Autologous monoclonal antibodies recognize tumour-associated antigens in X-irradiated C57BL/6 mice

Energy Technology Data Exchange (ETDEWEB)

Artus, A; Guillemain, B; Legrand, E; Astier-Gin, T; Mamoun, R; Duplan, J -F

1986-09-01

X-irradiation of C57BL/6 mice induces thymic lymphosarcomas which sometimes contain retroviruses which upon injection into normal mice mimic the effect of the irradiation. We examined whether specific antigenicities, viral or cellular, were expressed by tumour cells that could be recognized by antibodies from the irradiated animals. We developed monoclonal antibodies (MAbs) using splenocytes of the diseased animal. The reactivity of such MAbs towards thymoma cell lines established in vitro was investigated by means of an ELISA. At least 10 antibody specificities were detected on the 13 tumours investigated, allowing separation of the MAbs into three classes: (i) those recognizing the autologous tumour, heterologous tumours as well as normal thymic tissue, (ii) those specific for the autologous tumour, and (iii) those specific for one tumour, but not ones of autologous origin. The last two classes corresponded to specific tumour-associated antigens. Our panel of MAbs defined each tumour by the particular pattern of antigens harboured. It is striking that most of the antigens were present in the normal thymus and that only two tumours had additional antigenicities. Additionally, quantitative variations were observed in the levels of expression of these antigens.

Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando

1999-01-01

Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99m Tc at high yield. The resulting 99m Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required

Sequential use of indium-111 labeled monoclonal antibodies 96.5 and ZME-018 does not increase detection sensitivity for metastatic melanoma

International Nuclear Information System (INIS)

Frontiera, M.; Murray, J.L.; Lamki, L.

1989-01-01

Two indium-111 labeled anti-melanoma murine monoclonal antibodies (MAb), 96.5 and ZME-018, each recognizing separate antigens on melanoma cells, were administered intravenously to 17 patients with melanoma in a sequential fashion to determine whether: (1) additional tumor sites could be imaged with the combination compared to a single Mab; (2) the first MAb influenced the biodistribution and tumor localization of the second; and (3) significantly toxicity occurred with the combination. Patients were randomized to receive either 96.5, followed by ZME-018, ZME-018 followed by 96.5, or each MAb followed by itself (controls). Infusions of the second MAb occurred 10 days after the first infusion. Gamma camera images were obtained 72 hours after each antibody infusion. There were 139 known metastatic sites of which 72 lesions were localized by either MAb for an overall sensitivity of 52%. The detection rate was higher when lesions only greater than 1.5 cm were considered. Imaging results were independent of MAb administration sequence. When ZME-018 was given as the first infusion, when ZME-018 was given as a second infusion (p = NS). However, mean sensitivities using 96.5 as the first or second infusion were 48% and 66% respectively (p = NS). There was not a significant number of sites detected by MAb 2 that were missed by MAb 1. Human anti-murine antibody (HAMA) response occurred in seven of eight patients studied; two patients who experienced toxicity had levels of HAMA greater than 2000 ng/ml. We conclude that the use of these two murine anti-melanoma monoclonal antibodies given in sequential fashion did not significantly change the imaging sensitivity from that seen with each individual antibody

Novel Monoclonal Antibodies Recognizing Human Prostate-Specific Membrane Antigen (PSMA) as Research and Theranostic Tools.

