RESUMO
BACKGROUND: Computational methods provide approaches to identify epitopes in protein Ags to help characterizing potential biomarkers identified by high-throughput genomic or proteomic experiments. PEPOP version 1.0 was developed as an antigenic or immunogenic peptide prediction tool. We have now improved this tool by implementing 32 new methods (PEPOP version 2.0) to guide the choice of peptides that mimic discontinuous epitopes and thus potentially able to replace the cognate protein Ag in its interaction with an Ab. In the present work, we describe these new methods and the benchmarking of their performances. RESULTS: Benchmarking was carried out by comparing the peptides predicted by the different methods and the corresponding epitopes determined by X-ray crystallography in a dataset of 75 Ag-Ab complexes. The Sensitivity (Se) and Positive Predictive Value (PPV) parameters were used to assess the performance of these methods. The results were compared to that of peptides obtained either by chance or by using the SUPERFICIAL tool, the only available comparable method. CONCLUSION: The PEPOP methods were more efficient than, or as much as chance, and 33 of the 34 PEPOP methods performed better than SUPERFICIAL. Overall, "optimized" methods (tools that use the traveling salesman problem approach to design peptides) can predict peptides that best match true epitopes in most cases.
Assuntos
Biologia Computacional/métodos , Epitopos/química , Interface Usuário-Computador , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Cristalografia por Raios X , Epitopos/imunologia , Peptídeos/química , Peptídeos/imunologiaRESUMO
The occurrence of alloantibodies against Factor VIII (FVIII) is the main iatrogenic complication in haemophilia A (HA). Anti-FVIII autoantibodies may also spontaneously appear in non-HA patients, leading to acquired haemophilia A. In both contexts, the antibody response against FVIII is complex and difficult to analyse due to the lack of suitable tools. Our purpose was to comprehensively map, at the amino acid level, discontinuous epitopes of the C2 domain of FVIII targeted by patients' antibodies. We synthesized 33 synthetic peptides, which were predicted by the bioinformatic algorithm PEPOP to mimic C2 domain discontinuous epitopes. Using an inhibition assay based on the x-MAP technology, we evaluated their ability to block the binding to the C2 domain of anti-C2 domain antibodies from pooled plasma samples. Nine peptides were thus selected and tested again in individual plasma samples. Our results support the view that C2 domain epitopes are organized as an epitopic mosaic distributed around the molecule, showed that each patient displayed a specific anti-C2 epitopic profile, and confirmed the complexity and variability of the immune response against the C2 domain of FVIII. This ability to finely map epitopes could be further used to follow the antibody specificity modifications over time.
Assuntos
Epitopos/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Autoanticorpos/imunologia , Biologia Computacional , Mapeamento de Epitopos , Epitopos/análise , Fator VIII/química , Fator VIII/metabolismo , Hemofilia A/sangue , Humanos , Isoanticorpos/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/síntese química , Estabilidade ProteicaRESUMO
Autoimmune hepatitis (AIH) is a serious chronic inflammatory disease of the liver with yet unknown etiology and largely uncertain immunopathology. The hallmark of type 2 AIH is the generation of liver kidney microsomal-1 (LKM-1) autoantibodies, which predominantly react to cytochrome P450 2D6 (CYP2D6). The identification of disease initiating factors has been hampered in the past, since antibody epitope mapping was mostly performed using serum samples collected late during disease resulting in the identification of immunodominant epitopes not necessarily representing those involved in disease initiation. In order to identify possible environmental triggers for AIH, we analyzed for the first time the spreading of the anti-CYP2D6 antibody response over a prolonged period of time in AIH patients and in the CYP2D6 mouse model, in which mice infected with Adenovirus-human CYP2D6 (Ad-h2D6) develop antibodies with a similar specificity than AIH patients. Epitope spreading was analyzed in six AIH-2-patients and in the CYP2D6 mouse model using SPOTs membranes containing peptides covering the entire CYP2D6 protein. Despite of a considerable variation, both mice and AIH patients largely focus their humoral immune response on an immunodominant epitope early after infection (mice) or diagnosis (patients). The CYP2D6 mouse model revealed that epitope spreading is initiated at the immunodominant epitope and later expands to neighboring and remote regions. Sequence homologies to human pathogens have been detected for all identified epitopes. Our study demonstrates that epitope spreading does indeed occur during the pathogenesis of AIH and supports the concept of molecular mimicry as a possible initiating mechanism for AIH.
