狂犬病毒抗体ELISA检测试剂盒的改进研究

为了提高狂犬病毒抗体检测的灵敏度和特异性,采用狂犬病毒单克隆抗体包被酶标板,再分别加入重组的狂犬病毒糖蛋白或细胞培养抗原做固相层的方法(抗体捕捉法),用传统的间接ELISA法做对照,按常规方法检测抗狂犬病毒抗体。结果显示,抗体捕捉法的非特异性反应低于间接法,而灵敏度达到0．51U水平,高于间接法。在800份临床标本检测中,检出率明显高于间接法。用15份阳性血清作小鼠中和试验,并和抗体捕捉ELISA法比较具有高度的一致性。试验结果充分表明,该方法优于传统的ELISA间接法。因此可作为临床注射狂犬疫苗后检测血清中狂犬病毒抗体的常规方法。     

应用酶联免疫吸附试验(ELISA)快速检测狂犬病毒抗体的研究

国内检测狂犬病毒抗体一直用小鼠接种的方法,尚未见快速检测的研究报道,在建立了狂犬病毒组化免疫酶和免疫荧光方法后,由于防治狂犬病需要更敏感的方法。又建立了酶联吸附试验(ELISA)。 采用新鲜配制的磷酸铝凝胶吸附和释放,结合超速离心来提纯。浓缩组织培养的狂犬病毒,以此作为包被抗原,用辣根过氧化酶标记的SPA为第2抗体,底物为磷苯二胺,作间接ELISA测定小鼠血清中的     

改良抗体结合实验检测灭活狂犬病疫苗效价

目的:建立抗体结合试验检测狂犬病疫苗(aG株)效价的方法。方法:将待检测疫苗与疫苗标准品梯度稀释后分别加入人抗狂犬病毒免疫球蛋白国家标准品中和1 h,之后加入80%感染剂量的狂犬病毒CVS-11,体外中和1h后接种BSR细胞,培养24 h后免疫荧光染色,在显微镜下观察结果,通过检测剩余病毒量计算待检疫苗的效价,同时与小鼠中和试验法(NIH法)测定狂犬病疫苗效价进行比较。结果:2种方法对8个样品效价的检测结果无显著统计学差异(P=0.997,配对t检验)。结论:初步建立了改良抗体结合试验,可用于狂犬病疫苗中间产品的质量控制。     

狂犬病毒糖蛋白的克隆表达及其在中和抗体检测中的应用

目的:制备基因工程表达的狂犬病毒糖蛋白优势表位抗原,并评价其在疫苗免疫后中和抗体检测中的应用价值。方法:TRIzol法从狂犬病疫苗中提取总RNA,经RT-PCR获得糖蛋白目的基因片段,构建相应的原核表达质粒,转化大肠杆菌HB101,诱导表达获得纯化重组蛋白,以重组蛋白作为包被抗原,初步建立检测糖蛋白中和抗体的ELISA方法。结果:获得狂犬病毒糖蛋白优势表位区段抗原,建立了糖蛋白中和抗体ELISA检测方法。该检测方法对59例健康献血员血浆样本检测特异性为98.31%(58/59),接种狂犬疫苗免疫个体血浆样本抗体阳性率为98.95%(94/95)。结论:基因工程表达的狂犬病毒糖蛋白优势表位抗原可用于人接种狂犬疫苗后疫苗免疫效果评价。     

基于狂犬病病毒G蛋白scFv介导的靶向shRNA制备与鉴定

【目的】探讨以狂犬病病毒G糖蛋白单链抗体介导的载体表达shRNA靶向制剂,靶向抑制狂犬病毒复制的可行性。【方法】应用PCR技术获得狂犬病毒G糖蛋白单链抗体scFv(G)和绿脓杆菌跨膜区-酵母DNA结合结构域ETA-GAL4基因,通过搭桥PCR法获得scFv(G)-ETA-GAL4(SEG)嵌合基因;克隆至原核表达载体pET28a(+),构建重组表达质粒pET28a(+)-scFv(G)-ETA-GAL4(pET28a-SEG);在大肠杆菌BL21(DE3)中经IPTG诱导表达,利用镍柱亲和层析法纯化包涵体,经复性、鉴定制得SEG蛋白;ELISA法检测表达蛋白与狂犬病毒特异结合活性;将SEG蛋白与含shRNA的质粒(pRNATU6.3-shRNA)连接制成靶向shRNA,接入100 TCID50狂犬病毒感染BHK-21细胞,35 h观察细胞中绿色荧光蛋白(GFP)表达情况;48 h用直接免疫荧光抗体试验测定复合物抑制病毒效果。【结果】克隆得到1557 bp的SEG蛋白编码基因,大肠杆菌中成功表达57 KDa的SEG蛋白,能与抗His的单克隆抗体发生特异性反应,SEG蛋白经镍柱纯化、复性后得率为2.8 mg/mL。ELISA试验证明SEG蛋白在一定浓度范围内与RV结合呈正相关。细胞试验表明GFP在细胞内得到表达;直接免疫荧光试验测定该复合物能抑制76%病毒复制。【结论】SEG蛋白能与携带shRNA的质粒结合,可运送该质粒至RV感染BHK-21细胞中,抑制狂犬病毒的复制。     

