The use of peptide arrays for the characterization of monospecific antibody repertoires from polyclonal sera of psychiatric patients suspected of infection by Borna Disease Virus

Borna Disease Virus (BDV) is suspected to infect humans and to be associated with psychiatric disorders. To this date, BDV-reactive antibodies provide the only reliable markers to diagnose human BDV infection. Their diagnostic value, however, was recently questioned by the observation that these antibodies recognize BDV antigen with only low avidity, a typical feature of cross-reacting antibodies. This raised the possibility that the human BDV-reactive antibodies were triggered by other pathogens than BDV. The recent establishment of a peptide array-based screening test allowed the further characterization of these antibodies. It revealed the presence of small amounts of BDV-reactive antibodies in crude human sera that specifically recognized various epitopes of three major BDV proteins. Most importantly, the purified epitope-specific antibodies were shown to bind to BDV antigen with high avidity when assayed by conventional immunofluorescence assay (IFA) or by Western blot. These results are compatible with the view that the presence of BDV-reactive antibodies in human sera reflects an infection with BDV, although the poor affinity maturation remains unexplained. Furthermore, it demonstrates that peptide array-based screening tests are a reliable system for identifying monospecific antibodies from human polyclonal sera with high specificity and sensitivity.     

Use of peptide for selective and sensitive detection of an Anthrax biomarker via peptide recognition and surface‐enhanced Raman scattering

A short 16‐amino acid peptide has been used in place of an antibody to selectively detect the specific Anthrax biomarker, protective antigen (PA), using surface‐enhanced Raman scattering (SERS). Peptides are more stable than antibodies under various biological conditions and are easily synthesized for a specific target. A peptide that has high affinity to PA was conjugated onto gold nanoparticles along with a Raman reporter and then incubated in various concentrations of PA. Parallel studies in which the peptide sequence was replaced with an antibody were performed to compare the performance of the two methodologies. Both the peptide and antibody functionalized nanoparticles were able to specifically detect PA concentrations down to 6.1 fM . These results demonstrate that these short, robust peptides can be used in the place of traditional antibodies to specifically recognize target biomarkers in the field for the potential diagnosis of disease. Copyright © 2010 John Wiley & Sons, Ltd.     

Flexible antibodies with nonprotein hinges

There is a significant need for antibodies that can bind targets with greater affinity. Here we describe a novel strategy employing chemical semisynthesis to produce symmetroadhesins: antibody-like molecules having nonprotein hinge regions that are more flexible and extendible and are capable of two-handed binding. Native chemical ligation was carried out under mild, non-denaturing conditions to join a ligand binding domain (Aβ peptide) to an IgG1 Fc dimer via discrete oxyethylene oligomers of various lengths. Two-handed Aβ-Fc fusion proteins were obtained in quantitative yield and shown by surface plasmon resonance to bind an anti-Aβ antibody with a K(D) at least two orders of magnitude greater than the cognate Aβ peptide. MALDI-TOF MS analysis confirmed the protein/nonprotein/protein structure of the two-handed molecules, demonstrating its power to characterize complex protein-nonprotein hybrids by virtue of desorption/ionization mediated by peptide sequences contained therein. We anticipate many applications for symmetroadhesins that combine the target specificity of antibodies with the novel physical, chemical and biological properties of nonprotein hinges.     

Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays

Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.     

Evolutionary computation and multimodal search: A good combination to tackle molecular diversity in the field of peptide design

Summary The awesome degree of structural diversity accessible in peptide design has created a demand for computational resources that can evaluate a multitude of candidate structures. In our specific case, we translate the peptide design problem to an optimization problem, and use evolutionary computation (EC) in tandem with docking to carry out a combinatorial search. However, the use of EC in huge search spaces with different optima may pose certain drawbacks. For example, EC is prone to focus a search in the first good region found. This is a problem not only because of the undesirable and automatic rejection of potentially good search space regions, but also because the found solution may be extremely difficult to synthesize chemically or may even be a false docking positive. In order to avoid rejecting potentially good solutions and to maximize the molecular diversity of the search, we have implemented evolutionary multimodal search techniques, as well as the molecular diversity metric needed by the multimodal algorithms to measure differences between various regions of the search space.     

Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Aβ antibodies

Background  

Prior work from our group found that acid dissociation (pH 2.5 incubation) of serum from APP transgenic mice vaccinated against Aβ increased the apparent anti-Aβ titers, suggesting antibody masking by antigen in the ELISA assay. Subsequently, we found that pH 2.5 incubation of serum from unvaccinated non-transgenic mice showed antibody binding to Aβ1–42, but no increase when other proteins, including shorter Aβ peptides, coated the ELISA plate. To investigate further the effects of low pH incubation on apparent anti-Aβ1–42 signals, we examined normal sera from nonTg unvaccinated mice, nonTg mice vaccinated with Aβ peptide (to produce authentic anti-Aβ antibodies) or a monoclonal antibody against Aβ (6E10) using competitive-inhibition ELISA and Aβ epitope mapping assays. In addition, we examined use of a less stringent low pH procedure at pH 3.5, to ascertain if it had the same effects as the pH 2.5 procedure.     

High density peptide microarrays. In situ synthesis and applications

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.     

Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody

Summary Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteineconstrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.     

Mapping protein-protein contact sites using cellulose-bound peptide scans

Summary We have characterized the interaction of two monoclonal antibodies with their respective antigens using cellulose-bound sets of overlapping peptides (peptide scans). Both antibodies CB/RS/5 and CB/MT/1 recognize discontinuous epitopes present in human interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-). In addition, the interaction between TNF-a and its 55-kDa receptor (TNF-R) was investigated by the same approach. Both antibodies, as well as TNF-, interacted with two or more regions of the peptide scans. Antibody-binding competition studies between the native antigens and peptides, covering single parts of the binding regions, enabled us to distinguish between binding to the paratope or other regions of the antibody. The combination of these experimental approaches allowed the identification of short antigen-derived sequences that are separated on the primary sequence but close in space on the surface of IL-10 and TNF-, thus representing putative discontinuous epitopes. In the case of the TNF-R-derived peptide scans, two of the identified regions interact with the structurally similar TNF- in the TNF--TNF-R complex. These data indicate that this approach should be generally applicable for mapping nonlinear protein-protein contact sites.     

Counterstaining Improves Visualization of the Myenteric Plexus in Immunolabelled Whole-Mount Preparations

Immunocytochemistry has emerged as a powerful research tool in neurobiology. One of the widely used methods is an indirect fluorescence technique that uses FITC- conjugated IgG to visualise protein expression within tissues, but a major drawback of this technique is the high background fluorescence due to non-specific antibody binding. Gut innervation is complex and best visualized in three-dimensions in whole mount preparations. We describe a simple and easy to use counterstaining procedure in conjunction with an indirect immunofluorescence technique in gut whole mount preparations that largely eliminates background fluorescence and creates a contrasting background against the bright antigen-antibody complexes. Furthermore, this technique allows the detailed qualitative and quantitative study of myenteric plexuses in whole-mount preparations.     

Intra-arterial application of magnetic nanoparticles for targeted thrombolytic therapy: A rat embolic model

Targeted delivery of thrombolytic drug to the site of emboli exhibits potential to greatly reduce hemorrhagic side effect. A rat embolic model with an easy access of a magnet was established for study of the efficacy of magnetic drug targeting. In anesthetized rats, a whole blood clot produced in vitro was injected from the right iliac artery and lodged in the left iliac artery. Intra-arterial infusion of recombinant tissue plasminogen activator (rt-PA) thereafter significantly reversed the iliac flow within 15 min. Placement of an NdFeB magnet above the left iliac artery caused magnetic nanoparticle retention against hemodynamic dragging force in the presence and absence of the clot. Our results suggest the feasibility of this rat embolic model for the study of magnetic targeted delivery of thrombolytic drugs.     

