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1.
Nat Protoc ; 14(12): 3275-3302, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31723301

RESUMO

Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.


Assuntos
/métodos , Análise de Sequência de DNA/métodos , Animais , Especificidade de Anticorpos/imunologia , Caenorhabditis elegans/genética , Calibragem , Imunoprecipitação da Cromatina/métodos , Biologia Computacional , DNA , Drosophila melanogaster/genética , Biblioteca Gênica , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/genética , Histonas/imunologia , Humanos , Nucleossomos/genética , Nucleossomos/imunologia , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes
2.
Int J Nanomedicine ; 14: 7533-7548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571862

RESUMO

Background: The influenza A virus (IAV) is known for its high variability and poses a huge threat to the health of humans and animals. Pigs play a central role in the cross-species reassortment of IAV. Ectodomain of matrix protein 2 (M2e) is the most conserved protective antigen in IAV and can be used to develop nanovaccines through nanoparticles displaying to increase its immunogenicity. However, the high immunogenicity of nanoparticles can cause the risk of off-target immune response, and excess unwanted antibodies may interfere with the protective efficacy of M2e-specific antibodies. Therefore, it is necessary to select reasonable nanoparticles to make full use of antibodies against nanoparticles while increasing the level of M2e-specific antibodies. Porcine circovirus type 2 (PCV2) is the most susceptible virus in pigs and can promote IAV infection. It is meaningful to develop a vaccine that can simultaneously control swine influenza virus (SIV) and PCV2. Methods: In the present study, M2e of different copy numbers were inserted into the capsid (Cap) protein of PCV2 and expressed in Escherichia coli to form self-assembled chimeric virus-like particles (VLPs) nanovaccine. BALB/c mice and pigs were immunized with these nanovaccines to explore optimal anti-IAV and anti-PCV2 immunity. Results: Cap is capable of carrying at least 81 amino acid residues (three copies of M2e) at its C-terminal without impairing VLPs formation. Cap-3M2e VLPs induced the highest levels of M2e-specific immune responses, conferring protection against lethal challenge of IAVs from different species and induced specific immune responses consistent with PCV2 commercial vaccines in mice. In addition, Cap-3M2e VLPs induced high levels of M2e-specific antibodies and PCV2-specific neutralizing antibodies in pigs. Conclusion: Cap-3M2e VLP is an economical and promising bivalent nanovaccine, which provides dual protection against IAV and PCV2.


Assuntos
Circovirus/imunologia , Vírus da Influenza A/imunologia , Nanopartículas/uso terapêutico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Aves/virologia , Proteínas do Capsídeo/química , Proliferação de Células , Citocinas/metabolismo , Cães , Feminino , Humanos , Imunidade Humoral , Influenza Aviária/imunologia , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Linfócitos/citologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes/isolamento & purificação , Suínos , Vírion/imunologia , Vírion/ultraestrutura
3.
Mol Immunol ; 114: 389-394, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31454596

RESUMO

HLA-A2 is the most common serological HLA type among all ethnic groups. Through advances in DNA typing, more than 800 subtypes of HLA-A2 have been identified, and the existence of heterogeneity of antigen specificity among the HLA-A2 subtypes has been suggested by retrospective analyses of allogeneic transplantation patients and by studies of antigen amino acid structure. However, prior to this study, the antigenicity of a given subtype or the mismatch extent between two given subtypes could not be studied in vitro. Here, we used a modified autologous lymphocyte-monocyte coculture method to reveal heterogeneity of antigen specificity among HLA-A2 subtypes. The coculture was set up with HLA-A2 (non-A*02:01) lymphocytes and monocytes, and the monocytes were coated with an HLA-A*02:01/IgG1-Fc fusion protein (dimer) by high-affinity binding of the IgG1-Fc to FcgRI. Lymphocyte proliferation following coculture indicated that HLA-A*02:01 showed antigenicity against the HLA-A2 (non-A*02:01) subtype. Among the most frequent HLA-A2 subtypes in the Chinese population (HLA-A*02:01, -A*02:03, -A*02:06 and -A*02:07), we identified significant -A*02:01 antigenicity for T cells from -A*02:03 or -A*02:06 but not -A*02:07 individuals. Our findings were consistent with retrospective studies of allograft patients with a limited number of involved subtypes, indicating that this modified coculture method provides a practical and reliable means to study the antigenicity of HLA allele subtypes in vitro.