Assuntos
Autoanticorpos/biossíntese , Citocromo P-450 CYP2D6/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatite Autoimune/imunologia , Epitopos Imunodominantes/imunologia , Adenoviridae/química , Adenoviridae/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Autoanticorpos/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Criança , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Feminino , Vetores Genéticos/química , Vetores Genéticos/imunologia , Hepatite C Crônica/genética , Hepatite C Crônica/virologia , Hepatite Autoimune/genética , Hepatite Autoimune/virologia , Humanos , Epitopos Imunodominantes/genética , Camundongos , Camundongos Transgênicos , Mimetismo Molecular , Dados de Sequência MolecularRESUMO
BACKGROUND: In current comparative proteomics studies, the large number of images generated by 2D gels is currently compared using spot matching algorithms. Unfortunately, differences in gel migration and sample variability make efficient spot alignment very difficult to obtain, and, as consequence most of the software alignments return noisy gel matching which needs to be manually adjusted by the user. RESULTS: We present Sili2DGel an algorithm for automatic spot alignment that uses data from recursive gel matching and returns meaningful Spot Alignment Positions (SAP) for a given set of gels. In the algorithm, the data are represented by a graph and SAP by specific subgraphs. The results are returned under various forms (clickable synthetic gel, text file, etc.). We have applied Sili2DGel to study the variability of the urinary proteome from 20 healthy subjects. CONCLUSION: Sili2DGel performs noiseless automatic spot alignment for variability studies (as well as classical differential expression studies) of biological samples. It is very useful for typical clinical proteomic studies with large number of experiments.
Assuntos
Biologia Computacional/métodos , Eletroforese em Gel Bidimensional/métodos , Proteômica/métodos , Algoritmos , Análise por Conglomerados , Interpretação Estatística de Dados , Perfilação da Expressão Gênica , Humanos , Modelos Estatísticos , Reconhecimento Automatizado de Padrão/métodos , Proteoma , SoftwareRESUMO
BACKGROUND: Most methods available to predict protein epitopes are sequence based. There is a need for methods using 3D information for prediction of discontinuous epitopes and derived immunogenic peptides. RESULTS: PEPOP uses the 3D coordinates of a protein both to predict clusters of surface accessible segments that might correspond to epitopes and to design peptides to be used to raise antibodies that target the cognate antigen at specific sites. To verify the ability of PEPOP to identify epitopes, 13 crystallographically defined epitopes were compared with PEPOP clusters: specificity ranged from 0.75 to 1.00, sensitivity from 0.33 to 1.00, and the positive predictive value from 0.19 to 0.89. Comparison of these results with those obtained with two other prediction algorithms showed comparable specificity and slightly better sensitivity and PPV. To prove the capacity of PEPOP to predict immunogenic peptides that induce protein cross-reactive antibodies, several peptides were designed from the 3D structure of model antigens (IA-2, TPO, and IL8) and chemically synthesized. The reactivity of the resulting anti-peptides antibodies with the cognate antigens was measured. In 80% of the cases (four out of five peptides), the flanking protein sequence process (sequence-based) of PEPOP successfully proposed peptides that elicited antibodies cross-reacting with the parent proteins. Polyclonal antibodies raised against peptides designed from amino acids which are spatially close in the protein, but separated in the sequence, could also be obtained, although they were much less reactive. The capacity of PEPOP to design immunogenic peptides that induce antibodies suitable for a sandwich capture assay was also demonstrated. CONCLUSION: PEPOP has the potential to guide experimentalists that want to localize an epitope or design immunogenic peptides for raising antibodies which target proteins at specific sites. More successful predictions of immunogenic peptides were obtained when a peptide was continuous as compared with peptides corresponding to discontinuous epitopes. PEPOP is available for use at http://diagtools.sysdiag.cnrs.fr/PEPOP/.