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Nováková, Zora; Foss, Catherine A; Copeland, Benjamin T; Morath, Volker; Baranová, Petra; Havlínová, Barbora; Skerra, Arne; Pomper, Martin G; Barinka, Cyril

2017-05-01

Prostate-specific membrane antigen (PSMA) is a validated target for the imaging and therapy of prostate cancer. Here, we report the detailed characterization of four novel murine monoclonal antibodies (mAbs) recognizing human PSMA as well as PSMA orthologs from different species. Performance of purified mAbs was assayed using a comprehensive panel of in vitro experimental setups including Western blotting, immunofluorescence, immunohistochemistry, ELISA, flow cytometry, and surface-plasmon resonance. Furthermore, a mouse xenograft model of prostate cancer was used to compare the suitability of the mAbs for in vivo applications. All mAbs demonstrate high specificity for PSMA as documented by the lack of cross-reactivity to unrelated human proteins. The 3F11 and 1A11 mAbs bind linear epitopes spanning residues 226-243 and 271-288 of human PSMA, respectively. 3F11 is also suitable for the detection of PSMA orthologs from mouse, pig, dog, and rat in experimental setups where the denatured form of PSMA is used. 5D3 and 5B1 mAbs recognize distinct surface-exposed conformational epitopes and are useful for targeting PSMA in its native conformation. Most importantly, using a mouse xenograft model of prostate cancer we show that both the intact 5D3 and its Fab fragment are suitable for in vivo imaging. With apparent affinities of 0.14 and 1.2 nM as determined by ELISA and flow cytometry, respectively, 5D3 has approximately 10-fold higher affinity for PSMA than the clinically validated mAb J591 and, therefore, is a prime candidate for the development of next-generation theranostics to target PSMA. Prostate 77:749-764, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

OpenAIRE

Narat, Mojca; Biček, Ajda; Vadnjal, Robert; Benčina, Dušan

2004-01-01

Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs) that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY) shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of...

Freeze-dried formulation for direct {sup 99m}Tc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

Energy Technology Data Exchange (ETDEWEB)

Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando E-mail: normando@ict.sld.cu

1999-08-01

Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with {sup 99m}Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with {sup 99m}Tc at high yield. The resulting {sup 99m}Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

2018-03-01

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L 1 Mab-4 (IgG 2b , kappa), using cell-based immunization and screening (CBIS) method and investigated hPD-L1 expression in oral cancers. L 1 Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4) in flow cytometry and stained oral cancers in a membrane-staining pattern. L 1 Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of L 1 Mab-4. These results indicate that L 1 Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers.

Production, Characterization and Use of Monoclonal Antibodies Recognizing IgY Epitopes Shared by Chicken, Turkey, Pheasant, Peafowl and Sparrow

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Ajda Biček

2004-01-01

Full Text Available Chicken antibodies are not only a part of immune defense but are more and more popular commercial products in form of chicken polyclonal, monoclonal or recombinant antibodies. We produced and characterized mouse monoclonal antibodies (mAbs that recognize epitopes located on heavy or light chain of chicken immunoglobulin Y (chIgY shared also by some other Phasianidae birds. The use of mAbs 1F5 and 2F10 that recognize heavy chain on chIgY common epitopes was demonstrated on immunoglobulins of turkey, pheasant and peafowl. Chicken IgY light chain specific mAb 3E10 revealed the presence of common epitopes on immunoglobulins of turkey, pheasant and sparrow. Monoclonal antibody clone 1F5/3G2 was used to prepare horseradish peroxidase (HRP conjugate and immunoadsorbent column. Conjugated mAbs were demonstrated to be excellent secondary antibodies for diagnostics of certain infections in different avian species. Since they do not react with mammalian immunoglobulins using our mAbs as secondary antibodies in human serodiagnostics would minimize background staining that appears when using mouse detection system. In dot immunobinding assay (DIBA and immunoblot assay they recognized specific IgY antibodies against Mycoplasma synoviae, Mycoplasma gallisepticum and Newcastle disease virus in sera of infected or vaccinated birds. Immunoadsorption as a method for removal of IgY from samples in which Mycoplasma synoviae specific IgY was predominant immunoglobulin class enabled more exact demonstration of specific IgA and IgM antibodies. Herein we are presenting effective mAbs useful in diagnostics of avian and mammalian infections as well as in final steps of detection and purification of chicken antibodies and their subunits produced in vivo or in vitro as polyclonal, monoclonal or recombinant antibodies.