Assuntos
Especificidade de Anticorpos/imunologia , Antígeno HLA-A2/imunologia , Monócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Alelos , Técnicas de Cocultura/métodos , Humanos , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Estudos Retrospectivos
4.
BMC Biotechnol ; 19(1): 47, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315680

RESUMO

BACKGROUND: Interleukin-17 (IL-17), the characteristic cytokine secreted by T helper 17 lymphocytes (Th17 cells), plays a pivotal role in host defense and many inflammatory or autoimmune diseases. The aim of this study was to obtain purified protein caprine IL-17A (cIL-17A) as an antigen for preparing an IL-17A-specific monoclonal antibody (mAb). RESULTS: The coding sequence (CDS) region of cIL-17A was cloned from the peripheral blood mononuclear cells (PBMCs) of dairy goats and then inserted into the expression vector PET 32a and transformed into competent TransB (DE3) cells. Recombinant fusion protein obtained under optimized conditions was used to immunize BALB/c mice for preparing monoclonal antibodies. Finally, the supernatants of two hybridoma cell lines showing positive reaction with the recombinant fusion protein and negative reaction with fusion tags of PET 32a were collected for western blot, immunofluorescence (IF) and immunohistochemistry (IHC) analysis. Our results showed that the maximum amount of soluble protein could be obtained directly in the supernatant when the recombinant expression cells were induced by isopropyl-ß-d-thiogalactoside (IPTG) at a concentration of 0.3 mmol/L at 16 °C for 42 h. Western blot analysis showed that the mAb H8 could recognize the eukaryotically expressed cIL-17A in the supernatant of transfected HEK293T cells. Immunofluorescence and immunohistochemistry assays showed that mAb H8 could strongly recognize both the eukaryotically expressed and natural cIL-17A. CONCLUSIONS: The monoclonal antibody mAb H8 prepared in this study may be a potential tool for the detection of cIL-17A and beneficial for investigating the pathogenesis of various IL-17-associated diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Interleucina-17/imunologia , Leucócitos Mononucleares/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Indústria de Laticínios , Imunofluorescência , Cabras , Humanos , Imunização , Imuno-Histoquímica , Interleucina-17/genética , Interleucina-17/metabolismo , Leucócitos Mononucleares/metabolismo , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo
5.
J Immunol Res ; 2019: 3409371, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31240233

RESUMO

Immunogenicity of DNA vaccines can be efficiently improved by adding adjuvants into their formulations. In this regard, the application of nano- and microparticles as vaccines adjuvants, or delivery systems, provides a powerful tool in designing modern vaccines. In the present study, we examined the role of "Supramolecular Hacky Sacks" (SHS) particles, made via the hierarchical self-assembly of a guanosine derivative, as a novel immunomodulator for DNA plasmid preparations. These plasmids code for the proteins HIV-1 Gag (pGag), the wild-type vaccinia virus Western Reserve A27 (pA27L), or a codon-optimized version of the latter (pOD1A27Lopt), which is also linked to the sequence of the outer domain-1 (OD1) from HIV-1 gp120 protein. We evaluated the enhancement of the immune responses generated by our DNA plasmid formulations in a murine model through ELISpot and ELISA assays. The SHS particles increased the frequencies of IFN-γ-producing cells in mice independently immunized with pGag and pA27L plasmids. Moreover, the addition of SHS to pGag and pA27L DNA plasmid formulations enhanced the production of IFN-γ (Th1-type) over IL-4 (Th2-type) cellular immune responses. Furthermore, pGag and pA27L plasmids formulated with SHS, triggered the production of antigen-specific IgG in mice, especially the IgG2a isotype. However, no improvement of either of those adaptive immune responses was observed in mice receiving pOD1A27Lopt+SHS. Here, we demonstrated that SHS particles have the ability to improve both arms of adaptive immunity of plasmid coding "wild-type" antigens without additional strategies to boost their immunogenicity. To the best of our knowledge, this is the first report of SHS guanosine-based particles as DNA plasmid adjuvants.


