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1.
PLoS Negl Trop Dis ; 14(3): e0007803, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203503

RESUMO

Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.


Assuntos
Anticorpos Monoclonais/farmacologia , Imunoglobulina A/farmacologia , Imunoglobulina G/farmacologia , Salmonella/efeitos dos fármacos , Administração Oral , África , Animais , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Hibridomas , Imunização Passiva , Imunoglobulina A Secretora , Imunoglobulina G/genética , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados , Salmonella typhimurium/efeitos dos fármacos
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 1035-1041, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31879001

RESUMO

Objective To prepare the monoclonal antibodies specifically against P protein of human respiratory syncytial virus (HRSV) and identify it. Methods HRSV P protein prepared by prokaryotic expression in the form of overlapping peptides was used as the immunogen, and monoclonal antibodies (mAbs) were screened by hybridoma technology. Western blot analysis was used to verify the binding activity of the screened mAbs and P protein, and immunofluorescence cytochemical staining was used to determine whether the obtained mAbs could be used to detect the expression of P protein in HEp-2 cells infected with HRSV. Results P181-15A3 and P211-16D8 with great reactivity and specific recognition of HRSV P protein were screened. Both mAbs could bind to P protein by Western blot analysis and could be used for immunocytochemical detection of P protein in HEp-2 cells after HRSV infection. Conclusion We have successfully prepared the mAbs against HRSV P protein.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus Sincicial Respiratório Humano , Proteínas Estruturais Virais/imunologia , Animais , Western Blotting , Humanos , Hibridomas , Imuno-Histoquímica , Camundongos
3.
BMC Biotechnol ; 19(1): 102, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870349

RESUMO

BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.


Assuntos
Anticorpos Monoclonais/análise , Interferon gama/análise , Interferon gama/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Ectima Contagioso/sangue , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Hibridomas/metabolismo , Interferon gama/sangue , Interferon gama/genética , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Vírus do Orf/fisiologia
4.
Monoclon Antib Immunodiagn Immunother ; 38(5): 220-223, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31603742

RESUMO

Several members of enteroviruses (EVs) that belong to the EVs A and B species cause hand, foot, and mouth disease (HFMD) in infants and young children. The virus-specific protease 2Apro is conserved in all the EV species, thus developing a monoclonal antibody (mAb) against 2Apro may facilitate the identification from the HFMD-associated pathogens. In this study, we achieved a murine mAb, named 5A3, specifically toward EVA71 2Apro by using the traditional hybridoma technique. The mAb 5A3 recognizes 2Apro of both EVs A and B species, which was demonstrated by indirect fluorescent assay and Western blotting. Furthermore, a conserved N-terminal epitope on 2Apro recognized by mAb 5A3 was defined by using an overlapping peptide-based enzyme-linked immunosorbent assay (ELISA). Therefore, the unique mAb targeting conserved EVs 2Apro can be used as an important tool during both identifying the causative agent of HFMD and elucidating the pathological mechanism of HFMD.


Assuntos
Anticorpos Monoclonais/imunologia , Enterovirus Humano A/imunologia , Epitopos/imunologia , Peptídeo Hidrolases/imunologia , Animais , Antígenos Virais/imunologia , Western Blotting , Reações Cruzadas , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Hibridomas , Camundongos Endogâmicos BALB C , Células Vero
5.
Monoclon Antib Immunodiagn Immunother ; 38(5): 209-212, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31603743

RESUMO

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose-response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.


Assuntos
Enterotoxinas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Western Blotting , Enterotoxinas/imunologia , Enterotoxinas/metabolismo , Ensaio de Imunoadsorção Enzimática/instrumentação , Feminino , Hibridomas , Camundongos Endogâmicos BALB C
6.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1723-1735, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559754

RESUMO

To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12-200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%-5.6% and 5.6%-7.2%, respectively, and the fortified recovery was 89%-103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.


Assuntos
Ensaio de Imunoadsorção Enzimática , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Humanos , Hibridomas , Interleucinas , Camundongos , Camundongos Endogâmicos BALB C
7.
Monoclon Antib Immunodiagn Immunother ; 38(5): 185-189, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31486711

RESUMO

The separation of plasma from blood cells is critical for the accuracy of blood tests because cellular fractions can create discrepancies in analysis. The most common method to separate blood cells from the liquid part of the blood is centrifugation, which is not always applicable in resource-constrained areas and countries. In this study, we describe the generation of monoclonal antibodies (mAbs) against glycophorin A (GPA) of human erythrocytes. BALB/c mice were immunized with human erythrocytes followed by purified GPA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. Hybridoma clones were screened by hemagglutination assay and enzyme-linked immunosorbent assay (ELISA). Six hybridoma clones were obtained and subcloned. The characterization of the purified mAbs demonstrates that they are able to bind and retain erythrocytes in hemagglutination assay. Furthermore, one of the mAbs 1A9 recognizes purified GPA in ELISA, whereas the other mAb 1G7 is able to immunoprecipitate GPA from human erythrocyte lysates, and a band of 38 kDa is detected. In conclusion, the anti-GPA mAbs are useful tools in developing a quick and easy way to separate blood plasma from whole blood for clinical tests, and in developing bi-specific antibodies for other clinical applications.


