RESUMO
Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Toxinas Botulínicas Tipo A/análise , Toxinas Botulínicas/análise , Ensaio de Imunoadsorção Enzimática , Animais , Toxinas Botulínicas/imunologia , Toxinas Botulínicas Tipo A/imunologia , Botulismo/diagnóstico , Clostridium botulinum/isolamento & purificação , Microbiologia de Alimentos , Alimentos em Conserva/análise , Alimentos em Conserva/microbiologia , Carne/análise , Carne/microbiologia , Camundongos , Sensibilidade e Especificidade , Verduras/microbiologiaRESUMO
A method for the preparation of peptide mimetics of GMDP which could exhibit adjuvant activity without the negative effects of GMDP is described. The search for peptides with GMDP-like adjuvant activity was performed using highly specific monoclonal antibodies against GMDP and combinatorial peptide libraries in the format of phage display. Various elution methods were used for the immunoaffinity enrichment of the libraries during the course of the preparation of highly active and specific peptides. A sole peptide (Arg-Val-Pro-Pro-Arg-Tyr-His-Ala-Lys-Ile-Ser-Pro-Met-Val-Asn, RN) was obtained by the elution of phage particles from the immunosorbent with a 1 -microM solution of the natural ligand (GMDP). Elution with a buffer with a low pH value (0.1 M glycine-HCl, pH 2.2) gave two other peptides: Ser-Gly-Arg-Val-Ala-Val-Ser-Pro-Asp-Ser-Pro-Leu-Phe-Tyr-Pro (SP) and Arg-Tyr-Gly-Gly-Ser-Val-Leu-Asn-Ile-Glu-Cys-Gln-Phe-Tyr-Gly (RG). Affinity constants for the RN and SP peptides proved to be 3.6 x 10(8) and 3.5 x 10(8) M(-1), respectively. The specificity of the interaction with the monoclonal antibodies was checked by the competitive displacement of the peptides from the antigen-antibody complex by GMDP. The RN peptide exhibited adjuvant activity similar to that of GMDP, but had no pyrogenic effect characteristic of GMDP. The described method could be used for the search for mimetics of biologically active low-molecular compounds.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/isolamento & purificação , Anticorpos Monoclonais/química , Oligopeptídeos/isolamento & purificação , Biblioteca de Peptídeos , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/isolamento & purificação , Adjuvantes Imunológicos/química , Animais , Especificidade de Anticorpos , Bacteriófagos/química , Bacteriófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Oligopeptídeos/química , Oligopeptídeos/imunologia , VacinaçãoRESUMO
We describe here the cloning, expression, and production of specific single-chain antibodies (scFv) against the recombinant enterotoxin C1 of Staphylococcus aureus. High-affinity scFv were selected from the phage library of human mini antibodies; afterwards, the cells of E. coli trxA gor double mutant were infected with a product obtained by fusion of DNA encoding of these mini antibodies with the trxA gene to induce soluble scFv synthesis in cell cytoplasm. The scFv obtained displayed high enterotoxin C1 affinity. Analysis for cross reactivity showed that mini-antibodies interacted also with SEA- SEB-, SED-, SEE-, SEG-, and SEI-type enterotoxins, but they failed to interact with ricin, diphtheritic, and cholera toxins, or with both lethal and protective factors of the anthrax toxin. This property may be helpful in screening for staphylococcus enterotoxins.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Enterotoxinas/imunologia , Staphylococcus aureus , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Clonagem Molecular , Enterotoxinas/química , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306-332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/química , Vacinas Meningocócicas/síntese química , Neisseria meningitidis Sorogrupo A/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Fragmentos de Peptídeos/química , Polissacarídeos Bacterianos/química , Sequência de Aminoácidos , Animais , Bacteriemia/imunologia , Bacteriemia/microbiologia , Bacteriemia/prevenção & controle , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Meningocócicas/imunologia , Camundongos , Dados de Sequência Molecular , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologiaRESUMO
It is known that Aspergillus fumigatus secretes a serine protease ALP1 of the subtilisin family in the presence of extracellular protein substrates. We found conditions of A. fumigatus culturing that provide a high ALP1 activity inside cells without induction by extracellular proteins. The identity of the properties of the secreted and intracellular enzymes was shown. A thermostable protein inhibitor of the ALP1 protease was isolated from the plasmodium of the myxomycete Physarum polycephalum. Its molecular mass is 32-33 kDa. The inhibitor inhibits the ALP1 protease activity with IC50 of 0.14 microM. This protein was also shown to be a less efficient inhibitor of the activity of HIV-1 protease (IC50 2.5 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Physarum/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificação , Subtilisinas/química , Subtilisinas/isolamento & purificação , Animais , Proteínas Fúngicas/antagonistas & inibidores , Protease de HIV/química , Subtilisinas/antagonistas & inibidoresRESUMO
The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in a new crystal form (space group P2(1)2(1)2(1); unit cell parameters: a = 42.82, b = 90.68, and c = 139.82 A) was determined by the X-ray molecular replacement method at the resolution of 2.7 A. The protein folding and the stereochemistry of its antigen-binding site were comparatively analyzed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Interleucina-2/imunologia , Anticorpos Monoclonais/imunologia , Antígenos/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , ÁguaRESUMO
As shown in this work, the synthetic immunomodulator glucosaminylmuramyldipeptide (GMDP) can be included into acellular pertussis vaccine (APV). The optimal doses of GMDP, ranging from 0.001 to 0.0001 microg, have been found. These doses enhance the protective activity of APV, especially its low-active doses. GMDP decrease the manifestations of toxic, anaphylactogenic and pyrogenic properties of APV, which may lead to the decrease of the antigenic load of APV on the body of the vaccines and thus to lessening the side-effects of vaccination. GMDP has been shown to considerably increase, in comparison with common pertussis vaccine and APV, the percentage of phagocytizing leukocytes by day 14. The immunization of mice with APV with and without GMDP in doses of 0.01 and 0.001 microg leads to a change in T-lymphocyte/B-lymphocyte ratio in the population of spleen lymphocytes.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/administração & dosagem , Imunização , Fatores Imunológicos/administração & dosagem , Vacina contra Coqueluche/administração & dosagem , Coqueluche/prevenção & controle , Acetilmuramil-Alanil-Isoglutamina/imunologia , Animais , Animais não Endogâmicos , Relação Dose-Resposta Imunológica , Avaliação Pré-Clínica de Medicamentos , Feminino , Febre/induzido quimicamente , Fatores Imunológicos/imunologia , Injeções Intraperitoneais , Leucócitos/imunologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Vacina contra Coqueluche/efeitos adversos , Fagocitose , Baço/imunologia , Vacinas Acelulares/administração & dosagem , Coqueluche/imunologiaRESUMO
Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the bacterium Neisseria meningitidis were synthesized in order to create a synthetic vaccine against the meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with the free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.
Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Neisseria meningitidis/imunologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Meningite Meningocócica/mortalidade , Meningite Meningocócica/prevenção & controle , Vacinas Meningocócicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Fragmentos de Peptídeos/químicaRESUMO
Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Bacteriorodopsinas/imunologia , Sítios de Ligação , Epitopos , Glicina/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Octoxinol/química , Fragmentos de Peptídeos/imunologia , Fenilalanina/química , Conformação Proteica , Valina/químicaRESUMO
To develop a phage display of single-chain antibodies (scFv), fractions of total cell DNA and RNA were obtained from splenocytes of naive mice. The DNA fragments encoding variable regions of light and heavy immunoglobulin chains were amplified and isolated using primers specific to the conservative regions of these genes. The construction of the library was based on the principle of stochastic combining of the DNA fragments encoding the light and heavy antibody chains with the DNA linker, whose structure corresponded to the (Gly4Ser)3 sequence. The scFv library was constructed using the E. coli TG1 strain and the phagemid vector pHEN1. The repertoire of the library exceeded 5 x 10(7) independent recombinant clones. The clones producing antibodies to the granulocyte colony-stimulating human factor were isolated. The affinity constants of the resulting scFv were in the range of 2 x 10(4) to 1.8 x 10(7) M-1.