狂犬病病毒aG株糖蛋白膜外区的原核表达及纯化

旨在有效检测狂犬病毒抗原及抗体。以狂犬病病毒aG株为模板,反转录扩增糖蛋白膜外区基因,并进行序列测定。将目的片段与表达载体pET-32a(+)分别双酶切、胶回收后相连并转化至Rosseta(DE3)菌株。选取阳性克隆经IPTG诱导表达后,进行SDS-PAGE和Western blotting及质谱鉴定。结果显示,在大肠杆菌中成功表达了糖蛋白膜外区。本研究为建立检测狂犬病毒抗原及抗体的ELISA诊断方法奠定了基础。     

狂犬病病毒抗体胶体金检测试纸的制备

通过胶体金免疫层析技术建立一种特异、便捷、快速的狂犬病病毒抗体检测方法,对犬等动物免疫狂犬病疫苗后的抗体水平监测提供参考。用醋酸锌沉淀法沉淀狂犬病病毒CVS11,Sepharose 4FF进行层析纯化。用柠檬酸三钠还原法制备的胶体金,标记纯化的狂犬病病毒,喷于试纸的结合释放垫 (金标垫),将SPA (葡萄球菌表面A蛋白) 和纯化的兔抗狂犬病病毒IgG分别喷于试纸的T (检测线) 处和C (对照线) 处,组装试纸条。用制备的试纸条对261份犬血清进行检测,与快速荧光灶抑制试验 (RFFIT) 检测的结果一致;对已知效价的犬狂犬病毒中和抗体 (VNA) 大于0.5 IU,结果为阳性;对狂犬病病毒中和抗体 (VNA) 小于0.5 IU/mL的血清,结果为阴性。制备的狂犬病病毒抗体胶体金检测试纸检测犬血清抗体,具有特异、便捷、快速的特点,能够检测出狂犬病病毒中和抗体大于0.5 IU的血清,适用于临床犬血清抗体水平监测,具有良好的应用前景。     

狂犬病毒单克隆抗体的杂交瘤细胞系的建立

Wikter于1978年首先建立了狂犬病毒单克隆抗体(McAb),并用它发现了狂犬病毒株的抗原差异,而国内尚未见到有关狂犬病毒单克隆抗体的研究报道。为此,在建立快速检测狂犬病毒抗原和抗体的基础上又进行了狂犬病毒单克隆抗体的研究。 将感染狂犬病毒aG株(我国狂犬疫苗生产毒种)后发病的BALB/c乳鼠脑病毒免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞Sp2/0融合。融合率为95.7％(401/419),阳性克隆占26.8％。经克隆化     

人源抗狂犬病毒单克隆抗体载体的构建及烟草转基因研究(四)

为了构建高效表达人源抗狂犬病毒单克隆抗体载体,首先对人源抗狂犬病毒抗体(SO57)重链和轻链编码基因的密码子进行偏好性改造,添加增强外源基因表达的控制元件,然后分别与花椰菜花叶病毒35s启动子和木薯叶脉花叶病毒启动子融合,连接至植物表达载体pBI121上,然后将构建好的载体转入农杆菌LB4404,采用叶盘法转化烟草叶片.用分子生物学技术,对6株转基因烟草进行检测,电泳检测结果均为阳性.用酶联接免疫吸附剂法,检测6株烟草叶片中人源抗狂犬病毒单克隆抗体的表达.结果表明,6株烟草均成功表达人源抗狂犬病毒抗体.     

人源抗狂犬病毒二硫键稳定性Fv抗体的生物学特性研究

对抗狂犬病毒scFv进行稳定性改构 ,纯化制备有活性的人源抗狂犬病毒二硫键稳定性Fv抗体片段 (dsFv) ,然后对其生物学活性进行研究。将dsFvV_H 、V_L 基因在原核表达系统pET22b(+) BL21(DE3)中表达 ,将其包涵体分别在变性缓冲液中溶解 ,稀释后加入折叠缓冲液使其折叠形成有活性的dsFv抗体片段 ,上Ni-NTA柱进行纯化。然后以其亲本scFv作为对照 ,对dsFv的亲和力、稳定性以及体外中和活性进行评价。结果显示 ,与其母本抗体scFv相比 ,改构后的抗狂犬病毒dsFv保持了对狂犬病毒Vero疫苗的特异性识别能力 ,而且其亲和力明显提高 ;抗狂犬病毒dsFv在血清和BSA中的稳定性有明显的改进 ,而且其热稳定性和抵抗尿素化学变性的能力亦大大改进 ;蚀斑减少中和实验显示 ,抗狂犬病毒dsFv抗体片段能特异中和狂犬病毒 ,阻止狂犬病毒对Vero细胞的吸附 ,抑制狂犬病毒对靶细胞的感染 ,从而导致蚀斑减少甚至消失 ;scFv抗体片段仅可部分抑制蚀斑的形成 ,但不能全部抑制。这为进一步研究抗狂犬病毒dsFv基因工程抗体的免疫保护作用及其在临床的应用奠定了基础。     