Detection of high PD-L1 expression in oral cancers by a novel monoclonal antibody L1Mab-4

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Shinji Yamada

2018-03-01

Full Text Available Programmed cell death-ligand 1 (PD-L1, which is a ligand of programmed cell death-1 (PD-1, is a type I transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1 expression on tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb, L1Mab-4 (IgG2b, kappa, using cell-based immunization and screening (CBIS method and investigated hPD-L1 expression in oral cancers. L1Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 in flow cytometry and stained oral cancers in a membrane-staining pattern. L1Mab-4 stained 106/150 (70.7% of oral squamous cell carcinomas, indicating the very high sensitivity of L1Mab-4. These results indicate that L1Mab-4 could be useful for investigating the function of hPD-L1 in oral cancers. Keywords: Programmed cell death-ligand 1, Monoclonal antibody, Oral cancer

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

PMab-38 Recognizes Canine Podoplanin of Squamous Cell Carcinomas.

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Kaneko, Mika K; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Nakamura, Takuro; Saidoh, Noriko; Takagi, Michiaki; Kagawa, Yumiko; Konnai, Satoru; Kato, Yukinari

2016-10-01

Podoplanin, a type I transmembrane protein, is expressed in lymphatic endothelial cells. Although we previously developed an anticanine podoplanin monoclonal antibody (mAb), PMab-38, immunohistochemistry (IHC) showed that it did not react with canine lymphatic endothelial cells. Here, we determined whether PMab-38 recognizes canine podoplanin of squamous cell carcinomas (SCCs) and clarified its epitope. In IHC, PMab-38 reacted with 83% of SCCs (15/18 cases). Flow cytometry showed that the epitope of PMab-38 was different from that of the platelet aggregation-stimulating domain of the N-terminus, which was detected by almost all antipodoplanin mAbs such as D2-40 or NZ-1. PMab-38 is expected to be useful for investigating the function of podoplanin in canine tumors.

Radioimmunoscintigraphy with monoclonal antibody Technetium-99m-Anti-EGF-Receptor (R3-MAB) for the detection of head and neck tumours, metastasis and recurrence. Final report for the period 15 April 1995 - 15 April 1997

International Nuclear Information System (INIS)

Oliva Gonzalez, J.P.

1998-03-01

A clinical study was carried out to determine the sensitivity of radioimmunoscintigraphy (RIS) using indigenously produced mouse monoclonal antibody (MAB) against epidermal growth factor receptor in the detection of primary, recurrent and metastatic malignant epithelial tumours of the head and neck region in 13 patients. The MAB was labelled with 99m Tc and imaging was carried out using gamma camera and SPECT. The results were correlated with histopathological findings. RIS gave a sensitivity of 76.9%. This study showed that the indigenously produced MAB can be used for the detection of malignant epithelial tumours in the head and neck region but the MAB will be further characterized to improve its sensitivity in the detection of the neoplasia. (author)

Determination of critical epitope of PcMab-47 against human podocalyxin

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Shunsuke Itai

2018-07-01

Full Text Available Podocalyxin (PODXL is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA, flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

Determination of critical epitope of PcMab-47 against human podocalyxin.

Science.gov (United States)

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2018-07-01

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

The anti-(+-methamphetamine monoclonal antibody mAb7F9 attenuates acute (+-methamphetamine effects on intracranial self-stimulation in rats.