Assuntos
Anticorpos/genética , Biblioteca Gênica , Fator Estimulador de Colônias de Granulócitos/imunologia , Região Variável de Imunoglobulina/genética , Animais , Bacteriófagos/genética , Sequência de Bases , Primers do DNA , Escherichia coli/genética , Humanos , Camundongos , Dados de Sequência MolecularRESUMO
The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 +/- 200 per cell) and affinity (Kd = 10 +/- +/- 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitro with GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 +/- 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor alpha (TNF-alpha). The mechanism of the TNF-alpha action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Melanoma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Humanos , Hidrólise , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mice of various lines were immunized by 11 synthetic peptides that correspond to the sequences of fragments of the OpaB protein from the outer membrane of Neisseria meningitidis involving the known human T-helper epitopes and all the potential mouse T-helper epitopes calculated for the protein. The mice were immunized with the free peptides without their conjugation with a protein carrier. Most of the peptides were found to induce in mice the production of antipeptide antibodies. The mice protection against the experimental infection by a virulent strain of N. meningitidis of the B serotype was studied, and two peptides were shown to exert the most pronounced protective effect.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Imunidade/imunologia , Meningites Bacterianas/imunologia , Sequência de Aminoácidos , Animais , Imunização , Meningites Bacterianas/prevenção & controle , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologiaRESUMO
The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 was determined in two crystal forms by the X-ray method of molecular replacement at 2.2 and 2.9 A resolutions. The spatial structure of the protein and the stereochemistry of its antigen-binding site were analyzed.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Interleucina-2/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Conformação Proteica , EstereoisomerismoRESUMO
Fourteen peptides corresponding to sequences of all the exposed and some of the transmembrane protein regions of porin A from the outer membrane of Neisseria meningitidis strain B:15:P1.7,16 were synthesize. Mice of various lines were immunized with the free peptides not conjugated with any protein carrier. It was shown that the majority of the peptides possess immunogenic properties. Two peptides were identified binding to antibodies present in the serum of mice after meningitis. Protective properties of a number of the synthesized peptides were studied, and three peptide sequences inducing mice protection from an experimental infection with N. meningitidis were identified.
Assuntos
Meningite Meningocócica/imunologia , Neisseria meningitidis/imunologia , Fragmentos de Peptídeos/imunologia , Porinas/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Imunidade , Meningite Meningocócica/prevenção & controle , Camundongos , Camundongos Endogâmicos CBA , Neisseria meningitidis/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Porinas/síntese química , Porinas/química , Vacinas Sintéticas/administração & dosagemAssuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/farmacologia , Imunidade/efeitos dos fármacos , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico , Adjuvantes Imunológicos/uso terapêutico , Animais , Avaliação de Medicamentos , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Doenças do Sistema Imunitário/imunologia , Imunidade Celular/efeitos dos fármacos , Infecções/tratamento farmacológico , Infecções/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Resultado do TratamentoAssuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Melanoma Experimental/genética , Metástase Neoplásica/genética , Fenótipo , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Citotoxicidade Imunológica , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica/imunologia , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais Cultivadas , Evasão TumoralRESUMO
DNA fragments coding for the variable fragments of the heavy and light chains of the immunoglobulin G1 against human recombinant interleukin-2 were produced using reverse transcription of the total RNA isolated from the murine hybrid myeloma cell line LNKB-2 followed by amplification of the RNA-DNA duplexes with degenerated primers. The fragments obtained were cloned into the plasmid pGEM7-Zf(+) and their structures were determined. The fragments cloned were proved to encode the Fv-fragments by sequencing N-termini of the light and heavy chains of the antibody.
Assuntos
Anticorpos Monoclonais/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Interleucina-2/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Camundongos , Dados de Sequência Molecular , Plasmídeos , RNA Neoplásico/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
By means of radioligand analysis, murine peritoneal macrophages were shown to express several hundreds cell surface high-affinity GMDP-binding sites with a binding constant 350 pM. Photoaffinity labeling followed by SDS-PAGE enabled us to identify inside these cells 32-34 and 38 kDa proteins, specifically binding GMDP. Proteins 32-34 kDa were also detected by Western blotting analysis using biotinylated conjugate of polyacrylamide with immobilized GMDP-Lys [(GMDP-Lys)-PAA-(Bi)] in cell lysate of murine peritoneal macrophages.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/metabolismo , Macrófagos Peritoneais/metabolismo , Peptídeos/metabolismo , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Marcadores de Afinidade , Animais , Sítios de Ligação , Western Blotting , Sequência de Carboidratos , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ensaio RadioliganteRESUMO
Hybridoma E6/1.2 was produced by fusion of splenocytes from mice immunized with N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP), conjugated to methylated BSA, with SP2/0 myeloma cells. GMDP-specific monoclonal antibody was IgG1 subtype and had affinity constant 2.10(9) M-1. According to competitive ELISA, the presence of the intact disaccharide fragment and the alanyl residue was critical for the GMDP-antibody interaction.