Use Of Synthetic Peptides Research Articles

DAP1-2: a synthetic peptide targeting IL-1R1 receptor effectively suppresses IL-1β in vitro.

The pathological manifestation of the inflammatory process primarily stems from the heightened release of pro-inflammatory cytokines, with IL-1β standing out as a pivotal cytokine. The excessive presence of IL-1β disrupts immune signaling, thereby assuming a pathogenic and exacerbating role in the pathophysiology of numerous inflammatory diseases. Regulating IL-1β levels becomes crucial, and the IL-1Ra molecule serves this purpose by binding to the IL-1R1 receptor, thereby impeding the binding of IL-1β. Several pharmaceuticals have entered the market, aiming to neutralize IL-1β's biological function through diverse mechanisms. However, the existing IL-1β inhibitors are recombinant proteins, characterized by a high production cost and limited stability. Therefore, this study aimed to predict a peptide, named DAP1-2, based on the IL-1Ra molecule. DAP1-2 was designed to attenuate responses triggered by IL-1β by blocking the IL-1R1 receptor. The selection of amino acids from the IL-1Ra molecule (PDB: I1RA) that interact with the three domains of the IL-1R1 receptor was performed using Swiss PDB Viewer. After prediction, chemical synthesis was made using the Fmoc-Synthesis technique. The efficacy of DAP1-2 was assessed using RAW 264.7 cells, which were exposed to LPS (5μg/mL) for 24h to induce IL-1β expression and treated with the peptides in different concentrations. IL-1β levels were assessed using ELISA, and the gene expression of IL-1β was measured by RT-qPCR, additionally to the viability test. Results revealed a significant reduction in IL-1β levels and gene expression in cells stimulated by LPS and treated with DAP1-2 in different concentrations. Furthermore, the MTT assay confirmed the nontoxic nature of the peptides on the cell lineage. This alternative approach shows promise as an IL-1 inhibitor, due to the stability, ease of production, and cost-effectiveness provided by the use of synthetic peptides.

Synthetic Peptides: Promising Modalities for the Targeting of Disease-Related Nucleic Acids.

DNA and RNA play pivotal roles in life processes by storing and transferring genetic information, modulating gene expression, and contributing to essential cellular machinery such as ribosomes. Dysregulation and mutations in nucleic acid-related processes are implicated in numerous diseases. Despite the critical impact on health of nucleic acid mutations or dysregulation, therapeutic compounds addressing these biomolecules remain limited. Peptides have emerged as a promising class of molecules for biomedical research, offering potential solutions for challenging drug targets. This review focuses on the use of synthetic peptides to target disease-related nucleic acids. We discuss examples of peptides targeting double-stranded DNA, including the clinical candidate Omomyc, and compounds designed for regulatory G-quadruplexes. Further, we provide insights into both library-based screenings and the rational design of peptides to target regulatory human RNA scaffolds and viral RNAs, emphasizing the potential of peptides in addressing nucleic acid-related diseases.

Synthetic Peptides: Promising Modalities for the Targeting of Disease‐Related Nucleic Acids

AbstractDNA and RNA play pivotal roles in life processes by storing and transferring genetic information, modulating gene expression, and contributing to essential cellular machinery such as ribosomes. Dysregulation and mutations in nucleic acid‐related processes are implicated in numerous diseases. Despite the critical impact on health of nucleic acid mutations or dysregulation, therapeutic compounds addressing these biomolecules remain limited. Peptides have emerged as a promising class of molecules for biomedical research, offering potential solutions for challenging drug targets. This review focuses on the use of synthetic peptides to target disease‐related nucleic acids. We discuss examples of peptides targeting double‐stranded DNA, including the clinical candidate Omomyc, and compounds designed for regulatory G‐quadruplexes. Further, we provide insights into both library‐based screenings and the rational design of peptides to target regulatory human RNA scaffolds and viral RNAs, emphasizing the potential of peptides in addressing nucleic acid‐related diseases.

Melanocortin 1 Receptor (MC1R): Pharmacological and Therapeutic Aspects.