Mapping the detailed specificity of a calcium-dependent monoclonal antibody through the use of soluble positional scanning combinatorial libraries: Identification of potent calcium-independent antigens

Summary The detailed specificity of monoclonal antibody M1, which has been reported to bind in a calcium-dependent manner to the FLAG sequence DYKDDDDK-NH₂, was examined using soluble hexa- and decapeptide positional scanning synthetic combinatorial libraries (PS-SCLs) made up of 52×10⁶ and 4×10¹² different sequences, respectively. To study the influence of calcium on the specificity of this antigen-antibody interaction, each PS-SCL was screened in the presence and absence of calcium using a competitive ELISA. Overall, peptide mixtures had greater inhibitory activity against mAb M1 binding to FLAG in the absence of calcium. A total of 16 individual hexapeptides were identified, all of which contained the motif -DYK_K_-, and were recognized by mAb M1 in the absence of calcium with 50-to 100-fold higher affinity than the FLAG octapeptide (IC₅₀=273 nM). On average, the same set of peptides bound 10-fold less effectively in the presence of calcium. Upon screening the decapeptide PS-SCL in the absence of calcium, lysine was also more active in the fifth position than the original aspartic acid. Based on the screening results, 24 individual decapeptides were prepared and were found to have activities 10- to 100-fold higher than the FLAG octapeptide in the absence of calcium. The specificity of lysine at the fifth position in the antigen-antibody interaction was further examined by synthesizing and assaying substitution analogs at this position for the octapeptide and hexapeptide forms of the FLAG sequence, as well as for two hexapeptides identified from the PS-SCL. Truncation analog analysis was also carried out on the FLAG octapeptide to determine optimal antigen length for antibody binding. Overall, lysine at the fifth position could be substituted with ornithine with no significant loss in activity, and peptide length was not a critical factor for antibody binding in the absence of calcium. Also, the octapeptide having lysine at the fifth position in place of the aspartic acid had the same activity in the presence or absence of calcium. This study demonstrates the ease and effectiveness of PS-SCLs over individual peptide analogs for the examination of the degree of cross-reactivity for a given monoclonal antibody as well as for the identification of novel, high-affinity peptides.     

Confocal Raman microspectroscopy discriminates live human metastatic melanoma and skin fibroblast cells

Confocal Raman microspectroscopy (CRM) continues to develop as a promising technique with possible clinical applications for the diagnosis and treatment of skin cancers. CRM studies of single cells can provide information on the biochemical content of cancer cells in situ, potentially providing new biochemical signatures or markers of cancer cells. Here, we report a CRM study of single, living human metastatic melanoma cells (SK‐Mel‐2) and normal skin fibroblast cells (BJ) cultured and examined under identical experimental conditions. A total of almost 1200 Raman spectra were measured from more than 120 BJ and SK‐Mel‐2 cells using an inverted microscope with 647 nm laser excitation. Raman spectra were measured from within three distinct intracellular regions of the cells – cytoplasm, nucleoplasm, and nucleolus. When Raman spectra from each cell type were compared using principal components analysis (PCA) and linear discriminant analysis with leave‐one‐dish‐out cross‐validation (LDA‐CV), the two cell types were discriminated with 93% (cytoplasm), 98% (nucleolus), and 96% (nucleoplasm) accuracy. The main biochemical differences identified between the two cell types were higher RNA levels in the nucleoli of BJ cells and high amounts of lipid and collagen in the cytoplasm of SK‐Mel‐2 cells. For both cell types, higher levels of RNA were detected in the nucleoli versus the nucleoplasm. PCA with LDA‐CV was 98% (cytoplasm), 93% (nucleoplasm), and 73% (nucleolus) accurate in identifying the intracellular region based on the Raman spectra from both cell types. No significant trend was observed when the data were analyzed with respect to cell passage number. Thus, CRM with PCA and LDA‐CV successfully discriminated two skin cancer‐relevant cell lines while detecting different amounts of nucleic acids, lipids, and proteins in distinct intracellular regions, further underscoring its potential as a clinical diagnostic tool. Copyright © 2013 John Wiley & Sons, Ltd.     

