adult Turkish dyspeptic patients

AIM: To detect H pylori infection and to evaluate the anti CagA seropositivity in adult Turkish dyspeptic patients. METHODS: We evaluated anti-H pylori IgA, IgG and anti-CagA antibodies using commercial enzyme-linked immunoassay (ELISA) and Western blot in dyspeptic Turkish patients. H pylori status was determined by histology and rapid urease testing. RESULTS: Fifty-six patients were entered. Forty-eight (85.7%) out of the 56 patients were positive for H pylori. H pylori IgG seropositivity was 82.1%, IgA seropositivity 48.2%. CagA ELISA showed that IgG was positive in 50% and IgA in 30.4% of those with H pylori infections. Western blot showed that IgG seropositivity was 80.4% and IgA seropositivity 33.9%. Western blot detected IgG antibodies with reactivity to CagA in 50%, VacA in 62.5%, UreB in 87.5%, UreA in 80.4%, and OMP in 57.1%. None of the tests had a sensitivity and specifi city above 80%. CONCLUSION: None of these commercial tests seems clinically useful for H pylori detection in adult dyspeptic patients, while Western blot can give seropositivity and determine anti-CagA, VacA virulence factor status of Turkish dyspeptic patients in the Izmir region.

Expression of Elk-1 in Non-Small Cell Lung Cancer Detected by Western Blot and Tissue Microarray

Objective： The aim of this study was to investigate the Elk-1 （Ets like transcription factor-1） expression in non-small cell lung cancer （NSCLC） and normal lung tissues and the relationship between its expression and clinicopathological characters. Methods： To observe Elk-1 expression, western blot and immunochemistry （IHC） on tissue microarray （TMA） containing 118 lung cancers and their corresponding normal tissues were used. Results： In western blot and IHC on TMA, Elk-1 was highly expressed in NSCLC, while its expression was almost undetectable in normal lung tissues. Elk- 1 expression in NSCLC had no relationship with the patients＇ age, gender, smoking status and histological type, but had relationship with the differentiation degree, clinical stages and lymphonode metastasis. The expression was lower in early stage group （Ⅰ＋Ⅱ） than in advanced stage group （Ⅲ）, and lower in well-moderately differentiated group than in poorly differentiated group. The same trend was seen with lymphonode metastasis. Conclusion： The progression of NSCLC may be related with the increased Elk-1 expression, and Elk-1 may be regarded as a prognostic factor for NSCLC tissues.

Voltage profile generation for simultaneous multi-protein detection in western blot analysis

Western blotting is a popular technique for examining expression levels of proteins using gel-based electrophoretic fractionation followed by blotting and antibody reactions. Although this is a mature technique, one of the major limitations is the need to prepare an individual electrophoretic gel for each of the protein species to be analyzed. Since most analyses require the detection of multiple protein species, a procedure that allows utilization of a single gel for detecting multiple protein species should significantly save time and resources. In this paper, we developed a novel multiprotein detection device, which enabled simultaneous detection of several proteins species from a single electrophoretic gel. In this device, a protein transfer unit utilized a multi-anode plate that generated a non-uniform voltage profile. This voltage profile enabled uniform transfer regardless of molecular mass of proteins. In vitro experiments using samples, isolated from boneforming osteoblast cells, showed that the expression levels of 5 - 7 different proteins were detectable in the presence and absence of mechanical stimulation that activated genes necessary for bone formation. The result supports the notion that through simultaneous detection of multiple protein species, the described device contributes to reduction in procedural time and sample amounts, as well as a removal of variations among multiple gels.

Comparison of indirect immunofluorescence and western blot method in the diagnosis of hantavirus infections

BACKGROUND Serologic cross-reactivity between hantaviruses often complicates the interpretation of the results.AIM To analyze the diagnostic value of indirect immunofluorescence assay(IFA)and western blot(WB)in the diagnosis of hantavirus infections.METHODS One hundred eighty-eight serum samples from Puumala(PUUV)and Dobrava(DOBV)orthohantavirus infected patients were analyzed.Serology was performed using commercial tests(Euroimmun,Lübeck,Germany).RESULTS Using IFA,49.5%of acute-phase samples showed a monotypic response to PUUV,while 50.5% cross-reacted with other hantaviruses.The overall cross-reactivity was higher for immunoglobulin G(IgG)(50.0%)than for immunoglobulin M(IgM)(25.5%).PUUV IgM/IgG antibodies showed low/moderate reactivity with orthohantaviruses Hantaan(12.3%/31.5%),Seoul(7.5%/17.8%),DOBV(5.4%/28.1%),and Saaremaa(4.8%/15.7%).Both DOBV IgM and IgG antibodies were broadly reactive with Hantaan(76.2%/95.2%),Saaremaa(80.9%/83.3%),and Seoul(78.6%/85.7%)and moderate with PUUV(28.5%/38.1%).Using a WB,serotyping was successful in most cross-reactive samples(89.5%).CONCLUSION The presented results indicate that WB is more specific than IFA in the diagnosis of hantavirus infections,confirming serotype in most IFA cross-reactive samples.