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Andrew C Harris

Full Text Available Passive immunization with monoclonal antibodies (mAbs against (+-methamphetamine (METH is being evaluated for the treatment of METH addiction. A human/mouse chimeric form of the murine anti-METH mAb7F9 has entered clinical trials. This study examined the effects of murine mAb7F9 on certain addiction-related behavioral effects of METH in rats as measured using intracranial self-stimulation (ICSS. Initial studies indicated that acute METH (0.1-0.56 mg/kg, s.c. lowered the minimal (threshold stimulation intensity that maintained ICSS. METH (0.3 mg/kg, s.c. also blocked elevations in ICSS thresholds (anhedonia-like behavior during spontaneous withdrawal from a chronic METH infusion (10 mg/kg/day x 7 days. In studies examining effects of i.v. pretreatment with mAb7F9 (at 30, 100, or 200 mg/kg, 200 mg/kg blocked the ability of an initial injection of METH (0.3 mg/kg, s.c. to reduce baseline ICSS thresholds, but was less capable of attenuating the effect of subsequent daily injections of METH. MAb7F9 (200 mg/kg also produced a small but significant reduction in the ability of METH (0.3 mg/kg, s.c. to reverse METH withdrawal-induced elevations in ICSS thresholds. These studies demonstrate that mAb7F9 can partially attenuate some addiction-related effects of acute METH in an ICSS model, and provide some support for the therapeutic potential of mAb7F9 for the treatment of METH addiction.

First experiences with the AMERLEX-MAB FREE T4 assay

International Nuclear Information System (INIS)

Nijhof, W.A.; Penders, T.J.

1989-01-01

The new Amerlex-MAB FT 4 is a quick direct free T 4 assay with good reproducability. The correlation between the Amerlex-MAB FT 4 and the free T 4 of Byk is good. In the non-thyreoidal illness patient group no deviation for the values were found. Amerlex-MAB FT 4 is cheaper, because no total T4 has to be measured. More research has to be done for special patient sera. Disturbing influences as free fatty acids, heparin and auto-antibodies have to be checked. (R.B.). 3 refs.; 3 figs.; 4 tabs

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

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Guo Yajun

2006-01-01

Full Text Available Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested and for metastatic melanoma of 96% (146/151 tested in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.

Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies.

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Li Yu

Full Text Available BACKGROUND: SFTS virus (SFTSV is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs recognized the nucleocapsid (N protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase.

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Ingravallo, P; Lahser, F; Xia, E; Sodowich, B; Lai, V C; Hong, Z; Zhong, W

2001-06-01

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.

Structural and biochemical characterization of the cell fate determining nucleotidyltransferase fold protein MAB21L1.

Science.gov (United States)

de Oliveira Mann, Carina C; Kiefersauer, Reiner; Witte, Gregor; Hopfner, Karl-Peter

2016-06-08

The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.

Effect of exposure time reduction towards sensitivity and SNR for computed radiography (CR) application in NDT

International Nuclear Information System (INIS)

Sapizah Rahim; Khairul Anuar Mohd Salleh; Noorhazleena Azaman; Shaharudin Sayuti; Siti Madiha Muhammad Amir; Arshad Yassin; Abdul Razak Hamzah

2010-01-01

Signal-to-noise ratio (SNR) and sensitivity study of Computed Radiography (CR) system with reduction of exposure time is presented. The purposes of this research are to determine the behavior of SNR toward three different thicknesses (step wedge; 5, 10 and 15 mm) and the ability of CR system to recognize hole type penetrameter when the exposure time decreased up to 80 % according to the exposure chart (D7; ISOVOLT Titan E). It is shown that the SNR is decreased with decreasing of exposure time percentage but the high quality image is achieved until 80 % reduction of exposure time. (author)

Sorbitol crystallization-induced aggregation in frozen mAb formulations.

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Piedmonte, Deirdre Murphy; Hair, Alison; Baker, Priti; Brych, Lejla; Nagapudi, Karthik; Lin, Hong; Cao, Wenjin; Hershenson, Susan; Ratnaswamy, Gayathri

