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1.
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
2.
Ann Hematol ; 98(10): 2339-2346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250082

RESUMO

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Medula Óssea , Calreticulina , Mutação , Trombocitemia Essencial , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea/metabolismo , Medula Óssea/patologia , Calreticulina/genética , Calreticulina/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/patologia
3.
Gastroenterology ; 157(3): 720-730.e2, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31175863

RESUMO

BACKGROUND & AIMS: Although pancreatic cystic lesions (PCLs) are frequently and incidentally detected, it is a challenge to determine their risk of malignancy. In immunohistochemical and enzyme-linked immunosorbent assay (ELISA) analyses of tissue and cyst fluid from pancreatic intraductal papillary mucinous neoplasms, the monoclonal antibody Das-1 identifies those at risk for malignancy with high levels of specificity and sensitivity. We aimed to validate the ability of Das-1 to identify high-risk PCLs in comparison to clinical guidelines and clinical features, using samples from a multicenter cohort. METHODS: We obtained cyst fluid samples of 169 PCLs (90 intraductal papillary mucinous neoplasms, 43 mucinous cystic neoplasms, and 36 non-mucinous cysts) from patients undergoing surgery at 4 tertiary referral centers (January 2010 through June 2017). Histology findings from surgical samples, analyzed independently and centrally re-reviewed in a blinded manner, were used as the reference standard. High-risk PCLs were those with invasive carcinomas, high-grade dysplasia, or intestinal-type intraductal papillary mucinous neoplasms with intermediate-grade dysplasia. An ELISA with Das-1 was performed in parallel using banked cyst fluid samples. We evaluated the biomarker's performance, generated area under the curve values, and conducted multivariate logistic regression using clinical and pathology features. RESULTS: The ELISA for Das-1 identified high-risk PCLs with 88% sensitivity, 99% specificity, and 95% accuracy, at a cutoff optical density value of 0.104. In 10-fold cross-validation analysis with 100 replications, Das-1 identified high-risk PCLs with 88% sensitivity and 98% specificity. The Sendai, Fukuoka, and American Gastroenterological Association guideline criteria identified high-risk PCLs with 46%, 52%, and 74% accuracy (P for comparison to Das-1 ELISA <.001). When we controlled for Das-1 in multivariate regression, main pancreatic duct dilation >5 mm (odds ratio, 14.98; 95% confidence interval, 2.63-108; P < .0012), main pancreatic duct dilation ≥1 cm (odds ratio, 47.9; 95% confidence interval, 6.39-490; P < .0001), and jaundice (odds ratio, 6.16; 95% confidence interval, 1.08-36.7; P = .0397) were significantly associated with high-risk PCLs. CONCLUSIONS: We validated the ability of an ELISA with the monoclonal antibody Das-1 to detect PCLs at risk for malignancy with high levels of sensitivity and specificity. This biomarker might be used in conjunction with clinical guidelines to identify patients at risk for malignancy.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos/análise , Biomarcadores Tumorais/análise , Ensaio de Imunoadsorção Enzimática , Neoplasias Císticas, Mucinosas e Serosas/química , Cisto Pancreático/química , Neoplasias Intraductais Pancreáticas/química , Neoplasias Pancreáticas/química , Adulto , Idoso , Anticorpos/imunologia , Especificidade de Anticorpos , Biomarcadores Tumorais/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Císticas, Mucinosas e Serosas/imunologia , Neoplasias Císticas, Mucinosas e Serosas/patologia , Neoplasias Císticas, Mucinosas e Serosas/cirurgia , Cisto Pancreático/imunologia , Cisto Pancreático/patologia , Cisto Pancreático/cirurgia , Neoplasias Intraductais Pancreáticas/imunologia , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Intraductais Pancreáticas/cirurgia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Medição de Risco , Estados Unidos
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(5): 459-464, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31223114

RESUMO

Objective To prepare a polyclonal antibody against S3 of Nelson Bay virus (NBV). Methods The E.coli BL21 (DE3) competent cells were transfected with the constructed Pris His MB-S3 recombinant plasmid. Protein expression was induced by IPTG. The target protein was purified by Ni-NTA column affinity chromatography to obtain a large amount of fusion recombinant protein, which was tested and used as the antigen for producing the S3 polyclonal antibody in rats. Results The relative molecular mass (Mr) of Pris His MB-S3 protein was around 39 000, in the form of inclusion bodies. NBV S3 polyclonal antibody was successfully produced from the immunized rats. The titer of the antibody was up to 1:64 000 determined by indirect ELISA. The indirect immunofluorescence assay verified that the S3 protein was successfully expressed in cells and distributed in a granular form in the cytoplasm. Conclusion The highly reactive and specific S3 protein polyclonal antibody is successfully prepared.


Assuntos
Anticorpos , Proteínas do Capsídeo/isolamento & purificação , Orthoreovirus , Proteínas de Ligação a RNA/isolamento & purificação , Animais , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Plasmídeos , Proteínas de Ligação a RNA/imunologia , Ratos , Proteínas Recombinantes de Fusão
5.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 871-879, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223005

RESUMO

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.