Melanocortins play crucial roles in regulating the stress response, inflammation, and skin pigmentation. In this review, we focus on the melanocortin 1 receptor (MC1R), a G protein-coupled receptor primarily known for regulating skin pigmentation and exhibiting anti-inflammatory effects. First, we provide an overview of the structure, signaling pathways, and related diseases of MC1R. Next, we discuss the potential therapeutic use of synthetic peptides and small molecule modulators of MC1R, highlighting the development of various drugs that enhance stability through amino acid sequence modifications and small molecule drugs to overcome limitations associated with peptide characteristics. Notably, MC1R-targeted drugs have applications beyond skin pigmentation-related diseases, which predominantly affect MC1R in melanocytes. These drugs can also be useful in treating inflammatory diseases with MC1R expression present in various cells. Our review underscores the potential of MC1R-targeted drugs to treat a wide range of diseases and encourages further research in this area.

Use of synthetic peptides in the prevention of oral diseases

Among the protective factors of mixed saliva, antimicrobial peptides have received much attention. They have broad-spectrum antibacterial and antifungal activities. Synthetic peptides are found in hygiene products, such as mouthwashes, sprays, and toothpastes. According to their manufacturers, the use of these products helps prevent oral bacterial infections. Objective. To study the effect of toothpastes and mouth spray, which contain synthetic peptide complexes, on the physicochemical parameters of mixed saliva in order to substantiate their preventive antibacterial effect. Material and methods. Mixed saliva from patients was examined before and after the use of the synthetic peptide-containing spray and toothpaste. The following salivary physicochemical parameters (pH, surface tension, and base buffer capacity) were measured. Results. The peptide-containing spray and toothpaste caused changes in the oral fluid pH within the normal range, which assisted in maintaining oral homeostasis and in effectively exerting the functions of mixed saliva. When the spray or toothpaste was used, the base buffer capacity was slightly reduced, which suggested that the synthetic peptides were able to neutralize oral fluid acids and thereby to enhance the anti-inflammatory and antibacterial functions of mixed saliva. The surface tension of saliva was increased with the peptide-containing spray and that was decreased with the peptide-containing toothpaste. This effect indicated the active ability of the synthetic peptides to be absorbed into blood and to have a direct effect on cell metabolism at the site of lesion (when using the spray), as well as the ability of the complexes to integrate into the colloidal system of saliva, which enhanced its reparative and protective properties (when using the toothpaste). Conclusion. The study shows an increase in the antibacterial effect of mixed saliva when using oral hygiene products containing peptides, which contributes to the prevention of risk factors for inflammatory diseases of the oral mucosa and periodontium.

Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities.

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

Peptide vaccines designed with the aid of immunoinformatic against Caseous Lymphadenitis promotes humoral and cellular response induction in mice.

Caseous Lymphadenitis (CLA) is a chronic disease that affects also small ruminants. CLA is caused by Corynebacterium pseudotuberculosis and is responsible for high economic losses due to the formation of superficial and visceral granulomas, the latter is considered as asymptomatic CLA causing high levels of dissemination. Several vaccination strategies, in which the use of synthetic peptides stands out. Thus, this work aimed to evaluate the protective potential of peptide vaccines designed to determine the immunodominant epitopes of CP40 against CLA in mice. The animals were divided into eight groups separated in controls (G1—PBS, G2—Saponin and G9—rCP40) and experimental (G3—pep1, G4- pep2, G5-pep3, G6-pep4, G7-pep5 and G8-pep6), these were vaccinated on days 0 and 15 by a subcutaneous route. 60 days after the first immunization, all animals were challenged with C. pseudotuberculosis. On days 0, 15, 60, and 120 after the first immunization, blood samples were taken to measure immunoglobulins. On the same day of the challenge, the splenocytes were isolated and assayed for the production of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, and IL-10. After vaccinations, the animals were challenged and all of them were affected by the disease which led to their death. The G6 and G8 groups provided 10% protection and the G7 provided 20%. The G3 and G4 groups provided 30% and 40% protection respectively. The peptides showed the production of Total IgG antibodies and cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α), indicating a possible activation of the Th1 type response. However, groups G3, G5, G6, and G8 showed production of IL-17. None of the study groups showed IL-10 production. The immunogenicity of the peptides was not enough to protect these animals and it is believed that the use of adjuvants based on PAMPs may improve the immune response offered by these peptides.

Role of Peptides in Diagnostics.