2015-02-01

Sorbitol crystallization-induced aggregation of mAbs in the frozen state was evaluated. The effect of protein aggregation resulting from sorbitol crystallization was measured as a function of formulation variables such as protein concentration and pH. Long-term studies were performed on both IgG1 and IgG2 mAbs over the protein concentration range of 0.1-120 mg/mL. Protein aggregation was measured by size-exclusion HPLC (SE-HPLC) and further characterized by capillary-electrophoresis SDS. Sorbitol crystallization was monitored and characterized by subambient differential scanning calorimetry and X-ray diffraction. Aggregation due to sorbitol crystallization is inversely proportional to both protein concentration and formulation pH. At high protein concentrations, sorbitol crystallization was suppressed, and minimal aggregation by SE-HPLC resulted, presumably because of self-stabilization of the mAbs. The glass transition temperature (Tg ') and fragility index measurements were made to assess the influence of molecular mobility on the crystallization of sorbitol. Tg ' increased with increasing protein concentration for both mAbs. The fragility index decreased with increasing protein concentration, suggesting that it is increasingly difficult for sorbitol to crystallize at high protein concentrations. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

Science.gov (United States)

Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

2014-08-01

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The UC Davis/NIH NeuroMab Facility

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Federal Laboratory Consortium — The mission of the UC Davis/NIH NeuroMab facility is to generate and distribute high quality, validated mouse monoclonal antibodies against molecular targets found...

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

Science.gov (United States)

Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Analysis of Japanese Municipalities With Geopark, MAB, and GIAHS Certification

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Ryo Kohsaka

2015-11-01

Full Text Available We analyzed the discussions of Japanese municipalities in their process for obtaining certifications for the Geoparks by the United Nations Educational, Scientific and Cultural Organization (UNESCO, the Man and the Biosphere Programme (MAB by the UNESCO, and the Globally Important Agricultural Heritage systems (GIAHS by the Food and Agriculture Organization (FAO of the United Nations. The official records at the municipality diet were analyzed in a quantitative manner from 2011 to 2013. As the first step, we analyzed the eight municipalities of Noto and Sado for the GIAHS, the cities Itoigawa and Hakusan for the Geopark, and Katsuyama Yamanouchi village from Nagano for the MAB. As individual examples, we analyzed City of Suzu with GIAHS, Itoigawa (Geopark, and Yamanouchi town (MAB with the text-mining approach. For the GIAHS, it was clear that the larger municipalities with city status tended to discuss certification issues more frequently than the smaller towns and villages. Terms such as conservation and certification tended to be used with GIAHS at the Suzu City. The term brand was used with GIAHS and MAB but not for the Geopark. The findings using quantitative methods are at initial stage for analysis of municipality strategies and require further future research.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

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Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Dynamics of immature mAb glycoform secretion during CHO cell culture

DEFF Research Database (Denmark)

Jimenez del Val, Ioscani; Fan, Yuzhou; Weilguny, Dietmar

2016-01-01

Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy and immu......Ensuring consistent glycosylation-associated quality of therapeutic monoclonal antibodies (mAbs) has become a priority in pharmaceutical bioprocessing given that the distribution and composition of the carbohydrates (glycans) bound to these molecules determines their therapeutic efficacy...

Sensitivity improvement of an immuno-detection method for azaspiracids based on the use of microspheres coupled to a flow-fluorimetry system

Directory of Open Access Journals (Sweden)

