RESUMEN
Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A-56.36 and CB had overlapping binding sites on CA. By screening random phage-displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin-dissociating effect of A-56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage-displayed peptides corresponding to fragments of the two identified regions on CA reacted with A-56.36 and with CB. Our data support the hypothesis that A-56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.
Asunto(s)
Anticuerpos Monoclonales/química , Antitoxinas/inmunología , Mapeo Epitopo , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antitoxinas/química , Bacteriófagos/genética , Sitios de Unión/inmunología , Unión Competitiva , Crotalus , Crotoxina/química , Crotoxina/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Unión Proteica , Desnaturalización Proteica , Homología de Secuencia de AminoácidoRESUMEN
Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.
Asunto(s)
Factores de Crecimiento Endotelial/antagonistas & inhibidores , Linfocinas/antagonistas & inhibidores , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Células CHO , División Celular/efectos de los fármacos , Córnea/irrigación sanguínea , Córnea/efectos de los fármacos , Cricetinae , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Técnicas In Vitro , Linfocinas/genética , Linfocinas/fisiología , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Biblioteca de Péptidos , Unión Proteica , Conejos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The phage-displayed peptide CGRVCLRC (C15) has been isolated from a random library by affinity screening with the D14-3 monoclonal antibody, which was raised to the 42 kDa C-terminal fragment of the major merozoite surface protein 1 of Plasmodium vivax (Pv42). In order to investigate the use of such mimotopes as possible vaccine components, we studied the antibody response in Biozzi mice immunized with C15. High titers of antibodies cross-reacting with Pv42 were generated and the IC50 of all immune sera were in the 5 x 10(-9) M range. Two monoclonal antibodies that specifically bind the Pv42 fragment were isolated. Although these mAbs had a lower affinity for Pv42 when compared to D14-3, they reproduced the cross-reactivity of D14-3 with the equivalent protein in P. cynomolgi, a close relative of P. vivax. DNA sequence analysis showed similarities between the germline genes and the canonical CDR conformations of all three antibodies, but molecular modeling failed to reveal common structural features of their paratopes that could account for their cross-reacting patterns. These data demonstrate that mimotopes selected from random repertoires do not necessarily represent structural equivalents of the original antigen but provide functional images that could replace it for vaccine development.
Asunto(s)
Anticuerpos Monoclonales/química , Bacteriófagos/genética , Vacunas contra la Malaria/inmunología , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Plasmodium vivax/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Región Variable de Inmunoglobulina/inmunología , Proteína 1 de Superficie de Merozoito , Ratones , Ratones Endogámicos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Plasmodium cynomolgi/química , Plasmodium cynomolgi/inmunología , Precursores de Proteínas/inmunología , Proteínas Protozoarias/inmunología , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The invasion plasmid antigen B (IpaB), a 62-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells, is the bacterial antigen most strongly and consistently recognized by the host during infection. The strong systemic and mucosal immune responses observed against this invasin prompted us to map its B-cell epitopes. For this purpose, IpaB was first overexpressed in Shigella flexneri and used to raise rabbit polyclonal antiserum and murine monoclonal antibodies, which were subsequently used to screen a lambda gt11 ipaB library. Inserts of recombinant DNA clones that were specifically recognized by the antisera and antibodies were sequenced, and three distinct determinants were identified. Further characterization of these determinants showed that they were recognized by sera from patients convalescent from shigellosis, suggesting that they are relevant to the humoral response during natural infection. Moreover, the IpaB region comprising the three determinants was systematically recognized by all sera from infected patients that we tested, whereas other regions of the protein were not. These data suggest that this region, located between amino acid residues 147 and 258, is the major immunogenic domain of the invasin in the course of natural infection.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Disentería Bacilar/inmunología , Epítopos , Shigella flexneri/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Plásmidos , Conejos , Shigella flexneri/patogenicidadRESUMEN
Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection.