A bioinformatics method for predicting long noncoding RNAs associated with vascular disease

Long noncoding RNAs (lncRNAs) play important roles in human diseases including vascular disease. Given the large number of lncRNAs, however, whether the majority of them are associated with vascular disease remains unknown. For this purpose, here we present a genomic location based bioinformatics method to predict the lncRNAs associated with vascular disease. We applied the presented method to globally screen the human lncRNAs potentially involved in vascular disease. As a result, we predicted 3043 putative vascular disease associated lncRNAs. To test the accuracy of the method, we selected 10 lncRNAs predicted to be implicated in proliferation and migration of vascular smooth muscle cells (VSMCs) for further experimental validation. The results confirmed that eight of the 10 lncRNAs (80%) are validated. This result suggests that the presented method has a reliable prediction performance. Finally, the presented bioinformatics method and the predicted vascular disease associated lncRNAs together may provide helps for not only better understanding of the roles of lncRNAs in vascular disease but also the identification of novel molecules for the diagnosis and therapy of vascular disease.     

Maximum likelihood estimation of reviewers' acumen in central review setting: categorical data

Successfully evaluating pathologists' acumen could be very useful in improving the concordance of their calls on histopathologic variables. We are proposing a new method to estimate the reviewers' acumen based on their histopathologic calls. The previously proposed method includes redundant parameters that are not identifiable and results are incorrect. The new method is more parsimonious and through extensive simulation studies, we show that the new method relies less on the initial values and converges to the true parameters. The result of the anesthetist data set by the new method is more convincing.     

Using topic modeling to detect cellular crosstalk in scRNA-seq

Cell-cell interactions are vital for numerous biological processes including development, differentiation, and response to inflammation. Currently, most methods for studying interactions on scRNA-seq level are based on curated databases of ligands and receptors. While those methods are useful, they are limited to our current biological knowledge. Recent advances in single cell protocols have allowed for physically interacting cells to be captured, and as such we have the potential to study interactions in a complemantary way without relying on prior knowledge. We introduce a new method based on Latent Dirichlet Allocation (LDA) for detecting genes that change as a result of interaction. We apply our method to synthetic datasets to demonstrate its ability to detect genes that change in an interacting population compared to a reference population. Next, we apply our approach to two datasets of physically interacting cells to identify the genes that change as a result of interaction, examples include adhesion and co-stimulatory molecules which confirm physical interaction between cells. For each dataset we produce a ranking of genes that are changing in subpopulations of the interacting cells. In addition to the genes discussed in the original publications, we highlight further candidates for interaction in the top 100 and 300 ranked genes. Lastly, we apply our method to a dataset generated by a standard droplet-based protocol not designed to capture interacting cells, and discuss its suitability for analysing interactions. We present a method that streamlines detection of interactions and does not require prior clustering and generation of synthetic reference profiles to detect changes in expression.     

Authenticity of ancient-DNA results: a statistical approach

Although there have been several papers recommending appropriate experimental designs for ancient-DNA studies, there have been few attempts at statistical analysis. We assume that we cannot decide whether a result is authentic simply by examining the sequence (e.g., when working with humans and domestic animals). We use a maximum-likelihood approach to estimate the probability that a positive result from a sample is (either partly or entirely) an amplification of DNA that was present in the sample before the experiment began. Our method is useful in two situations. First, we can decide in advance how many samples will be needed to achieve a given level of confidence. For example, to be almost certain (95% confidence interval 0.96-1.00, maximum-likelihood estimate 1.00) that a positive result comes, at least in part, from DNA present before the experiment began, we need to analyze at least five samples and controls, even if all samples and no negative controls yield positive results. Second, we can decide how much confidence to place in results that have been obtained already, whether or not there are positive results from some controls. For example, the risk that at least one negative control yields a positive result increases with the size of the experiment, but the effects of occasional contamination are less severe in large experiments.     