Assuntos
Guanosina , Nanopartículas , Plasmídeos , Vacinas de DNA/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Especificidade de Anticorpos/imunologia , Citocinas/biossíntese , ELISPOT , Feminino , Guanosina/química , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Humanos , Imunidade Humoral , Imunização , Imunogenicidade da Vacina , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Camundongos , Nanopartículas/química , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/imunologia , Vacinas de DNA/química , Vacinas de DNA/genética
6.
Bull Exp Biol Med ; 167(1): 120-122, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31183643

RESUMO

We optimized the method of isolation of antibodies from placental tissue of a conventionally healthy patient. Four protocols of antibody isolation were evaluated and a protocol with tissue grinding (without homogenization) and successive elution of the antibodies with acidic and alkaline buffers was recommended for use. The repertoire of the isolated antibodies was characterized using a glycan array. Partial coincidence of the specificity of the isolated antibodies with antibodies in the peripheral blood was demonstrated, which indicates their possible association with carbohydrate antigens in the placenta. Identification of potential molecular targets of resident antibodies in the placenta is necessary for understanding the mechanisms of formation of immunological tolerance to the fetus.


Assuntos
Imunoglobulina G/isolamento & purificação , Placenta/imunologia , Adulto , Especificidade de Anticorpos/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Placenta/metabolismo , Polissacarídeos/imunologia , Gravidez , Trofoblastos/metabolismo
7.
Clin Lab ; 65(6)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31232019

RESUMO

BACKGROUND: While all modalities used for diagnosis of Helicobacter pylori (H. pylori) have demonstrated sufficient sensitivity and specificity, each test has advantages and limitations. The serum test for anti-H. pylori antibody with the latex method is noninvasive, easy, and inexpensive; it is thus a useful tool for mass-screening for H. pylori. In this study, we evaluated the utility of a newly developed latex kit, in comparison with other serum diagnostic kits based on enzyme-linked immunosorbent assay (ELISA). METHODS: In total, 187 subjects (77: H. pylori-positive, 75: H. pylori-negative, 35: previous infection with H. pylori) seen at Oita University Hospital during the period from January 1988 to September 2014 were enrolled in the study. All subjects were evaluated with 4 types of serum H. pylori antibody kits. One modality was based on the use of latex (Denka Kit, Denka Seiken Co., Ltd., Tokyo, Japan). Three kits were based on the use of ELISA. The E-Plate II Eiken (Eiken Chemical Co., Ltd., Tokyo, Japan) is henceforth referred to as Kit A. The Premier H. pylori kit (Meridian Bioscience, Inc., USA) is referred to as Kit B. The Platelia H. pylori IgG (Bio-Rad, Marnes-la-Coquette, France) is referred to as Kit C. RESULTS: Evaluation of 152 study participants, including some who were positive for H. pylori and some who were negative, sensitivity, specificity, and accuracy values were as follows: for the Denka kit, these values were, respec-tively, 92.2%, 93.3%, and 92.8%. For Kit A, these values were 88.3%, 100.0%, and 194.1%. For Kit B, these values were 98.7%, 76.0%, and 87.5%. For Kit C, these values were 98.7%, 80.0%, and 89.5%. The specificity of Kit A was > 90%. Sensitivity was > 90% for Kits B and C. For the Denka kit, both sensitivity and specificity were > 90%. Among the 35 subjects previously infected with H. pylori, the rate of positive diagnosis was 48.6% (17/35) with the Denka kit, 17.1% (6/35) with Kit A, 54.3% (19/35) with Kit B, and 54.3% (19/35) with Kit C. The rate of positive diagnosis was significantly higher with the Denka kit than with Kit A (p < 0.05). CONCLUSIONS: An assay based on use of the latex method, H. pylori-latex Seiken, demonstrated satisfactory sensitivity and specificity for detecting serum levels of H. pylori antibody. The performance of this kit was equivalent to that of ELISA kits currently used for the same purpose. This kit is therefore considered to be extremely suitable for diagnosis of H. pylori and mass-screening of patients at high risk for gastric cancer.


Assuntos
Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos/imunologia , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/imunologia , Imunoturbidimetria/métodos , Kit de Reagentes para Diagnóstico/normas , Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , França , Infecções por Helicobacter/sangue , Infecções por Helicobacter/virologia , Helicobacter pylori/fisiologia , Humanos , Japão , Látex , Kit de Reagentes para Diagnóstico/classificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados Unidos
8.
J Immunol Res ; 2019: 2754920, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31223627