Assuntos
Algoritmos , Biologia Computacional/métodos , Epitopos/genética , Peptídeos/genética , Peptídeos/imunologia , Engenharia de Proteínas/métodos , Estrutura Quaternária de Proteína , Anticorpos/metabolismo , Peptídeos/metabolismo , Sensibilidade e EspecificidadeRESUMO
Four IgE-binding epitopes have been characterized that cover a large area (40%) of the molecular surface of lipid transfer protein allergens of Rosaceae (apple, peach, apricot, and plum). They mainly correspond to electropositively charged regions protruding on the molecular surface of the modeled apple (Mal d 3), apricot (Pru ar 3), and plum (Pru d 3) allergens. Two of these epitopes consist of consensus epitopes structurally conserved among the lipid transfer protein allergens from the Rosaceae. Their occurrence in different lipid transfer protein allergens presumably accounts for the IgE-binding cross-reactivity often observed among different Rosaceae fruits. In this respect, LTP consist of phylogenetically- and structurally-related pan allergens. However, the IgE-binding cross-reactivity due to fruit lipid transfer protein has varying degrees of clinical relevance and this cross-reactivity is not necessarily accompanied by a cross-allergenicity to the corresponding fruits.
Assuntos
Alérgenos/imunologia , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Epitopos/imunologia , Frutas/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Animais , Sequência Consenso/imunologia , Reações Cruzadas/imunologia , Humanos , Ligação Proteica , CoelhosRESUMO
The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.
Assuntos
Actinas/química , Ligação Competitiva , Reagentes de Ligações Cruzadas/metabolismo , Destrina/química , Análise Serial de Proteínas , Mapeamento de Interação de Proteínas , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Animais , Desoxirribonuclease I/metabolismo , Gelsolina/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Fatores de Transcrição/metabolismoRESUMO
The development of antibodies (Abs) against infused factor VIII (FVIII) is currently one of the most serious complications in the treatment of patients suffering from haemophilia A. Improved prevention and eradication of these anti-FVIII Abs remain a challenge for both clinicians and scientists. Here we describe an immunoassay to simultaneously detect and map the epitope specificity of haemophilia A patients' inhibitors by screening plasma against both heavy and light chains (HC and LC) of human plasma-derived FVIII (pFVIII). The format used was a two-site sandwich assay, where one monoclonal antibody (mAb) specific for the HC or LC was first immobilized on beads, and then incubated with the different forms of pFVIII. After incubation with patients' plasma samples, binding was revealed by a phycoerythrin-labeled secondary Ab. Samples from haemophilia patients with autoantibodies (autoAb) or alloantibodies (alloAb) were screened in this format. The former preferentially recognized the LC, whereas the latter were directed against both LC and HC. This technology appears attractive as it is fast and requires only 100 microl of patient's plasma. Furthermore, not only are anti-FVIII Abs detected, but information on their epitopic specificity is also obtained.
Assuntos
Anticorpos Monoclonais , Autoanticorpos/sangue , Mapeamento de Epitopos , Fator VIII/imunologia , Hemofilia A/imunologia , Imunoensaio/métodos , Isoanticorpos/sangue , Animais , Reações Antígeno-Anticorpo , Estudos de Viabilidade , França , Humanos , Camundongos , SuíçaRESUMO
The development of antibodies directed against factor VIII (FVIII) represents a major hurdle in the treatment of hemophilia A. Most anti-FVIII antibodies are identified through their ability to inhibit the FVIII procoagulant activity. Many of them, however, do not interfere with the functional properties of FVIII. Antibodies directed against the B domain belong to this latter category. Here, we characterized B domain-specific human monoclonal Abs (mAbs) at the molecular level. A series of human mAbs directed against FVIII was produced upon immunization of transgenic XenoMouse mice with human recombinant FVIII (rFVIII). Selection of the hybridoma with epitope specificity for the B domain was performed by differential recognition of full-length and B domain-deleted rFVIII. None of the anti-B domain mAbs demonstrated inhibitory activity against FVIII. Three of the mAbs recognized linear epitopes: mAb 25H3 bound to the (1014)HIDGPSLLIEN(1024) sequence; mAbs 8E3 and 22B6 shared the same epitope, composed of residues (1534)KWNEANR(1540). The corresponding soluble peptides inhibited the binding of their respective mAbs to FVIII. mAbs 8E3 and 22B6 displaced the binding of FVIII to vonWillebrand factor. Moreover, some of them (in particular mAbs 4G6 and 8E3) were able to compete for binding to the B domain with the anti-FVIII Abs from hemophilia A patients without inhibitor or with low Bethesda titers. Further investigation will allow to better characterize their clinical relevance.