疯牛病和羊痒病Western blotting检测方法的建立

以朊蛋白单抗AH6和碱性磷酸酶标记的马抗鼠酶标二抗建立了疯牛病和羊痒病的Western blotting检测方法。对Western blotting各种反应条件进行摸索,并确定了最佳工作条件,结果表明:匀浆缓冲液为RIPA时的最佳反应条件包括浓缩胶电泳电压为恒压90V,分离胶电泳电压为恒压160V,转印的最佳电压和时间为恒压100V1.5h;封闭液为3%BSA时,封闭15min,封闭效果最好;AH6的最佳稀释度为1∶4000,4℃下孵育过夜,马抗鼠二抗的最佳稀释度1∶1000,室温下孵育30min。采用已确立的反应条件对样品进行检测并与Prionics-Check WEST-ERN进口试剂盒的检测结果比较,发现其敏感性为100%,特异性为99.4%,与进口试剂盒(100%,100%)无显著差异,这为国产试剂盒研制提供了条件。

拟南芥DBB1a(double B-box 1a)蛋白的表达、纯化及Western blotting检测

PCR扩增拟南芥(Arabidopsis thaliana)DBB1a cDNA的保守区段(GenBank登录号:AT2G21320),转化到冷诱导表达载体pCold TF上,构建pCold-DBB1a重组质粒,转化大肠杆菌DH5a。15℃下IPTG诱导表达融合蛋白,并通过SDS-PAGE检测。证实目的蛋白以可溶形式在约20 kD处高效表达,与预期蛋白大小相吻合。表达蛋白经Ni琼脂糖凝胶亲和层析纯化,SDS-PAGE及Western blotting检测证实纯化后获得高纯度融合蛋白,这为进一步研究DBBl a功能奠定了基础。

快慢转法及不同滤膜和显色检测法在Western blotting中的应用分析

目的探讨快、慢转法及不同滤膜和显色检测法在Western blotting中的应用及各实验环节分析。方法采用MDA-MB-231细胞制备细胞蛋白,应用不同印迹膜[硝酸纤维素膜、聚偏乙烯二氟(PVDF)膜和阳离子尼龙膜]分别采用快转法和慢转法进行增强化学发光法(ECL)和DAB化学显色法检测,并对Western blotting的各实验环节进行了分析。结果(1)在快、慢转法中硝纤膜的蛋白预染marker条带略强于尼龙膜,而尼龙膜又略强于PVDF膜;PVDF膜和尼龙膜的正反面易于混淆,而硝纤膜的正反面不易混淆。(2)在快、慢转法中,PVDF膜的DAB化学显色法图像略强于尼龙膜和硝纤膜,而尼龙膜和硝纤膜无明显差异;与慢转法相比,快转的硝纤膜和尼龙膜中的蛋白条带略呈波浪状。(3)在慢转法中三种膜化学发光法的图像无明显背景,但在快转法中尼龙膜的背景较明显,而硝纤膜和PVDF膜亦无明显背景。结论应根据实验需要选用不同的印迹膜;慢转法通常优于快转法,增强化学发光法优于DAB化学显色法;增强化学发光法特异胜强、灵敏度高,是分析蛋白质表达较为理想的方法。

采用Western blotting的方法分析蚕蛹致敏原成份

目的 应用Western blotting的方法检测蚕蛹（silkworm chrysalis）浸出液中致敏原的成份，为蚕蛹致敏原的研究和临床诊断提供依据。方法取蚕蛹浸出液，经SDS-聚丙烯酰胺凝胶分离，再转印到PVDF膜上，封闭后将血清与膜条共同孵育，后与辣根过氧化物酶标记的羊抗人IgE抗体反应，显色底物用对氨基联苯胺。结果SDS-PAGE显示蚕蛹可辨条带有12条，分子量在14～94kD之间，其中主带有8条，分子量依次为80kD、68kD、62kD、50kD、29kD、23KD、18kD、16kD。Western blotting结果表明，22例蚕蛹过敏患者血清全部呈阳性反应，浸出液中共有6条致敏条带，其中分子量68kD和29kD是主要致敏组份，阳性反应率均为100％。结论蚕蛹分子量68KD和29kD的组份为主要致敏组份，结果可为开发适舍我国特色的蚕蛹变应原提供依据。