Assuntos
Proteínas de Escherichia coli , Peptídeos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1117-1125, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232008

RESUMO

To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.


Assuntos
Escherichia coli , Animais , Anticorpos , Especificidade de Anticorpos , Proteínas de Bactérias , Western Blotting , Proteínas do Citoesqueleto , Ensaio de Imunoadsorção Enzimática , Plasmídeos , Ratos
7.
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
8.
Anal Bioanal Chem ; 411(20): 5255-5265, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31119346

RESUMO

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 µg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp. Graphical abstract.


Assuntos
Anticorpos Monoclonais/imunologia , Crustáceos/química , Dietilestilbestrol/análise , Ensaio de Imunoadsorção Enzimática/métodos , Estrogênios não Esteroides/análise , Haptenos/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Feminino , Concentração Inibidora 50 , Limite de Detecção , Coelhos
9.
J Clin Pathol ; 72(8): 536-541, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31055472

RESUMO

AIMS: Very recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs. METHODS AND RESULTS: The histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series. CONCLUSIONS: Alternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.


Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Biomarcadores Tumorais/análise , DNA Nucleotidilexotransferase/análise , Imuno-Histoquímica , Neoplasias do Mediastino/enzimologia , Neoplasias Embrionárias de Células Germinativas/enzimologia , Neoplasias Ovarianas/enzimologia , Neoplasias Testiculares/enzimologia , Biomarcadores Tumorais/imunologia , Reações Cruzadas , DNA Nucleotidilexotransferase/imunologia , Feminino , Humanos , Itália , Masculino , Neoplasias do Mediastino/imunologia , Neoplasias do Mediastino/patologia , Neoplasias Embrionárias de Células Germinativas/imunologia , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Neoplasias Testiculares/imunologia , Neoplasias Testiculares/patologia
10.
Arch Virol ; 164(6): 1639-1646, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30982935

RESUMO

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Doença Hemorrágica de Coelhos/imunologia , Rotavirus/imunologia , Vírus Sendai/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Imunoensaio/veterinária , Coelhos , Kit de Reagentes para Diagnóstico
11.
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
12.
Blood Coagul Fibrinolysis ; 30(3): 127-132, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30958453

RESUMO

: We hypothesized that inhibitor specificity may predict the outcome of antifactor VIII autoantibodies eradication treatment in acquired hemophilia A. Our objective was to analyze the association between factor VIII domains recognized by inhibitors and outcome of the immunosuppressive therapies (ISTs) in a prospective, observational study. 16 patients were recruited. Inhibitor specificities were assessed at diagnosis and throughout the study. Their association with IST outcome was addressed. First-line IST succeeded in 56% of patients. Inhibitors reacted mainly with light chain domains (69%) and/or the A2 domain (44%). 31% inhibitors recognized more than one domain. Significantly, the number of patients whose inhibitors recognized the light chain was significantly higher in the group of those who did not reach complete remission after first line IST when compared with those who did [6/7 (85.7%) vs. 4/9 (44.4%), P < 0.05]. Therefore, inhibitor specificity could predict the success of IST in acquired hemophilia A.


Assuntos
Especificidade de Anticorpos , Autoanticorpos/imunologia , Fator VIII/imunologia , Hemofilia A/tratamento farmacológico , Humanos , Imunossupressores/uso terapêutico , Estudos Prospectivos , Domínios Proteicos , Resultado do Tratamento
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(3): 271-276, 2019 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-31030722

RESUMO

Objective To prepare monoclonal antibodies (mAbs) against bovine tumor necrosis factor-alpha (BoTNF-α). Methods Recombinant BoTNF-α with His tag (rHis-BoTNF-α) was expressed in a prokaryotic system as immunogen, and recombinant BoTNF-α with GST tag (rGST-BoTNF-α) was expressed as detection antigen. The mAbs were developed by hybridoma cell technology. The reactivity of mAbs with rHis-BoTNF-α and rGST-BoTNF-α was detected by Western blot analysis. Indirect ELISA was used to detect the titer and specificity of mAbs, and the reactivity to commercialized recombinant BoTNF-α (rBoTNF-α) was also evaluated. The reactivity of mAbs against natural antigens was identified by flow cytometry. Results Seven hybridomas stably secreting anti-rBoTNF-α mAbs were successfully obtained. The results showed that all seven mAbs had good reactivity and specificity. Flow cytometry analysis also showed that mAb 4G4 had good reactivity with natural BoTNF-α antigens. Conclusion The anti-rBoTNF-α mAbs are successfully achieved.