RESUMO

Transferon® is an immunomodulator made of a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs). Development of surrogate antibodies directed to hDLE is an indispensable tool for studies during process control and preclinical trials. These antibodies are fundamental for different analytical approaches, such as identity test and drug quantitation, as well as to characterize its pharmacokinetic and mechanisms of action. A previous murine study showed the inability of the peptides of Transferon® to induce antibody production by themselves; therefore, in this work, two approaches were tested to increase its immunogenicity: chemical conjugation of the peptides of Transferon® to carrier proteins and the use of a rabbit model. Bioconjugates were generated with Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA) through maleimide-activated carrier proteins. BALB/c mice and New Zealand rabbits were immunized with Transferon® conjugated to KLH or nonconjugated Transferon®. Animals that were immunized with conjugated Transferon® showed significant production of antibodies as evinced by the recognition of Transferon®-BSA conjugate in ELISA assays. Moreover, rabbits showed higher antibody titers when compared with mice. Neither mouse nor rabbits developed antibodies when immunized with nonconjugated Transferon®. Interestingly, rabbit antibodies were able to partially block IL-2 production in Jurkat cells after costimulation with Transferon®. In conclusion, it is feasible to elicit specific and functional antibodies anti-hDLE with different potential uses during the life cycle of the product.


Assuntos
Isoanticorpos/imunologia , Fator de Transferência/efeitos adversos , Adjuvantes Imunológicos , Animais , Formação de Anticorpos , Especificidade de Anticorpos/imunologia , Antígenos/administração & dosagem , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Isoanticorpos/isolamento & purificação , Masculino , Camundongos , Peptídeos/administração & dosagem , Peptídeos/imunologia , Coelhos , Fator de Transferência/imunologia , Fator de Transferência/uso terapêutico
9.
Mol Immunol ; 112: 338-346, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31254774

RESUMO

Therapeutic antibodies have transformed the clinical practice. Not surprisingly, development of antibody therapeutics is currently the main focus of the biotechnology industry. Nonetheless, the development process is complex, and many antibodies do not reach the clinic. Reasons for the failures include, undesired binding behavior (polyreactivity), low stability, poor expression yields, unfavorable pharmacokinetics etc. Numerous studies have proposed different analytical methods for assessment of physicochemical parameters of antibodies and identification of problematic molecules at early stages of the development process. These studies, however, have not addressed the basic mechanistic question of how sequence features of variable regions determinate the different biophysical characteristics and the binding behavior of the antibodies. In a recent study, Jain et al assessed 12 biophysical qualities of 137 monoclonal therapeutic antibodies. We used the raw data from this comprehensive study to perform correlation analyses of different biophysical measurables with various sequence features of variable regions of the antibodies - number of mutations, length of hypervariable loops, and frequency of amino acid residue types. The obtained data reveled significant relationships between the sequence characteristics of the variable domains and different physiochemical properties of antibodies. The data from this study can assist in design of a set of criteria for early identification of antibodies with developability issues. Moreover, our findings provide novel fundamental insights into the sequence-function relationship of antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Região Variável de Imunoglobulina/imunologia , Aminoácidos/imunologia , Humanos
10.
BMC Res Notes ; 12(1): 331, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186065

RESUMO

OBJECTIVE: The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFß) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD). RESULTS: Using a modified, cell-free immunoprecipitation assay, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Consistent with this, 3F6 blocks endogenous BMPR2 function in the BMP-responsive cell line HEK293T. The specificity of 3F6 action was confirmed by demonstrating that this antibody has no effect on BMP-responsiveness in HEK293T cells in which BMPR2 expression is knocked-down. Our results provide important proof-of-concept data for future studies interrogating BMPR2 function.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos/imunologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células HEK293 , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
11.
Monoclon Antib Immunodiagn Immunother ; 38(3): 104-107, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31161964

RESUMO

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.


Assuntos
Células Epiteliais Alveolares/metabolismo , Anticorpos Monoclonais/imunologia , Células Endoteliais/metabolismo , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Phocoena/metabolismo , Podócitos/metabolismo , Células Epiteliais Alveolares/imunologia , Animais , Especificidade de Anticorpos/imunologia , Células Endoteliais/imunologia , Humanos , Imuno-Histoquímica , Phocoena/imunologia , Podócitos/imunologia
12.
Molecules ; 24(10)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117255

RESUMO

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.