Asunto(s)
Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Epítopos/análisis , Plásmidos , Shigella flexneri/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Proteínas Bacterianas/genética , Unión Competitiva , Clonación Molecular , Escherichia coli/genética , Biblioteca de Genes , Haplorrinos , Humanos , Ratones , Conejos , Shigella flexneri/patogenicidadRESUMEN
The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.
Asunto(s)
Alérgenos/inmunología , Epítopos/inmunología , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Ácaros/inmunología , Animales , Anticuerpos Monoclonales , Antígenos Dermatofagoides , Basófilos/inmunología , Unión Competitiva , Histamina/metabolismoRESUMEN
Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice. Three of these could inhibit the hemolytic activity of LLO. One of them inhibited binding of LLO to erythrocyte membranes. The two other antibodies blocked the activity of LLO at a step subsequent to membrane binding. Only two of the nine MAbs recognized three other purified SH-activated toxins, streptolysin O, alveolysin, and pneumolysin. Western blot (immunoblot) analysis of culture supernatants of Listeria ivanovii and Listeria seeligeri, two hemolytic species of the genus Listeria, revealed that two MAbs recognized ivanolysin and seeligerolysin. The latter was also recognized by two other MAbs, including one of the neutralizing antibodies. MAbs raised against a peptide, ECTG LAWEWWR, present in all thiol-activated toxins sequenced to date, recognized all toxins and were not neutralizing. Taken together, these results demonstrate the existence of regions important for hemolytic activity that are unique to hemolysins of the genus Listeria and show that regions outside the conserved peptide are important for activity of LLO.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Toxinas Bacterianas , Proteínas de Choque Térmico/inmunología , Proteínas Hemolisinas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Hemólisis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
Two anti-nucleoside monoclonal antibodies (A-16 and G-K21) were raised after immunizing mice with adenosine or guanosine coupled to bovine serum albumin by periodate oxidation. They were selected for their ability to detect these immunogens and single-stranded DNA in an enzyme-linked immunosorbent assay test. The antibodies were purified from ascitic fluids, their isotypes were determined and their ability to detect DNAs and RNAs on nitrocellulose membranes was tested. They belonged to the IgG1 subclass and were both able to recognize picogram amounts of single-stranded DNAs on nitrocellulose sheets, whatever the origin of the nucleic acid, but were unable to detect RNA efficiently. The same monoclonal antibodies were used to estimate minute amounts of target staphylococci DNAs to permit standardization of non-radioactive hybridization experiments for detection of antibiotic resistance genes.
Asunto(s)
Adenosina/inmunología , Anticuerpos Monoclonales/inmunología , ADN/análisis , Guanosina/inmunología , Especificidad de Anticuerpos , ADN/inmunología , ADN de Cadena Simple/inmunología , Inmunoglobulina G/inmunología , Hibridación de Ácido NucleicoRESUMEN
A method is described for achieving the efficient production of proteins of biological interest by the establishment of hybridomas from lymphocytes of transgenic animals carrying a fusion gene having promoter-regulatory sequences functional in lymphocytes fused to the coding sequences of the protein to be produced. While possible improvements in the method are discussed, it is anticipated that the method will ultimately be applicable to the production of any protein of interest.