Assuntos
Anticorpos Monoclonais/imunologia , Animais , Especificidade de Anticorpos , Bovinos , Ensaio de Imunoadsorção Enzimática , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa
14.
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
15.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(2): 174-179, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30975284

RESUMO

Objective To develop high-sensitivity and high-specificity monoclonal antibody against tiamulin (TML). Methods Using oximation and carbodiimide method, TML was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare the immunogen TML-BSA and coating antigen TML-OVA, respectively. BALB/c mice were immunized with TML-BSA. The anti-TML monoclonal antibody hybridoma cell lines were screened by indirect ELISA after cell fusion. The ascites were prepared by in vivo induction method. The monoclonal antibody was purified from ascites by caprylic acid-ammonium sulfate method and identified by SDS-PAGE. Results The hybridoma cell lines of 3B3 and 4A7 were successfully obtained. Their titers of cell supernatant were 1:1600 and 1:6400, respectively. After purification, the titer of monoclonal antibody 4A7 was 5.12×105. The half inhibitory concentration (IC50) of 4A7 was 0.049 ng/mL. The affinity constant Kaff of 4A7 was 1.47×109 L/mol. The cross-reaction rate of 4A7 was 2.7% with analog retapamulin, whereas no cross-reaction was detected with other antibiotics such as valnemulin and fleroxacin. Conclusion Anti-TML monoclonal antibody has been successfully developed.


Assuntos
Anticorpos Monoclonais , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Reações Cruzadas , Diterpenos/metabolismo , Ensaio de Imunoadsorção Enzimática , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C
16.
Appl Microbiol Biotechnol ; 103(8): 3453-3464, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30863876

RESUMO

This study described the production, characterization, and application of monoclonal antibodies (mAbs) against porcine circovirus type 2 (PCV2). Twelve stable hybridomas were produced by immunization with purified PCV2a/LG strain and characterized by immunoperoxidase monolayer assay (IPMA), Western blotting, and neutralization assays. All mAbs could react with the PCV2 Cap protein and neutralize PCV2a/LG strain. One of them, mAb 3A5, reacted to all PCV2 strains from PCV2a, PCV2b, and PCV2d and it could be applied to detect PCV2 antigen and antibodies. It was shown that the mAb 3A5 could be used to locate PCV2 antigen in PK15 cells and the inguinal lymph nodes of PCV2b/YJ stain-infected piglets. Furthermore, this mAb could immunoprecipitate the Cap protein in PCV2-infected PK15 cells. Meanwhile, a capture ELISA based on mAb 3A5 was developed and used to specifically test PCV2 antigen from cultures; a linear relationship was observed between the optical density at 405 nm of the ELISA and viral titers (200-12,800 TCID50/mL), with a correlation coefficient of 0.9999. Finally, a competitive ELISA based on mAb 3A5 was developed to specifically detect antibodies in PCV2-infected and immunized pigs, and its sensitivity was higher than that of the blocking ELISA. This study suggested that the mAb 3A5 could be used in several convenient and efficient methods for PCV2 clinical and pathological studies, as well as surveillance in pigs and seroconversion monitoring in the vaccinated pigs.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Doenças dos Suínos/diagnóstico , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Linhagem Celular , Infecções por Circoviridae/diagnóstico , Circovirus/genética , Genótipo , Imunoensaio , Suínos
17.
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
18.
Mol Cell ; 73(5): 1075-1082.e4, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849388

RESUMO

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.


Assuntos
Anticorpos/metabolismo , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligopeptídeos/metabolismo , Análise Serial de Proteínas/métodos , Afinidade de Anticorpos , Especificidade de Anticorpos , Automação Laboratorial , Sítios de Ligação de Anticorpos , Catálise , Análise Mutacional de DNA/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Cinética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligopeptídeos/genética , Análise Serial de Proteínas/instrumentação , Ligação Proteica , Engenharia de Proteínas , Fluxo de Trabalho
19.
Transplant Proc ; 51(2): 488-491, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30879574

RESUMO

Results of 773 actual flow crossmatches (aFXMs) and virtual flow crossmatches (vFXMs) performed for living and deceased donor kidney transplantation in our center were analyzed retrospectively and evaluated for their concordance. Prediction of vFXMs was based on antibody identification using single antigen bead assay and locally established mean fluorescence intensity cutoff point compared with donor HLA antigens. The vast majority of aFXMs were in concordance with vFXMs with an overall concordance of 97%. Twenty-three predicted to be negative showed positive aFXMs; 12 of them had 0% calculated panel-reactive antibody, and 11 were found in patients with multiple non-donor-specific HLA antibodies. Three predicted positive vFXMs yielded negative aFXMs; 2 of them had allele-specific antibodies. CONCLUSIONS: vFXMs based on precise characterization of antibody specificities detected by single antigen bead assay using our cutoff point accurately predicted FXMs in the majority of patients and can be used safely to allocate kidney offers without performing physical crossmatches in selected patients.


Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , Isoanticorpos/análise , Transplante de Rim/métodos , Transplante de Pâncreas/métodos , Especificidade de Anticorpos , Feminino , Citometria de Fluxo , Antígenos HLA/imunologia , Humanos , Masculino , Estudos Retrospectivos , Doadores de Tecidos
20.
Phytochemistry ; 162: 99-108, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877900

RESUMO

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.


Assuntos
Especificidade de Anticorpos , Cucumovirus/fisiologia , Engenharia Genética/métodos , RNA Replicase/imunologia , Anticorpos de Cadeia Única/imunologia , Tabaco/genética , Tabaco/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Cucumovirus/enzimologia , Plantas Geneticamente Modificadas , Anticorpos de Cadeia Única/química , Transformação Genética
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