Assuntos
Anticorpos Monoclonais/imunologia , Epitopos , Fator VIII/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacologia , Ligação Competitiva , Criança , Pré-Escolar , Fator VIII/metabolismo , Hemofilia A/imunologia , Humanos , Hibridomas/imunologia , Lactente , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Fator de von Willebrand/metabolismoRESUMO
This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface. Introduction of polyethylene glycol (EG(6)) chains into mixed SAM layers and addition of an anionic surfactant to the human serum reduced non-specific binding without modifying the viscoelasticity properties of the layer. Under our conditions, the antibody SPR detection limit was determined to be 0.2 nM in diluted human serum. This value is in agreement with the reported rank distribution of IA-2 antibodies in diabetic patient sera. Label-free and real-time technologies such as SPR and/or QCM-D could be precious tools in future diagnostic assays.
Assuntos
Reações Antígeno-Anticorpo , Autoanticorpos/sangue , Quartzo/química , Ressonância de Plasmônio de Superfície/métodos , Sequência de Aminoácidos , Humanos , Dados de Sequência MolecularRESUMO
We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium-binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine-glutamine exchange at position 29 in the nonfunctional calcium-binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N-terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107-112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A-dependent phosphorylation of cTnI at serine 22 and serine 23 in wild-type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+-free and Ca2+-saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+-dependent actomyosin subfragment 1 (actoS1)-ATPase activity and the sliding velocity of thin filaments. The maximum actoS1-ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A-dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1-ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A-dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.
Assuntos
Cardiomiopatia Hipertrófica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miocárdio/química , Transdução de Sinais/fisiologia , Troponina C/genética , Troponina C/metabolismo , Troponina I/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Mutação Puntual , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
With the fast growth of high-throughput strategies in Biology, there is a strong need to accelerate knowledge acquisition and organization of molecular functions. Unfortunately, although we know that there is a correlation between protein molecules and their functions, we are unable to clearly identify this link. Here, we revisit the current views of protein functions as well as their annotation, and we show that they are incompatible with unambiguous interpretations and the use of this knowledge. We describe herein a description scheme for biological processes based on elementary bricks of action that may be associated with biological molecules. To retrieve the descriptive quality found in annotations of other kinds of biological data, it was decided to develop a scheme involving four levels of abstraction: Basic Elements of Action, Biological Activities, Biological Functionalities and Biological Roles. This multi-level organization is a generic method; it allows for a description of biological processes by using a limited number of elementary bricks of action. Moreover, by using this description of biological processes, it should now be possible to clearly identify unambiguous relationships between the organization of biological processes and the structural or functional organizations of biological molecules.