Assuntos
Anticorpos Monoclonais/imunologia , Separação Celular/métodos , Eritrócitos/imunologia , /imunologia , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Eritrócitos/citologia , Testes de Hemaglutinação , Humanos , Hibridomas/imunologia , Camundongos
8.
Monoclon Antib Immunodiagn Immunother ; 38(4): 179-182, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31403910

RESUMO

Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol (DG) to phosphatidic acid (PA). As both DG and PA serve as lipidic second messengers, DGK plays a pivotal role in controlling the balance of two signaling pathways mediated by DG and PA in cellular functions. DGKζ, one member of the mammalian DGK family, is reported to contain a nuclear localization signal, which suggests its functional role in the nucleus. Previously, morphological studies using tagged expression vectors and immunostaining of rat tissues or cells have revealed that DGKζ localizes mainly to the nucleus. However, a limited number of studies reported the detailed localization of native protein of DGKζ in human tissues and cells. In this study, we developed a novel anti-human DGKζ monoclonal antibody, DzMab-1, which is very advantageous in immunocytochemistry of human cultured cells.


Assuntos
Anticorpos Monoclonais/imunologia , Diacilglicerol Quinase/imunologia , Hibridomas/imunologia , Imuno-Histoquímica/métodos , Animais , Células HeLa , Humanos , Imunização , Ratos , Ratos Endogâmicos WKY
9.
Monoclon Antib Immunodiagn Immunother ; 38(4): 162-170, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31260385

RESUMO

Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.


Assuntos
Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Hibridomas/imunologia , Cinesina/antagonistas & inibidores , Cinesina/imunologia , Neoplasias Laríngeas/metabolismo , Proteínas Recombinantes/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos , Humanos , Neoplasias Laríngeas/diagnóstico , Neoplasias Laríngeas/imunologia , Células Tumorais Cultivadas
10.
Monoclon Antib Immunodiagn Immunother ; 38(4): 145-156, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31305212

RESUMO

Tumor necrosis factor-α (TNFα), one of the major proinflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders, such as rheumatoid arthritis, psoriasis, Crohn's disease, etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody, which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNFα. Four mAbs rescued L929 fibroblast cells from TNFα-triggered cell death and one of these, namely C8, was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNFα-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (glutamine, arginine, glutamic acid) form the core epitope. The observation was supported by bioinformatics analyses of an antigen/antibody complex model. In addition, the binding affinity of mAb C8 to TNFα was found to be comparable with that of infliximab, which is a commercially available anti-TNFα mAb.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Fibroblastos/imunologia , Hibridomas/imunologia , Imunoglobulina G/imunologia , Proteínas Recombinantes/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/farmacologia , Formação de Anticorpos , Células Cultivadas , Feminino , Fibroblastos/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C
11.
Molecules ; 24(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269647

RESUMO

The structure of hapten determines the performance of the antibody and the corresponding detection method. A new type of antigen was designed and synthesized to expose the spatial and characteristic structure of dinotefuran molecule, and a type of high-quality antibody was obtained. The IC50 value of the monoclonal antibody was 5.30 ng/mL and its cross-reactivity (CRs) was less than 2% when reacting with other structurally related analytes. The effects of spatial configurations of hapten on the antibody were visually analyzed while using the appropriate software according to the quality of the antibodies, which showed that the specificity of the antibody is closely related with the exposed structure of hapten. An ELISA assay with an IC50 of 5.66 ng/mL and a linear range of 1.95 to 16.29 ng/mL was developed. The results that were obtained from the ELISA and HPLC methods were equivalent. The results showed that spatial simulation is a crucial method that is used in the designing of hapten to obtain a sensitive and specific antibody. The application of this method will highlight the potential aim and improve the detection efficiency of ELISA.