Generating neutralizing antibodies,Th1 response and MHC non restricted immunogenicity of HIV-I <Emphasis Type="Underline">env</Emphasis> and <Emphasis Type="Underline">gag</Emphasis>peptides in liposomes and ISCOMs with in-built adjuvanticity

For enhancing immunogenicity and develop vaccine strategies using peptide based constructs against HIV-1, a chimeric peptide containing V3 loop and transmembrane sequence of gp41 with two glycine motifs as spacer was constructed. The V3-gp41, gp41 peptide and p17 and p24 peptides separately or in a cocktail were entrapped with or without MA729 as an immunoadjuvant in liposomes or ISCOMs. The immunogenicity, antigen induced T-cell proliferation and cytokine profiles of various formulations were studied in four different inbred strains of mice of H-2^d, H-2^b, H-2^k and H-2^q haplotypes, keeping alum as a control adjuvant. Both liposomes and ISCOM preparations elicited high titer and long lasting antibody response (60 days and above). When compared to the alum formulation, the liposomes co-entrapped with MA729 produced high antibody levels, comparable with that induced by ISCOMs. Peptide in alum, liposomes and ISCOMs enhanced both antigen specific IgG2a and IgG2b isotypes and high T-cell stimulation index. Peptide formulations also induced antibodies with high affinity and in vitro neutralizated the formation of HIV-1 syncytia. T-cell supernatants contained high levels of IFN-γ and IL-2. Thus formulation in these adjuvants induced a predominant Th1 like response with MA729 as a versatile novel delivery vehicle for stimulating the appropriate arm of the immune response that can selectively modulate MHC class I or MHC class II response. The above peptide can be of wide vaccination interest as a means to improve immune responses to several other HIV-1 antigens and may serve as candidates for vaccine development.     

基于可调控多肽纳米管和石墨烯复合纳米结构的光吸收催化平台

近年来, 自组装纳米结构因为其容易制备、稳定、环保以及与各种功能基团、粒子等的多样结合能力吸引了科学家们的目光, 成为人们研究的热点课题, 在光电池、光催化、水凝胶、药物缓释等方面的实验科学领域得到了广泛的应用. 尤其是光催化方面, 自组装结构的重复性为激子的传递创造了比较良好的条件, 成为众多激子传递平台中的佼佼者. 本文报道了一种以苯丙氨酸二肽纳米管和羧基石墨烯为基础的自组装光吸收催化平台, 对其结构进行研究, 并使用该体系进行了烟酰胺腺嘌呤二核苷酸到它的还原态的催化实验. 该体系的微观结构由纳米管和石墨烯膜复合而成, 羧基石墨烯的存在能够降低纳米管直径, 实现纳米管的形态操控, 石墨烯与多肽纳米管复合纳米结构的存在实现了多通道协同激子传递, 降低了激子传递的距离, 极大增强了催化中心对于激子的接受和使用效率. 在复合了光敏剂和催化中心之后, 该体系具有较高的稳定性, 均一的分散性, 很强的光能吸收和转化能力等性质. 对于从NADP⁺往NADPH转变的催化实验表明, 该体系有较高的反应速率和催化效率, 并且比两种单一结构催化平台效果之和更好, 实现了一加一大于二的效应, 展现了复合纳米结构光吸收催化平台的巨大潜力和广阔应用前景.     