Assuntos
Proteínas/classificação , Proteínas/fisiologia , Receptor de Insulina/fisiologia , Análise de Sequência de Proteína/métodos , Transdução de Sinais , Algoritmos , Biologia Computacional , Bases de Dados de Proteínas , Humanos , Proteínas/química , ProteomaRESUMO
The development of anti-factor VIII (FVIII) antibodies is currently one of the most serious complications in the treatment of haemophilia A patients. Numerous studies in literature report on their epitope specificity, their mechanism of FVIII inactivation, and their relationship with FVIII genetic alterations. During the last two years, however, a particular effort has been made to better understand their generation, with particular emphasis on the interplay of T cells and B cells specific for FVIII and the generation of anti-FVIII antibodies. Moreover, novel strategies to improve the management or treatment of patients with anti-FVIII antibodies have been recently proposed: the use of less immunogenic engineered recombinant FVIII molecules, neutralization of inhibitors by blocking their deleterious activity either by low molecular weight peptide decoys or by anti-idiotypic antibodies, and attempts to suppress the T-cell response involved in the antibody formation. All of these represent promising therapeutic approaches. This review attempts to sum up current knowledge of the nature and properties of anti-FVIII antibodies, their mechanism of action, their neutralization by anti-idiotypic antibodies, and the role of T cells in FVIII inhibitor formation. In the final part, some of the new strategies susceptible to improve the management or the eradication of anti-FVIII antibodies are presented.
Assuntos
Autoanticorpos/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Epitopos/imunologia , Hemofilia A/genética , HumanosRESUMO
A non-toxic protein (TsNTxP) isolated from the venom of the noxious scorpion Tityus serrulatus (Ts) induces polyclonal antibodies cross-reactive with several toxins from the venom, in sharp contrast to anti-toxin antibodies which are toxin specific. To try to uncover the molecular basis for these unusual properties, peptide scanning experiments were performed and indicated that the N- and C-terminal parts of TsNTxP enclose continuous epitopes (residues 1-15 and 47-61). Antibodies raised against peptides corresponding to these two regions were found to have neutralizing properties against a mixture of all toxic proteins from the T. serrulatus venom, indicating that residues 1-15 and 47-61 correspond to neutralizing epitopes. The identification of key antigenic residues within these two epitopes revealed that several of them are well conserved in the amino-acid sequences of the three main toxins (Ts II, Ts IV and Ts VII) from the venom: Glu 3, Tyr 5, Asp 8, Asp 50, Trp 55 and Lys 61. A single key-residue (Glu 58) is unique to TsNTxP. By using homology modeling, a model of the three-dimensional structure of TsNTxP was obtained. The antigenically important residues from TsNTxP were found to be surface exposed, with five of them clustered on the facet of the protein reported to enclose the active site of toxins. Residues equivalent to the seven key-residues of the anatoxin were also found to be exposed in the active toxins from T. serrulatus venom. These results show that antibodies elicited by the non-toxic protein TsNTxP recognized, within the N- and C-terminal parts of toxins of T. serrulatus, conserved and surface exposed residues which might also be involved in the toxic action of the proteins.
Assuntos
Anticorpos/imunologia , Venenos de Escorpião/imunologia , Toxoides/imunologia , Sequência de Aminoácidos , Animais , Reações Cruzadas , Mapeamento de Epitopos , Feminino , Cavalos , Camundongos , Dados de Sequência Molecular , Coelhos , Venenos de Escorpião/química , Toxoides/químicaRESUMO
ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL.
Assuntos
Doenças do Cão/diagnóstico , Doenças do Cão/imunologia , Imunoensaio/veterinária , Leishmaniose Visceral/diagnóstico , Biblioteca de Peptídeos , Testes Sorológicos/veterinária , Animais , Cães , Mapeamento de Epitopos/veterinária , Feminino , Leishmaniose Visceral/imunologia , Masculino , Peptídeos/imunologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Leishmania parasites can cause visceral or cutaneous disease and are found in subtropical and tropical regions of the Old and New World. The pathology of the infection is determined by both host immune factors and species/strain differences of the parasite. Dogs represent the major reservoir of Leishmania infantum (syn. L. chagasi) and vaccines are considered the most cost-effective control tools for canine disease. METHODS: Selection of immunodominant peptides was performed by Phage Display to identify sequences recognized by L. infantum naturally infected animals. Sera from Leishmania infected animals were used in the biopanning to selection of specific peptides. Serum samples from T. cruzi infected and healthy animals were used as control. After selection, synthetic peptides were produced in membrane (spot-synthesis) in soluble form and blotting and ELISA were performed for validation of serum reactivity. Selected peptide was formulated with aluminum hydroxide and liposomes and immunization was performed in BALB/c mice. Protection was determined by qPCR after challenge infection with virulent L. infantum. RESULTS: We reported the selection of Peptide 5 through Phage Display technique and demonstrate its ability to promote a state of immunity against L. infantum infection in murine model after immunization using liposomes as vaccine carrier. Our results demonstrate that immunization with Peptide 5 when formulated with aluminum hydroxide and liposomes is immunogenic and elicited significant protection associated with the induction of mixed Th1/Th2 immune response against L. infantum infection. CONCLUSION: Peptide 5 is a promising vaccine candidate and the findings obtained in the present study encourage canine trials to confirm the effectiveness of a vaccine against CVL.