Assuntos
Antígenos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Guanidinas/análise , Neonicotinoides/análise , Nitrocompostos/análise , Animais , Anticorpos/imunologia , Reações Cruzadas , Feminino , Guanidinas/química , Haptenos/química , Hibridomas , Camundongos Endogâmicos BALB C , Neonicotinoides/química , Nitrocompostos/química
12.
Immunohorizons ; 3(7): 341-351, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31356163

RESUMO

γδNKT cells are an abundant γδT cell population with restricted Vγ1.1 Vδ6.3 gene usage and phenotypic and functional similarity to conventional αß-invariant NKT cells. The γδNKT population responds to Listeria infections, but specific ligands are not known. In this work, we studied the CDR3 requirements of the γδNKT TCR, Vγ1.1Vδ6.3 for recognizing naive macrophages, and macrophages infected with Listeria We expressed four different variants of the Vγ1.1Vδ6.3 TCR in TCR-deficient hybridomas, one with germline-encoded sequences and three with nongermline-encoded sequences. All of the hybridomas were activated when cultured in the presence of macrophages, and the activation was increased when the macrophages were infected with Listeria This indicates that these TCRs can recognize a self-ligand present in macrophages and suggests that the ligand is modified or upregulated when the cells are infected with Listeria One of the three nongermline-encoded Vγ1.1 variants induced a lower activation level compared with the other variants tested in this study, suggesting that recognition of the Listeria-induced ligand involves the CDR3γ region of the TCR.


Assuntos
Regiões Determinantes de Complementaridade/genética , Células Germinativas/química , Listeria/imunologia , Listeriose/microbiologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Hibridomas/imunologia , Hibridomas/microbiologia , Interleucina-2/metabolismo , Linfócitos Intraepiteliais/imunologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/classificação , Transfecção
13.
Biotechnol Lett ; 41(8-9): 963-977, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325004

RESUMO

OBJECTIVES: The relationships of manipulation of culture temperature and medium circulation rate on the metabolic parameters were regressed by multiple linear regression analysis in hollow fiber bioreactors (HFB). RESULTS: The high circulation rate could significantly enhance the oxygen consumption of the hybridoma cells and the medium's oxidation-reduction potential. A mildly hypothermic condition of 36 °C and a circulation rate of 182.5 mL/min could support the hybridoma had the maximal antibody titer of 60.75 µg/mL for 20 days. When the ammonium ion was 65 ppm or lactate close to 2.6 g/L, the medium was replaced to maintain the stable and healthy cells at the high cell concentration of 3.33 × 108/mL for continuous antibody production. Two serum-free media could be successfully applied to this perfusion system and maintain hybridoma growth and antibody production. CONCLUSION: The single-use HFBs could provide the advantages including high cell density, low shear stress, and continuous antibody production.


Assuntos
Anticorpos/metabolismo , Reatores Biológicos , Contagem de Células , Hibridomas/metabolismo , Anticorpos/genética , Meios de Cultura/química , Análise Multivariada , Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
14.
J Pharm Biomed Anal ; 174: 263-269, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31181489

RESUMO

To produce specific antibodies for the detection and quantification of copper ions, bifunctional chelators (BFCs) are commonly applied in the preparation of copper conjugates. However, some copper-chelator complexes exhibit limited stability under in vivo conditions. In this study, Cu2+ was coupled with carrier proteins via three different macrocyclic BFCs: p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, and p-SCN-Bn-TETA. The stability in plasma and the immunogenicity of three copper immunoconjugates were compared. The chelators other than p-SCN-Bn-DOTA were very stable in plasma, with <9% dissociation of Cu2+ over 96 h. The immune response varied depending on the choice of chelator; notably, antisera from the Cu2+-NOTA-KLH conjugate demonstrated the best reactivity toward chelated Cu2+. p-SCN-Bn-NOTA, which showed significant advantages over the other chelators, was used for antibody production. The efficiency of immune-positive hybridoma production was satisfactory, and the resultant monoclonal antibodies (McAbs) 4B7 showed sensitivity (half-maximal inhibitory concentration (IC50) of 8.9 ng/mL) to chelated Cu2+, with a working range from 1.21 to 48.9 ng/mL. The recovery of Cu2+ from water samples was 85.7-108%, and the intra- and inter-assay coefficients of variation were 4.0-10.1% and 7.1-11.4%, respectively. Compared with previously reported McAb specific to Cu2+, DF4, the sensitivity of the newly developed assay was improved 100-fold. The results of this study indicate the utility of NOTA for the efficient generation of highly sensitive McAbs against Cu2+.