The specificity of a diagnostic assay depends upon the purity of the biomolecules used as a probe. To get specific and accurate information of a disease, the use of synthetic peptides in diagnostics have increased in the last few decades, because of their high purity profile and ability to get modified chemically. The discovered peptide probes are used either in imaging diagnostics or in non-imaging diagnostics. In non-imaging diagnostics, techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), lateral flow devices (i.e., point-of-care testing), or microarray or LC-MS/MS are used for direct analysis of biofluids. Among all, peptide-based ELISA is considered to be the most preferred technology platform. Similarly, peptides can also be used as probes for imaging techniques, such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The role of radiolabeled peptides, such as somatostatin receptors, interleukin 2 receptor, prostate specific membrane antigen, αβ3 integrin receptor, gastrin-releasing peptide, chemokine receptor 4, and urokinase-type plasminogen receptor, are well established tools for targeted molecular imaging ortumor receptor imaging. Low molecular weight peptides allow a rapid clearance from the blood and result in favorable target-to-non-target ratios. It also displays a good tissue penetration and non-immunogenicity. The only drawback of using peptides is their potential low metabolic stability. In this review article, we have discussed and evaluated the role of peptides in imaging and non-imaging diagnostics. The most popular non-imaging and imaging diagnostic platforms are discussed, categorized, and ranked, as per their scientific contribution on PUBMED. Moreover, the applicability of peptide-based diagnostics in deadly diseases, mainly COVID-19 and cancer, is also discussed in detail.

↵Peptide based Indirect ELISA for Detection of Antibodies against Peste des petits ruminants virus

Background: Infectious virus antigen is not recommended for disease monitoring in global Peste des petits ruminants eradication strategies. Native virus antigens are gradually being replaced with recombinant or synthetic peptide antigens. The focus of the present study is to optimize and develop peptide-based immunoassay for the detection of antibodies to PPRV N and H proteins. Methods: Epitopes of PPRV H and N proteins were selected based on prediction with bioinformatics tools and from previous studies. Two peptides each were synthesized for N and H proteins and peptide ELISA developed. The peptide ELISA’s sensitivity and specificity were tested with sera samples collected at different time intervals of vaccination (goat =73, sheep= 62) and 88 random serum samples (goat =47, sheep=41). The collected sera were screened using cELISA before proceeding to peptide ELISA. Result: In competitive ELISA, 106 goat serum samples and 96 sheep serum samples were found to be positive. Fourteen goat serum samples and seven sheep serum sample were shown to be negative. Among120 goat serum samples tested, 114 were found to be positive by peptide ELISA. Similarly, out of 103 sheep serum samples analyzed, 96 were found to be positive with peptide ELISA. The peptide ELISA based on the highly conserved and antigenic N and H epitope detected antibodies to PPRV in precise manner. This study demonstrated the effective use of synthetic peptides as an antigen in the detection of antibodies to PPRV.

Peptide Allergen Immunotherapy: A New Perspective in Olive-Pollen Allergy.

Allergic diseases are highly prevalent disorders, mainly in industrialized countries where they constitute a high global health problem. Allergy is defined as an immune response “shifted toward a type 2 inflammation” induced by the interaction between the antigen (allergen) and IgE antibodies bound to mast cells and basophils that induce the release of inflammatory mediators that cause the clinical symptoms. Currently, allergen-specific immunotherapy (AIT) is the only treatment able to change the course of these diseases, modifying the type 2 inflammatory response by an allergenic tolerance, where the implication of T regulatory (Treg) cells is considered essential. The pollen of the olive tree is one of the most prevalent causes of respiratory allergic diseases in Mediterranean countries, inducing mainly nasal and conjunctival symptoms, although, in areas with a high antigenic load, olive-tree pollen may cause asthma exacerbation. Classically, olive-pollen allergy treatment has been based on specific immunotherapy using whole-olive pollen extracts. Despite extracts standardization, the effectiveness of this strategy varies widely, therefore there is a need for more effective AIT approaches. One of the most attractive is the use of synthetic peptides representing the B- or T-cell epitopes of the main allergens. This review summarizes experimental evidence of several T-cell epitopes derived from the Ole e 1 sequence to modulate the response to olive pollen in vitro, associated with several possible mechanisms that these peptides could be inducing, showing their usefulness as a safe preventive tool for these complex diseases.

New trends in the development of multifunctional peptides to functionalize biomaterials.

Improving cell-material interactions is a major goal in tissue engineering. In this regard, functionalization of biomaterials with cell instructive molecules from the extracellular matrix stands out as a powerful strategy to enhance their bioactivity and achieve optimal tissue integration. However, current functionalization strategies, like the use of native full-length proteins, are associated with drawbacks, thus urging the need of developing new methodologies. In this regard, the use of synthetic peptides encompassing specific bioactive regions of proteins represents a promising alternative. In particular, the combination of peptide sequences with complementary or synergistic effects makes it possible to address more than one biological target at the biomaterial surface. In this review, an overview of the main strategies using peptides to install multifunctionality on biomaterials is presented, mostly focusing on the combination of the RGD motif with other peptides sequences. The evolution of these approaches, starting from simple methods, like using peptide mixtures, to more advanced systems of peptide presentation, with very well defined chemical properties, are explained. For each system of peptide's presentation, three main aspects of multifunctionality-improving receptor selectivity, mimicking the extracellular matrix and preventing bacterial colonization while improving cell adhesion-are highlighted.