Assuntos
Doenças do Cão/imunologia , Leishmania infantum/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/veterinária , Peptídeos/imunologia , Animais , Colesterol , Doenças do Cão/parasitologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , EsfingomielinasRESUMO
Overlapping peptide scans prepared by Spot synthesis have been used to map interaction sites in several systems. Here we report our experience with this approach to identify peptides from the variable parts of anti-hapten, anti-peptide and anti-protein antibodies that retain their specific antigen-binding capacity in the Spot format. In general, the identification by the Spot method of antigen-reactive peptides was confirmed by using soluble peptides which demonstrated antigen-binding capacity in ELISA or Biacore and, biological activity for some peptides derived from anti-CD4 antibodies. The Spot method was also used to map precisely key residues from the antibody paratope. The identification of critical residues from an anti-troponin I antibody of diagnostic interest is reported as well as the compiled results from the analysis of five other antibodies of various specificities. A critical assessment of our results is provided by comparing results obtained by our approach in the mapping of antibody residues critical for antigen binding with data from the literature concerning the structural analysis of antigen-antibody complexes.
Assuntos
Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo , Sítios de Ligação , Humanos , Hibridomas/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Scorpion neurotoxins acting on ion channels share some structural features but differ in antigenic and immunogenic properties. They are highly structured peptides, 60-70 amino acids long. Monoclonal antibodies have been obtained for Androctonus australis hector scorpion venom neurotoxin II (AahII) and a nontoxic synthetic analog ((Abu)(8) AahII). In this study, no antibody response was elicited in mice of various strains injected with AahI, the other important toxin of the venom, in a native or an inactive ((Abu)(8) AahI) form. We found that AahI was only immunogenic in BALB/c or C57BL/6 mice if it was coupled to a carrier protein. The helper protein molecule could be BSA, KLH, or the nontoxic analog of AahII. We obtained a panel of high-affinity mAbs with these immunogens. Two of these mAbs, including the very high-affinity antibody 9C2 (K(D)=0.11x10(-11) M), were used to set up a two-site ELISA, sensitive enough for the quantification of AahI in the biological fluids of envenomed animals. The detection limit of the assay was 75 pg/ml.
Assuntos
Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Neurotoxinas/imunologia , Radioimunoensaio/métodos , Venenos de Escorpião/imunologia , Escorpiões/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Antivenenos/imunologia , Proteínas de Transporte/imunologia , Imunoglobulina G/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Coelhos , Ressonância de Plasmônio de SuperfícieRESUMO
Anti-p53 antibodies have been detected in the sera of patients with various types of cancers. In this report, we describe the development of a new ELISA aimed at detecting anti-p53 antibodies using two peptides belonging to immunodominant epitopes of the p53 N-terminal region. We first tested the reactivity of the sera by an indirect ELISA using the peptides as a capture system. Then, the specificity of the reaction was confirmed by an inhibition assay. Two systems of peptide presentation, phage display and the streptavidin/biotin system, were evaluated. Using a panel of sera from cancer patients, both systems were found to be equally reliable, demonstrating that both peptide-based ELISAs can be used for the specific detection of anti-p53 antibodies. The presence of anti-p53 antibodies was associated with p53 alteration whether it be mutation or accumulation.