Assuntos
Anticorpos Monoclonais/química , Quelantes/farmacologia , Cobre/química , Imunoconjugados/química , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Haptenos/química , Hemocianinas/química , Hibridomas , Imunoensaio , Concentração Inibidora 50 , Íons , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Compostos Radiofarmacêuticos/química , Reprodutibilidade dos Testes , Soroalbumina Bovina , Água/química
15.
J Biosci Bioeng ; 128(5): 578-584, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31147218

RESUMO

Until now, various kinds of monoclonal antibodies have been raised against many antigens. Nevertheless, the production of these monoclonal antibodies was usually limited to only one antigen. If simultaneous generation of monoclonal antibodies against multiple antigens were available at one time, we could reduce not only laborious work, but also experimental animals. Here, we developed a multitargeting (MT) method that enables simultaneous production of monoclonal antibodies against multiple antigens on the basis of strict selection of sensitized B lymphocytes by the target antigens via B-cell receptors. After immunization using multiple antigens, monoclonal antibodies against four different antigens containing lower antigenic one were successfully generated only in one experiment. At maximum, more than 90 % of ELISA-positive wells to hybridoma-positive ones was obtained by this advanced technology. This must be attributed to strict selection of sensitized B lymphocytes by different antigens. In the MT method, sensitized B lymphocytes were selected by means of each desired antigen regardless of their antigenic differences. Selective fusion of B cell-myeloma cell complexes by electrical pulses was also of critical importance for efficient generation of hybridoma cells secreting desired monoclonal antibodies. This study strongly suggests that simultaneous production of novel monoclonal antibodies directed against multiple antigens of interest by the MT method can be feasible.


Assuntos
Anticorpos Monoclonais/biossíntese , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática , Hibridomas/imunologia
16.
Methods Mol Biol ; 1988: 59-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147932

RESUMO

The major histocompatibility complex (MHC) class I restricted pathway of antigen processing allows the presentation of intracellular antigens to cytotoxic T lymphocytes. The proteasome is the main protease in the cytoplasm and the nucleus, which is responsible for the generation of most peptide ligands of MHC-I molecules. Peptides produced by the proteasome can be further trimmed or destroyed by numerous cytosolic or endoplasmic reticulum (ER) luminal proteases. Small molecule inhibitors are useful tools for probing the role of proteases in MHC class I antigen processing. Here, we describe different methods to test the impact of protease inhibitors in antigen presentation assays.


Assuntos
Apresentação do Antígeno/efeitos dos fármacos , Imunoensaio/métodos , Inibidores de Proteases/farmacologia , Animais , Linhagem Celular , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Hibridomas/metabolismo , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Coloração e Rotulagem , Linfócitos T/metabolismo , beta-Galactosidase/metabolismo
17.
Vet Microbiol ; 234: 83-91, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213277

RESUMO

Since 2011, there have been outbreaks of pseudorabies (PR) in several pig farms despite vaccination coverage, which causes substantial economic loss to the swine industry in China. The emergence of a pseudorabies virusvariant strain with high virulence and antigenic variation (e.g., PRV ZJ01), is considered to be the primary cause. In this study, truncated gB, gC, and gE of PRV ZJ01 was expressed and used to generate seven monoclonal antibodies (mAbs) against gB, gC, or gE. An indirect immunofluorescence assay (IFA) revealed that these mAbs were specific against PRV. Subsequently we identified the B cell epitopes recognized by these mAbs by Western blot. The mAbs 5A2 and 6G5 against gB recognized the same B cell linear epitope at 576SAVATAA582, the mAb 5D10 against gC recognized the B cell linear epitope at 134GETFE138, mAb 7C5 against gC recognized the B cell linear epitope at 143RRGRFRSPDAD153, and mAbs 3E1, 3H8, and 4D2 against gE recognized the same B cell linear epitope at 151IGDYL155 of gE. Biological information analysis showed that these B cell linear epitopes are highly conserved among different PRV isolates and the epitope 143RRGRFRSPDAD153 with a high antigenic index and high hydrophilicity, fully exposed on the surface of the gC, is likely to be an important B cell epitope. These mAbs and their defined epitopes may provide useful tools for the study of the structure and function of the PRV protein, analysis of antigenic epitope characteristics, and establishment of antibody detection methods.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos de Linfócito B/imunologia , Pseudorraiva/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linhagem Celular , Feminino , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Suínos
18.
Redox Biol ; 26: 101252, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31254735

RESUMO

Methylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases.


Assuntos
Anticorpos Monoclonais/química , Meios de Cultura/química , Ensaio de Imunoadsorção Enzimática/métodos , Aldeído Pirúvico/sangue , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Butionina Sulfoximina/farmacologia , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática/normas , Células HEK293 , Humanos , Hibridomas/química , Hibridomas/imunologia , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Cultura Primária de Células , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem
19.
PLoS One ; 14(6): e0218717, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31233538

RESUMO

The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5' end of the antibody cDNA. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.


Assuntos
Anticorpos Monoclonais/genética , Região Variável de Imunoglobulina/genética , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Reações Antígeno-Anticorpo , Primers do DNA/genética , DNA Complementar/genética , Células HEK293 , Humanos , Hibridomas/imunologia , Imunoglobulina G/genética , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fluxo de Trabalho
20.
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
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