Design and validation of a frugal, automated, solid-phase peptide synthesizer.

Solid phase peptide synthesis (SPPS) has enabled widespread use of synthetic peptides in applications ranging from pharmaceuticals to materials science. The demand for synthetic peptides has driven recent efforts to produce automated SPPS synthesizers which utilize fluid-handling components common to chemistry laboratories to drive costs down to several thousand dollars. Herein, we describe the design and validation of a more ‘frugal’ SPPS synthesizer that uses inexpensive, consumer-grade fluid-handling components to achieve a prototype price point between US$300 and $600. We demonstrated functionality by preparing and characterizing peptides with a variety of distinct properties including binding functionality, nanoscale self-assembly, and oxidation-induced fluorescence. This system yielded micromoles of peptide at a cost of approximately $1/residue, a cost which may be further reduced by optimization and bulk purchasing.

Molecular Characterization of Tc964, A Novel Antigenic Protein from Trypanosoma cruzi.

The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosis.

Mass spectrometry detection of protein allergenic additives in emulsion-type pork sausages

The application of various functional protein additives in the meat industry is still increasing. Soy, milk and egg white proteins are applied to various types of meat product to improve their structural properties or as good substitutes for meat and fat components. It is common practice to add these preparations to reduce production costs. New methods are needed for reliable detection of small amounts of these additives in complex and processed food matrices regarding the potential for food adulteration and allergen detection. In this work, a mass spectrometry method was implemented for the detection of soy (Glycine max), milk (Bos taurus) and egg white (Gallus gallus) in smoked and cooked pork sausages. Specific peptide markers for glycinin and β-conglycinin, α-S1-casein, β-lactoglobulin, ovotransferrin and lysozyme C were monitored in the sausage samples. The proteins were detectable at the low concentration of 0.53% (w/w) of a given additive, which corresponds to a limit of detection of 0.14% for milk proteins and 0.45% for soy and egg white proteins in the final meat product. The results were validated by the use of synthetic peptides labelled with stable isotopes. Considering the universal nature of the presented peptide markers, the method has the potential to be implemented for other complex and processed foodstuffs for authenticity purposes.

Identification of a linear B-cell epitope in the catalytic domain of bothropasin, a metalloproteinase from Bothrops jararaca snake venom

Bothropasin is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca venom, the snake responsible for most bites in Southeastern Brazil. SVMPs, such as bothropasin, are involved in the main bothropic envenoming symptoms, which include hemorrhage, inflammation, necrosis and blood coagulation deficiency. B-cell epitope mapping of SVMPs can lead to the identification of peptides capable of inducing neutralizing antibodies without causing toxic effects, therefore improving anti-venom production. Here, using the SPOT synthesis technique, we have identified an epitope located in the catalytic domain of bothropasin (202KARMYELANIVNEILRYLYMH222) which was synthesized and named BotEp1. The peptide was used to immunize Swiss mice and Anti-BotEp1 serum cross-reacted with bothropasin and crude venoms from B. jararaca and B. atrox venoms. Furthermore, Anti-BotEp1 antibodies were able to completely neutralize the hemorrhagic activity of a chromatographic fraction from B. jararaca venom, which contains hemorrhagic SVMPs. In addition, the coagulation activity of the hemorrhagic fraction showed to be diminished when tested in serum from rabbit immunized with BotEp1 (compared to serum from non-immunized animal). Our results show the identification of neutralizing epitopes in bothropasin and provide basis for the use of synthetic peptides to improve the production of immunotherapeutics.

Monitoring of Plant Protein Post-translational Modifications Using Targeted Proteomics.