Assuntos
Anticorpos/imunologia , Benzofuranos/isolamento & purificação , Depsídeos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Salvia miltiorrhiza/química , Anticorpos/química , Especificidade de Anticorpos/imunologia , Benzofuranos/química , Benzofuranos/imunologia , Benzofuranos/uso terapêutico , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Depsídeos/química , Depsídeos/imunologia , Depsídeos/uso terapêutico , Cardiopatias/tratamento farmacológico , Humanos , Medicina Tradicional Chinesa , Proteínas de Plantas/química , Proteínas de Plantas/imunologia
13.
Mol Biol Rep ; 46(4): 4027-4037, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31073914

RESUMO

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Clonagem Molecular/métodos , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos/genética , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Proteínas Reguladoras de Apoptose/genética , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica/genética , Engenharia Genética/métodos , Humanos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Anticorpos de Cadeia Única/imunologia
14.
Vet Immunol Immunopathol ; 211: 10-18, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084888

RESUMO

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94+ cells were CD3+/-, CD8+/-, CD4-, CD21-, CD5low, NKp46+, and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94+ lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.


Assuntos
Anticorpos Monoclonais/imunologia , Cães/imunologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Especificidade de Anticorpos/imunologia , Clonagem Molecular , Feminino , Citometria de Fluxo/veterinária , Células Matadoras Naturais/imunologia , Masculino , Camundongos/imunologia , Células T Matadoras Naturais/imunologia , Reação em Cadeia da Polimerase/veterinária
15.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064767

RESUMO

There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene-transfected T-cell line lacking TCR as well as of Vγ1- Vγ4- Vγ5- Vγ7- γδ T cells and the CD3high TCRδint γδ T cells in various organs. The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.


Assuntos
Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Receptores de Antígenos de Linfócitos T gama-delta/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Feminino , Imunização , Imunofenotipagem , Camundongos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
16.
Monoclon Antib Immunodiagn Immunother ; 38(3): 129-132, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31112076

RESUMO

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Tigres/metabolismo , Animais , Tigres/imunologia
17.
Monoclon Antib Immunodiagn Immunother ; 38(2): 79-84, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30939066

RESUMO

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Cavalos , Camundongos
18.
Monoclon Antib Immunodiagn Immunother ; 38(2): 89-95, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31009336

RESUMO

Podoplanin (PDPN), also known as T1alpha, has been used as a lung type I alveolar cell marker in the pathophysiological condition. Although we have established several monoclonal antibodies (mAbs) against mammalian PDPNs, mAbs against tiger PDPN (tigPDPN), which are useful for immunohistochemical analysis, remain to be developed. In this study, we immunized mice with tigPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/tigPDPN) and screened hybridomas producing mAbs against tigPDPN using flow cytometry. One of the mAbs, PMab-231 (IgG2a, kappa), specifically detected CHO/tigPDPN cells using flow cytometry as well as recognized tigPDPN protein using western blotting. In addition, PMab-231 was found to cross-react with cat PDPN (cPDPN). The dissociation constants (KD) of PMab-231 for CHO/tigPDPN and CHO/cPDPN cells were determined to be 1.2 × 10-8 and 1.9 × 10-8, respectively, indicating moderate affinity for CHO/tigPDPN and CHO/cPDPN cells. PMab-231 stained type I alveolar cells of the feline lungs and podocytes of the feline kidneys using immunohistochemistry. Our findings suggest the potential usefulness of PMab-231 for the functional analyses of tigPDPN and cPDPN.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Hibridomas/imunologia , Rim/metabolismo , Pulmão/metabolismo , Glicoproteínas de Membrana/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Células CHO , Gatos , Cricetinae , Cricetulus , Rim/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Tigres
19.
Iran J Immunol ; 16(1): 26-42, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30864553

RESUMO

BACKGROUND: We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. OBJECTIVE: To describe chimerization and functional characterization of 2A8 mAb. METHODS: The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. RESULTS: Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. CONCLUSION: The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Macaca fascicularis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transfecção , Trastuzumab/farmacologia
20.
Proc Natl Acad Sci U S A ; 116(14): 6812-6817, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894493

RESUMO

Aberrant activation of Wnt/ß-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.


Assuntos
Especificidade de Anticorpos , Antineoplásicos Imunológicos , Receptores Frizzled/antagonistas & inibidores , Neoplasias Pancreáticas , Engenharia de Proteínas , Animais , Especificidade de Anticorpos/genética , Especificidade de Anticorpos/imunologia , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/farmacocinética , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Feminino , Receptores Frizzled/genética , Receptores Frizzled/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
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