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Tiago Ant?nio de Oliveira Mendes Jo?o Luís Reis Cunha Rodrigo de Almeida Lourdes Gabriela Flávia Rodrigues Luiz Lucas Dhom Lemos Ana Rita Rocha dos Santos Ant?nia Cláudia Jácome da Camara Lúcia Maria da Cunha Galv?o Caryn Bern Robert H. Gilman Ricardo Toshio Fujiwara Ricardo Tostes Gazzinelli Daniella Castanheira Bartholomeu 《PLoS neglected tropical diseases》2013,7(10)
Background
The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.Methodology
By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.Findings and Conclusions
A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping. 相似文献3.
Bj?rn Forsstr?m Barbara Bis?awska Axn?s Klaus-Peter Stengele Jochen Bühler Thomas J. Albert Todd A. Richmond Francis Jingxin Hu Peter Nilsson Elton P. Hudson Johan Rockberg Mathias Uhlen 《Molecular & cellular proteomics : MCP》2014,13(6):1585-1597
Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody''s linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.Antibodies are used in proteomics both as imaging reagents for the analysis of tissue specificity (1) and subcellular localization (2) and as capturing agents for targeted proteomics (3), in particular for the enrichment of peptides for immunoaffinity methods such as Stable Isotope Standards and Capture by Anti-peptide Antibodies (4). In fact, the Human Proteome Project (5) has announced that one of the three pillars of the project will be antibody-based, with one of the aims being to generate antibodies to at least one representative protein from all protein-coding genes. Knowledge about the binding site (epitope) of an antibody toward a target protein is thus important for gaining basic insights into antibody specificity and sensitivity and facilitating the identification and design of antigens to be used for reagents in proteomics, as well as for the generation of therapeutic antibodies and vaccines (1, 6). With over 20 monoclonal-antibody-based drugs now on the market and over 100 in clinical trials, the field of antibody therapeutics has become a central component of the pharmaceutical industry (7). One of the key parameters for antibodies includes the nature of the binding recognition toward the target, involving either linear epitopes formed by consecutive amino acid residues or conformational epitopes consisting of amino acids brought together by the fold of the target protein (8).A large number of methods have therefore been developed to determine the epitopes of antibodies, including mass spectrometry (9), solid phase libraries (10, 11), and different display systems (12–14) such as bacterial display (15) and phage display (16). The most common method for epitope mapping involves the use of soluble and immobilized (tethered) peptide libraries, often in an array format, exemplified by the “Geysen Pepscan” method (11) in which overlapping “tiled” peptides are synthesized and used for binding analysis. The tiled peptide approach can also be combined with alanine scans (17) in which alanine substitutions are introduced into the synthetic peptides and the direct contribution of each amino acid can be investigated. Maier et al. (18) described a high-throughput epitope-mapping screen of a recombinant peptide library consisting of a total of 2304 overlapping peptides of the vitamin D receptor, and recently Buus et al. (19) used in situ synthesis on microarrays to design and generate 70,000 peptides for epitope mapping of antibodies using a range of peptides with sizes from 4-mer to 20-mer.So far it has not been possible to investigate on- and off-target binding in a proteome-wide manner, but the emergence of new methods for in situ synthesis of peptides on ultra-dense arrays has made this achievable. Here, we describe the design and use of peptide arrays generated with parallel in situ photolithic synthesis (20) of a total of 2.1 million overlapping peptides covering all human proteins with overlapping peptides. Miniaturization of the peptide arrays (21) has led to improved density of the synthesized peptides and consequently has improved the resolution and coverage of the epitope mapping. This has allowed us to study the specificity and cross-reactivity of both monoclonal and polyclonal antibodies across the whole “epitome” with the use of both proteome-wide arrays and focused-content peptide arrays covering selected antigen sequences to precisely map the contribution of each amino acid of the target protein for binding recognition of the corresponding antibodies. The results show the usefulness of proteome-wide epitope mapping, showing a path forward for high-throughput analysis of antibody interactions. 相似文献
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目的:建立一种高灵敏度、高特异性、操作简单快捷、通量高的重组人血清白蛋白(rHSA)抗体检测方法。方法:采用桥连ELISA法,即将rHSA包被于96孔板,加入待测血样及阳性对照,用辣根过氧化物酶标记的rHSA检测,显色读取D_450nm/D_570nm值:用此方法确定临界值、方法灵敏度、精密度、血药浓度对检测方法的影响,再以免疫清除法进行确证。结果:通过桥连ELISA法确定临界值为0.0492,方法灵敏度为352ng/mL,方法板间、板内精密度均小于20%,且血药中的rHSA浓度为20μg/mL时不影响抗体的检测;经免疫清除法可将假阳性样本排除,从而提高了方法的特异性.结论:建立的方法可以准确、快速地检测出rHSA的特异性抗体。 相似文献
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重组蛋白/多肽类药物在人体血清内半衰期较短, 很大程度上限制了其临床应用。人血清白蛋白(HSA)具有半衰期长、生物相容性好、低免疫原性等优点,是理想的药物载体。各种基于HSA的蛋白质药物长效化技术得到了广泛的应用和发展,目前主要包括构建HSA融合蛋白,通过共价化学键与HSA偶联,通过非共价键与HSA可逆性结合。总结了近年来基于白蛋白药物长效化技术的发展,各项技术的优缺点及药物开发现状。 相似文献
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《Molecular & cellular proteomics : MCP》2019,18(4):642-656
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- •Profiling antibody responses of patients with naturally acquired malaria immunity.
- •High-density peptide arrays featuring linear epitopes.
- •Epitope mapping of known and potential novel vaccine candidates.
- •Novel immunogenic epitopes discovered, and known antibody target motifs confirmed.