Asunto(s)
Hibridomas , Animales , Femenino , Hormona del Crecimiento/genética , Antígenos H-2/genética , Hibridación Genética , Linfocitos , Masculino , Ratones , MicroinyeccionesRESUMEN
Spleen cells from 6-day-old unimmunized male and female (CBA/N X BALB/c)F1 mice, fused with the nonsecreting hybrid SP2/0, gave 184 hybrids secreting immunoglobulins (183 IgM class; 115 from males and 69 from females) which were screened for antibody (Ab) activity against actin, tubulin, myosin, TNP-BSA, dsDNA, and denatured DNA. Eleven hybrids from the male series (9.65%) and eight hybrids from the female series (11.55%) secreted immunoglobulins with significant Ab activity against at least one of the six antigens tested. Four of these clones reacted with a single antigen, while 15 others reacted simultaneously with at least two antigens. Three hybrid clones exhibited Ab activity against all of the antigens tested. The monoclonality and specificity of the immunoglobulins from tissue culture supernatants were respectively assessed by two-dimensional gel electrophoresis after biosynthetic labeling and competitive enzyme-linked immunosorbent assay. These results confirm our previous findings with newborn and adult BALB/c mice, demonstrating a high frequency of this auto-Ab repertoire, especially in newborn mice, and widespread polyspecificity found among these natural auto-Ab. Xid mice are characterized by the absence of a Lyb-3+,5+ B cell population; nevertheless, these studies suggest--but do not prove--that the Lyb-3-,5- lymphocyte population seems to carry genetic information for the production of natural auto-Ab, because no marked differences were observed in the secretory patterns of hybrids obtained from male mice bearing the Xid defect and from normal female mice. Hence, we postulate that the Xid gene might be a regulatory gene because Xid mice, in contrast with normal mice, are unable to secrete anti-DNA auto-Ab on mitogenic stimulation, despite carrying the genetic information.
Asunto(s)
Animales Recién Nacidos/inmunología , Autoanticuerpos/biosíntesis , Proteínas del Citoesqueleto/inmunología , ADN/inmunología , Síndromes de Inmunodeficiencia/genética , Nitrobencenos/inmunología , Trinitrobencenos/inmunología , Animales , Anticuerpos Antinucleares/análisis , Anticuerpos Antinucleares/biosíntesis , Especificidad de Anticuerpos , Células Productoras de Anticuerpos/metabolismo , Autoanticuerpos/análisis , Sitios de Unión de Anticuerpos , Femenino , Hibridomas/metabolismo , Síndromes de Inmunodeficiencia/inmunología , Masculino , Ratones , Ratones Endogámicos CBA , Cromosoma X/inmunologíaRESUMEN
A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1-8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase. Nevertheless, using the C1-8 monoclonal antibody, it was possible to show that they are different proteins. The C1-8 binding protein appears to be specific for the SPM in Torpedo electric organ since it was not detected in plasma membranes from the electroplaque, the electric nerve trunks or the electric lobe. The hydrophobic AChE and the C1-8 binding protein appear therefore to be useful markers of the SPM. Pronase treatment of intact synaptosomes removes most of the ectocellularly exposed proteins of the SPM, which amount to 35% of the SPM protein. Presynaptic AChE and the C1-8 binding protein are detached. But ACh release can still be induced by depolarization of the Pronase treated synaptosomes. This demonstrates that the two 67 kdalton presynaptic proteins are not directly involved in the release of the neurotransmitter.
RESUMEN
Spleen cells from nonimmunized BALB/c mice were fused with two nonsecreting myeloma lines. The hybrids were selected in HAT medium and screened for Ig production and for antibody activity against actin, tubulin, myosin, thyroglobulin, myoglobin, spectrin, dsDNA, fetuin, and transferrin. Among 161 hybrids secreting Ig, three were found to react with DNA, one with thyroglobulin, and one mainly with myosin. Two of these hybrids could be propagated and further characterized. On the basis of inhibition experiments, one was found to be directed against dsDNA; the other was directed mainly against myosin but at the same time reacted significantly with actin, tubulin, spectrin, and dsDNA. Reactivity with myosin seemed to be concentrated in the light meromyosin subfragment, known to be rich in alpha-helical structure. These results indicate: 1) There are reactive B cell clones directed against self antigens. 2) The antibody specificities found for these antibodies are very similar to those found for natural antibodies in normal human serum and for human monoclonal Ig. 3) The widespread reactivity found for the clone mainly reacting with myosin raises the possibility that the determinant recognized by this antibody is a conformational structure that possibly is associated with alpha-helical structures.