医学检验专业开设Western Blotting综合性实验初探

分子生物学是当前生命科学发展的主流，其理论与技术已广泛应用于各个领域。Western Blottlng作为分子生物学的常规技术，在医学领域具有不可忽视的地位。因此，在医学检验专业开设Western Blotting实验课程，已成为一项非常必要的实验教学内容。其一，Western Blotting实验是分子生物学与免疫学的有效结合体，有助于学生对前沿的、跨学科知识加深了解；

用western blotting方法检测乙醇固定细胞中细胞周期素的表达

用 western blotting法测定乙醇固定细胞中细胞周期素 (cyclin)的表达情况 ,探索 western blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后对同批标本进行蛋白同步分析的可行性。采用对数生长期的 Molt- 4细胞 ,经两种常规固定方法固定后 ,用 western blotting方法检测 cyclin A、B1 、D和 E的表达。另取乙醇固定经流式细胞仪分选的 G1 期和 G2 / M期细胞裂解后 ,用 western blotting方法检测不同时相细胞中 cyclin B1 的表达。结果显示从固定细胞中提取的蛋白经 western blotting检测可得到清晰且分子量正确的条带 ,两种不同方法固定的细胞中检测到的 cyclins表达未见明显差异。乙醇固定细胞经分选后提取蛋白行 western blotting检测可见 G2 / M期细胞的 cyclin B1 有明显表达而 G1 期细胞不明显。细胞进行固定、洗涤、染色和分选等处理后不影响 western blotting对其 cyclins表达的分析 ,说明用 western

Western blotting检测survivin封闭载体转染大肠癌SW620细胞的实验研究

【目的】探讨KNA interferenece(RNAi)对survivin在大肠癌细胞SW620表达的影响及siRNA干扰后诱导survivin蛋白表达降低的机制。【方法】构建survivin特异性的RNA封闭性载体survivin,通过培养大肠癌细胞SW620,survivin封闭性载体转染SW620细胞;Hoechest染色大肠癌细胞,通过荧光显微镜观察细胞形态变化;Western blotting分析肿瘤细胞的蛋白表达情况。【结果】DNA测序证实表达质粒构建成功;免疫荧光显微镜可见细胞凋亡小体;Western blotting显示:大肠癌survivin的蛋白表达明显弱于对照组。【结论】Survivin与大肠癌的发生存在一定的关系,RNAi能够下调survivin的表达。

高内涵分析及Western blotting法在蛋白质核质分布研究中的应用及比较研究

目的使用不同方法对蛋白质的核质分布进行检测并比较差异。方法细胞经免疫荧光染色后使用高内涵分析(high content analysis,HCA)对蛋白质的细胞质或细胞核总荧光强度进行分析;分别提取胞质及胞核蛋白,使用Western blotting法对核质组分中的蛋白质含量进行鉴定。结果两种方法均可检测到蛋白质的出核转运;经定量分析,HCA法检测到的HuR蛋白质/核比升高倍数小于Western blotting法。结论使用HCA法检测蛋白质核质定位具有准确、客观、成本低、快捷、多参数等特点,不失为Western blotting的有效补充及替代方法。

肝脏特异性表达载体的构建及Western blotting检测

研究靶向于肝细胞的组织特异性siRNA,实现siRNA基因治疗的组织特异性,可用于特异性治疗乙肝,突破RNA干扰技术在临床应用的一大障碍。用靶向于EGFP的siRNA(以下简称siEGFP)替代靶向于乙肝病毒保守区的siRNA,构建可以在肝细胞中特异性表达的载体,用来表达siRNA。将目标载体分别转染肝癌细胞HepG2、乳腺癌细胞MDB-MB-231、人胚肾细胞293,在蛋白质水平上检验siRNA抑制的组织特异性。Western blotting结果证实,siEGFP在肝组织来源的细胞系HepG2中抑制效率明显高于其他两组细胞。构建的载体可以在肝癌细胞系中特异性的表达siRNA,而在其他组织细胞中不表达,实现了组织特异性。进一步将siEGFP替换为抑制HBV表达的siRNA,同样可以实现肝脏特异性的表达。

Western blotting中低丰度蛋白转膜方法改进

目的:改进Western blotting转膜方法,提高低丰度蛋白转膜效率。方法:在Western blotting转膜过程中,采用不同孔径的PVDF膜、不同的转膜时间及不同的甲醇含量进行转膜。转膜后,考马斯亮蓝染色观察PAGE胶上蛋白残留情况,ECL发光法检测FGFR1曝光强度。结果:通过改变膜孔径大小和转膜时间所得实验结果可见,孔径为0.22μm的PVDF膜转移2h的转膜效率更高。通过改变转膜液中甲醇含量所得实验结果可见,甲醇含量为10%转膜效率更高。结论:本实验对Western blotting转膜过程中低丰度蛋白的转膜方法进行了成功改进,改进后能提高蛋白显影强度,具有广泛的应用前景,是一种方便可行的操作方法。