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《Bioscience, biotechnology, and biochemistry》2013,77(11):2232-2236
Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin. 相似文献
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Buffalo milk safety was highlighted with the increase in dietary consumption, and a little information is available on buffalo milk allergy except for cross-reactivity between buffalo and cow milk. In this work, linear epitopes and critical amino acids of buffalo β-lactoglobulin were defined by 4 rabbit's sera using SPOTTM peptide arrays approach based on the defined mimotopes. The eight epitopes on buffalo β-lactoglobulin were located in the position of A6(21-30), A7(25-34), A8 (29-38), B4 (73-82), B5(77-86), C(87-96), F4(134-143) and F8(150-159), respectively. Among them, four epitopes (A7, A8, F4 and F8) were described as the most major epitopes and peptide (A6, B4, B5 and C) as the second major epitopes. Following single AA substitutions (Alanine or Glycine) at each position of the major epitopes, 2,3,2,3,5 and 3 of critical amino acids were identified on epitopes of A6, A8, B5, C , F4 and F8, respectively, which vary in distribution among the epitopes, such as in C terminal or N terminal and in continuous or discontinuous forms, characteristics including hydrophobicity, polar and charge, and existed frequency. 相似文献
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Kai Fu Qin Cheng Zhenwei Liu Zhen Chen Yan Wang Honggang Ruan Lu Zhou Jie Xiong Ruijing Xiao Shengwu Liu Qiuping Zhang Daichang Yang 《PloS one》2014,9(8)
Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4+ and CD8+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development. 相似文献
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人血清白蛋白纯化技术研究进展 总被引:6,自引:0,他引:6
本文综述了国内外关于血浆人血清白蛋白(pHSA)和基因重组人血清白蛋白(rHSA)分离纯化的进展和发展趋势。以冷乙醇沉淀为主的pHSA分离方法仍是目前多数工业采用的工艺,但近年来发展的离子交换色谱、凝胶过滤色谱、亲和色谱等多种色谱分离技术具有自动化程度高、生产周期短、更符合GMP等特点,正在逐步取代传统的冷乙醇沉淀法。当前用基因工程技术表达的人血清白蛋白对分离纯化提出了新的挑战。为获得高纯度、安全、稳定的产品,各种色谱分离技术得到更多的应用,其中扩张床离子交换色谱可以省去离心、过滤等传统固液分离操作。尽管rHSA的纯化技术已有一定的发展,但纯化过程仍需进一步优化以提高产品纯度及收率。 相似文献
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Xing Yi; Ouyang Zhengqing; Kapur Karen; Scott Matthew P.; Wong Wing Hung 《Molecular biology and evolution》2007,24(7):1577
doi:10.1093/molbev/msm061 Mol. Biol. Evol. 相似文献
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人或牛血清白蛋白溶液调至pH10并在4℃左右放置30h,诱导出重复性良好的极谱峰。然后把试样溶液调至适当pH并记录极谱电流。在浓度低于1.0×10-5mol/L时,峰电流对浓度作图是直线,试样浓度可从图上直接读出。对影响白蛋白测定的诸因素作了详细研究和讨论。结果对富含硫的蛋白质的极谱研究有参考价值。 相似文献
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Jason E. Chung Hannah R. Joo Jiang Lan Fan Daniel F. Liu Alex H. Barnett Supin Chen Charlotte Geaghan-Breiner Mattias P. Karlsson Magnus Karlsson Kye Y. Lee Hexin Liang Jeremy F. Magland Jeanine A. Pebbles Angela C. Tooker Leslie F. Greengard Vanessa M. Tolosa Loren M. Frank 《Neuron》2019,101(1):21-31.e5
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研究了人血清白蛋白中色氨酸的荧光红际激发效应,阐明了人血清白蛋白的红际激发效应与PH,温度及尿素浓度存在一定的关系,外界条件的改变使得色氨酸残基所处的微环境发生了变化,使得色氨酸残基的基态和激发态的能级产生不同的分布,从而得到不同的红际激发效应。 相似文献
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Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 1 of Wheat 总被引:25,自引:2,他引:23
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We studied the distribution of genes and recombination in wheat (Triticum aestivum) group 1 chromosomes by comparing high-density physical and genetic maps. Physical maps of chromosomes 1A, 1B, and 1D were generated by mapping 50 DNA markers on 56 single-break deletion lines. A consensus physical map was compared with the 1D genetic map of Triticum tauschii (68 markers) and a Triticeae group 1 consensus map (288 markers) to generate a cytogenetic ladder map (CLM). Most group 1 markers (86%) were present in five clusters that encompassed only 10% of the group 1 chromosome. This distribution may reflect that of genes because more than half of the probes were cDNA clones and 30% were PstI genomic. All 14 agronomically important genes in group 1 chromosomes were present in these clusters. Most recombination occurred in gene-cluster regions. Markers fell at an average distance of 244 kb in these regions. The CLM involving the Triticeae consensus genetic map revealed that the above distribution of genes and recombination is the same in other Triticeae species. Because of a significant number of common markers, our CLM can be used for comparative mapping and to estimate physical distances among markers in many Poaceae species including rice and maize. 相似文献
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Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat 总被引:20,自引:2,他引:18
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The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice. 相似文献
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人血白蛋白是人血浆中最丰富的蛋白质,具有许多重要的生理特性,用途广泛。目前主要以毕赤酵母作为宿主表达的重组人血白蛋白,开发了重组人血白蛋白的纯化技术,同时对重组人血白蛋白结构进行了分析,结果表明与人血浆白蛋白基本一致。临床研究结果表明重组人血白蛋白与人血浆白蛋白有着几乎相同的疗效和安全性。综述了重组人血白蛋白的性质结构分析及酵母表达系统;重点介绍了重组人血白蛋白在临床方面研究进展。 相似文献