Protein post-translational modifications (PTMs) are among the fastest and earliest of plant responses to changes in the environment, making the mechanisms and dynamics of PTMs an important area of plant science. One of the most studied PTMs is protein phosphorylation. This review summarizes the use of targeted proteomics for the elucidation of the biological functioning of plant PTMs, and focuses primarily on phosphorylation. Since phosphorylated peptides have a low abundance, usually complex enrichment protocols are required for their research. Initial identification is usually performed with discovery phosphoproteomics, using high sensitivity mass spectrometers, where as many phosphopeptides are measured as possible. Once a PTM site is identified, biological characterization can be addressed with targeted proteomics. In targeted proteomics, Selected/Multiple Reaction Monitoring (S/MRM) is traditionally coupled to simple, standard protein digestion protocols, often omitting the enrichment step, and relying on triple-quadruple mass spectrometer. The use of synthetic peptides as internal standards allows accurate identification, avoiding cross-reactivity typical for some antibody based approaches. Importantly, internal standards allow absolute peptide quantitation, reported down to 0.1 femtomoles, also useful for determination of phospho-site occupancy. S/MRM is advantageous in situations where monitoring and diagnostics of peptide PTM status is needed for many samples, as it has faster sample processing times, higher throughput than other approaches, and excellent quantitation and reproducibility. Furthermore, the number of publicly available data-bases with plant PTM discovery data is growing, facilitating selection of modified peptides and design of targeted proteomics workflows. Recent instrument developments result in faster scanning times, inclusion of ion-trap instruments leading to parallel reaction monitoring- which further facilitates S/MRM experimental design. Finally, recent combination of data independent and data dependent spectra acquisition means that in addition to anticipated targeted data, spectra can now be queried for unanticipated information. The potential for future applications in plant biology is outlined.

Targeting HOX-PBX interactions causes death in oral potentially malignant and squamous carcinoma cells but not normal oral keratinocytes

BackgroundHigh HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers.MethodsActivity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay.ResultsPMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9.ConclusionInhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically.

New Treatments for Allergy: Advances in Peptide Immunotherapy.

Nowadays, allergen-specific immunotherapy (AIT) is the only treatment able to modulate the course of allergic diseases. Although it has been applied for the last 100 years, treatment with whole allergen extracts is not without its drawbacks: AIT can cause local and systemic adverse events and may produce new IgE sensitization against other allergens present in the extract. Furthermore, the lengthy treatment duration (3-5 years), frequent administration, and high cost of treatment are other disadvantages. For these reasons, there is a need for safer and more effective AIT strategies. One promising approach is the use of synthetic peptides representing the B- or T-cell epitopes of allergens. This review summarizes the main advances in peptide immunotherapy, from preclinical models to early clinical trials, focusing on house dust mite, bee venom, cat allergy, and Oleaceae pollinosis. Following an extensive review of the relevant literature, we summarize how peptide therapies may change the course of allergic diseases and promote allergen tolerance, thereby ameliorating the main disadvantages of AIT. Although the molecular mechanisms involved are not yet fully defined, they seem to depend on structure, length, peptide sequence, and route of administration. This novel immunotherapy has been demonstrated to modulate the immune system, promoting regulatory T-cell induction and Th2 inhibition. This tolerance-inducing potential has led this therapy to be termed SPIRE (synthetic peptide immuno-regulatory epitopes). Experimental models and clinical trials have demonstrated the usefulness of SPIRE treatment to cure these diseases, opening a new era in allergen therapeutics.

The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity. ACPA levels were determined with respect to sensitivity and specificity in 332 serum samples using the newly developed peptide panel, which was compared to the commercial assays CCPlus (Eurodiagnostica) and CCP3.1 (Inova Diagnostics).A primary panel (peptides 814, 33062 and 33156) was identified, which obtained a sensitivity of 71%, while the complete peptide panel reacted with 79% of RA sera screened. Total specificities of 89% and 80% were obtained for the primary peptide panel and the complete peptide panel. Sensitivities for the commercial assays ranged between 71% and 76% and specificities between 88% and 90%. These findings indicate that the generated peptide panel is optimal for ACPA detection and able to compete with commercial available assays. Collectively, this study may contribute to characterize autoimmunity towards citrullinated proteins and to the development of new and improved diagnostic assays for detection of ACPA and determination of RA.

Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes.

Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up of vector-borne cases and to prevent transmission of the disease by way of blood transfusions and organ transplantation. Diagnosis is routinely performed using serological methods, some of which require the production of parasite lysates, parasite antigenic fractions or purified recombinant antigens. Although available serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. Short peptides spanning linear B-cell epitopes have proven ideal serodiagnostic reagents in a wide range of diseases. Recently, we have conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects). The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96.3% and a specificity of 99.15%. Therefore, the use of synthetic peptides as diagnostic tools are an attractive alternative in Chagas’ disease diagnosis.