An improved algorithm for the prediction of optimum and suboptimum folding structures of long single-stranded RNA

We have previously reported an algorithm (Yamamoto and Yoshikura,1985) for the prediction of optimum and suboptimum RNA foldingstructures. The calculation data was presented as an ‘informationmap’. However, the result was affected by the startingpoint of calculation. In this paper, we have improved the methodso that the result will not be affected by the starting point.In addition, we present a method of converting the informationmap into a set of pictures of optimal and suboptimal molecularstructures. Received on May 19, 1986; accepted on December 22, 1986     

Can the false-discovery rate be misleading?

The decoy-database approach is currently the gold standard for assessing the confidence of identifications in shotgun proteomic experiments. Here, we demonstrate that what might appear to be a good result under the decoy-database approach for a given false-discovery rate could be, in fact, the product of overfitting. This problem has been overlooked until now and could lead to obtaining boosted identification numbers whose reliability does not correspond to the expected false-discovery rate. To overcome this, we are introducing a modified version of the method, termed a semi-labeled decoy approach, which enables the statistical determination of an overfitted result.     

Prediction of binding hot spot residues by using structural and evolutionary parameters

In this work, we present a method for predicting hot spot residues by using a set of structural and evolutionary parameters. Unlike previous studies, we use a set of parameters which do not depend on the structure of the protein in complex, so that the predictor can also be used when the interface region is unknown. Despite the fact that no information concerning proteins in complex is used for prediction, the application of the method to a compiled dataset described in the literature achieved a performance of 60.4%, as measured by F-Measure, corresponding to a recall of 78.1% and a precision of 49.5%. This result is higher than those reported by previous studies using the same data set.     

Matrilateral biases in the investment of aunts and uncles

In a study of the kin investment of aunts and uncles we show that the laterality effect expected as a result of paternity uncertainty is statistically reliable but somewhat smaller than the sex effect. Matrilateral aunts invest significantly more than patrilateral aunts, and the same is true for uncles. Regardless of laterality, however, aunts invest significantly more than uncles. Multivariate controls show that the matrilateral bias is fully independent of any age or distance confounds that might result from sex differences in age at marriage or dispersal. We discuss our results in relation to recent findings on the kin investment of grandparents (Euler and Weitzel 1996). In addition, we propose a simple method for estimating the level of paternity uncertainty from kin investment data; application of this method to our data on aunts and uncles suggests that between 13% and 20% of children are not the offspring of their putative father. Our parallel analyses of Euler and Weitzel’s (1996) data on grandparental investment suggest a similar estimate, that paternity uncertainty lies between 9% and 17%.     

Mutual information and linkage disequilibrium based SNP association study by grouping case-control

Two main reasons for the difficulties to search for susceptibility single-nucleotide polymorphisms (SNPs) underlying genetic diseases are that the findings are not easy to be confirmed and the interactions between potential susceptibility SNPs are not clear. Many available association studies usually presented results with significance levels but did not illustrate the stability of the results. In some sense, their performances might be unclear in real practice. In this paper, we develop a novel method based on mutual information theory and linkage disequilibrium by grouping case-control. Mutual information (MI) is used to test multiple SNPs in combining with disease status. Those SNPs contributing the maximum MI are selected as potential susceptibility SNPs. Linkage disequilibrium (LD) analysis is used to extend MI detected result so that both direct and indirect factors can be included in the final result. The purpose of case-control grouping is to generate a number of data groups by randomly sampling from target samples. Each group is assumed to have almost the same number of individuals (cases and controls), and overlap is allowed among the groups. We apply the method to each data group, and then make comparisons and intersections between the results obtained from each of the groups so as to give the final result. We implement the method by continuously grouping until the final result reaches a stable state and a highly significance level. The experimental results indicate that our method to detect susceptibility SNPs in simulated and real data sets has shown remarkable success.     

Physico-chemical and biological study of excision-repair of UV--irradiated phiX174 RF DNA in vitro.

We have studied excision-repair of UV-irradiated phiX174 RFI DNA in vitro with UV-specific endonuclease from Micrococcus luteus (UV-endo), DNA polymerase I from Escherichia coli and DNA ligase from phage T4 infected E. coli. Excision-repair was measured a) by physico-chemical methods, i.e. by determination of the conversion of RF I DNA into RF II DNA by UV-endo and by the subsequent conversion of RF II DNA ligase, b) by biological methods i. e. by measuring the ability of the reaction product to form phages upon incubation with spheroplasts from the appropriate strains of E. coli. Using the first method, we have shown, that more than 90% of the pyrimidine dimers can be repaired in vitro; with the latter method we have shown, that the molecules which are repaired as defined by method a) have regained full biological activity. Exonuclease III was found to be not essential for excision-repair in vitro and also did not stimulate repair. From this result we conclude that UV-endo generates 3'OH endgroups, in agreement with results obtained by Hamilton et al. (1974). The usefulness of the method presented in this paper with regard to the study of excision-repair is discussed.