表面等离子体共振技术检测重组户尘螨变应原rDer p2与抗体的结合

利用表面等离子体共振技术对屋尘螨重组变应原(rDer p2)与单克隆抗体之间的结合作用进行了研究,并且运用该技术对尘螨过敏患者血清与变应原之间的结合作用进行了初步探讨。变应原和抗体之间的结合通常是通过免疫吸附技术或者酶免法来进行研究的,这些方法一般需要对分析物进行标记,由于背景的影响和低灵敏度,目前对抗原和抗体的反应检测所用的免疫吸附技术的准确性还远不如表面等离子共振技术。用氨基偶联法将重组变应原rDer p2固定在CM5传感片表面上后,表面等离子体共振(SPR)技术检测的结果显示,rDer p2与尘螨过敏患者血清的结合作用比单克隆抗体要弱得多。而且,尘螨过敏不同患者血清与rDer p2的结合有很大的差异性。本研究为临床上对过敏疾病的诊断提供了一个简单、 快速和实时监控的检测手段。     

New planar substrates for the in situ synthesis of peptide arrays

The performance of new cellulose membranes, aminofunctionalized via a PEG spacer, as a solid support in the synthesis of peptide arrays is described. The new membranes are stable to trifluoroacetic acid (TFA) and strong aqueous base for days. These properties extend the scope of synthesis considerably, e.g., more efficient side chain cleavage protocols can be applied which yielded peptides of improved purity. For the first time, cellulose membranes with a loading as high as 5 micromol/cm2 were accessible. Additionally, newly developed polypropylene membranes with hydroxy- or amino functionalities were successfully employed for the SPOT synthesis of peptides and phosphopeptides. The membranes are compatible with antibody binding as well as enzymatic phosphorylation assays.     

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage

Summary We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 × 10⁹ members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of -endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v -ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.     

Combination therapy with normobaric oxygen (NBO) plus thrombolysis in experimental ischemic stroke

Background  

The widespread use of tissue plasminogen activator (tPA), the only FDA-approved acute stroke treatment, remains limited by its narrow therapeutic time window and related risks of brain hemorrhage. Normobaric oxygen therapy (NBO) may be a useful physiological strategy that slows down the process of cerebral infarction, thus potentially allowing for delayed or more effective thrombolysis. In this study we investigated the effects of NBO started simultaneously with intravenous tPA, in spontaneously hypertensive rats subjected to embolic middle cerebral artery (MCA) stroke. After homologous clot injection, animals were randomized into different treatment groups: saline injected at 1 hour; tPA at 1 hour; saline at 1 hour plus NBO; tPA at 1 hour plus NBO. NBO was maintained for 3 hours. Infarct volume, brain swelling and hemorrhagic transformation were quantified at 24 hours. Outcome assessments were blinded to therapy.     

A gamma-tubulin antibody against a plant peptide sequence localises to cell division-specific microtubule arrays and organelles in plants

Gamma tubulin (gamma-tubulin) is involved in microtubule initiation in the eukaryotes. In animal cells it is localised to centrosomes and to other, non-centrosomal sites of microtubule initiation. In addition, cytoplasmic complexes containing gamma-tubulin (gamma-TuRCs; gamma-somes) have been described, which are multiprotein complexes involved in microtubule initiation. Most localisations of gamma-tubulin in plants have previously been achieved using an antibody directed towards a conserved peptide sequence found in animal cells, showing co-localisation with all microtubule arrays throughout the cell cycle. Because different antibodies may give various patterns of subcellular localisation, in the present study we raised a polyclonal antibody ('Hayley') to the plant peptide sequence EDFATQGGDRKDVFFY (bold letters indicate plant-specific amino acids) to further investigate the subcellular distribution in plants. Immunoblotting using wheat root tip protein extracts revealed a 58 kDagamma-tubulin-like peptide as has been described before. Immunofluorescence microscopy of wheat root-tip cells, however, revealed localisation of gamma-tubulin to a subset of mitotic microtubule arrays and the cytokinetic phragmoplast, but not to interphase cortical arrays or the preprophase band of microtubules. This lack of labelling may be caused by a restriction of antibody access during interphase, but more likely by a cell division-specific conformational change in the gamma-tubulin molecule. Our antibody also gave an organelle-like labelling, not described before, which may represent storage forms or precursors of gamma-tubulin, perhaps related to plastid-based microtubule initiation in hepatics and hornworts.