María Fraga Corral

2014-06-01

These results demonstrate the high capability in terms of sensitivity of the microsphere-based immuno-detection assay for AZAs. The immobilization of AZA-1 instead of the synthetic AZA-2 used in Rodríguez et al (Rodriguez et al., 2014, combined with a lower mAb 8F4 concentration provided a remarkable improvement of sensitivity. The ON protocol used in Rodríguez et al. (Rodriguez et al., 2014 displayed a similar IC50 than the new short assay (around 1 nM while the new ON protocol provided an IC50 5-fold more sensitive (0.3 nM. Therefore, the new short assay allows a reduction of the experimental time. Additionally, the increase of sensitivity could help to avoid shellfish matrix interferences. Previously published works using immunoassays for the detection of phycotoxins present in shellfish avoided matrix interference by further extract dilution in combination with an increase of assay sensitivity (Fraga et al., 2012;Fraga et al., 2013. The extraction protocol described by Rodríguez et al. (Rodriguez et al., 2014 will probably be suitable for this newly optimized AZA-detection method since many reagents are the same and the higher sensitivity will allow higher extract dilution. Considering the extraction protocol recovery, sensitivity of the current assay and the regulated limit, shellfish extracts could be diluted up to 1:30 or 1:150 (v/v for detection with the short or long protocols, respectively. Additionally, mAb 8F4 was demonstrated to recognize AZA-2 and AZA-3 with cross-reactivities of 42 and 138 %, respectively. Presumably, this optimized assay will detect these analogs with similar cross-reactivity. The sensitivity of the microsphere-based assay for AZAs is enough to detect these compounds at the regulated levels in shellfish. This microsphere-based multi-detection method provides an easy-to-perform, highly sensitive and rapid method for the detection of AZAs. It could be included in a multi-detection method, which would allow time and sample volume

Development of at-line assay to monitor charge variants of MAbs during production.

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St Amand, M M; Ogunnaike, B A; Robinson, A S

2014-01-01

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers.

47-mG2a: A Mouse IgG2a-Type of PcMab-47 Useful for Detecting Podocalyxin in Esophageal Cancers by Immunohistochemistry.

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Kaneko, Mika K; Itai, Shunsuke; Yamada, Shinji; Kato, Yukinari

2018-04-09

Esophageal cancer is one of the highly malignant cancers. It comprises two of the most common histological tumor types: squamous cell carcinoma (SCC) and adenocarcinoma. SCC accounts for about 90% of esophageal cancers. Despite developments in treatment strategies, the prognosis and survival rate remain poor. Podocalyxin (PODXL) is a highly glycosylated type-I transmembrane protein. It is expressed in normal tissues such as kidney, heart, breast, and pancreas. Upregulation of PODXL correlates with tumor progression, invasion, and metastasis. Therefore, this glycoprotein could be a potential biomarker for predicting the prognosis of some cancers, for instance, brain, colorectal, oral, lung, bladder, prostate, and ovarian cancers. We previously developed a specific and sensitive anti-PODXL monoclonal antibody (mAb), PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ). We showed their utility in immunohistochemical analysis of oral cancers. Herein, we demonstrate that PcMab-47 and 47-mG 2a can also be used to detect esophageal squamous cell carcinoma (ESCC) with this technique. These two antibodies, respectively, stained 123/130 (94.6%) and 127/130 (97.7%) ESCC cases, indicating that they can detect PODXL with high sensitivity in this carcinoma. Of more than 3+ cases, 47-mG 2a was more effective than PcMab-47, respectively, staining 56/127 (44.1%) and 41/123 (33.3%). Therefore, 47-mG 2a can be used for the detection of PODXL in ESCC using immunohistochemical analysis.

The expression analysis of ICOS-L on activated T cells and immature dendritic cells as well as malignant B cells and Grave's-disease-derived thyroid tissues by two novel mAbs against human ICOS-L.

Science.gov (United States)

Wang, F; Zhu, W; Liu, T; Sun, Z; Ju, S; Ju, S; Yu, G; Xie, W; Deng, Z; Lu, B; Zhang, X

2007-01-01

ICOS-L, a newly identified member of B7 superfamily, plays an important role in immune responses. In this article, we report on two novel mouse anti-human ICOS-L monoclonal antibodies (mAbs) named as 11C4 and 12B11, whose specificities were verified by methods of flow cytometry, western blotting, and epitope competition assay. The two mAbs bound to distinct ICOS-L epitopes on B cells. Interestingly, mAb 11C4 could well recognize ICOS-L molecule on activated T cells and Jurkat cell lines, which is different from commercial anti-ICOS-L mAb (clone number MIH12) and the other mAb 12B11. In addition, we found that the expression of ICOS-L molecule was only detected on the surface of immature monocyte-derived dendritic cells (Mo-DCs) and was sharply decreased after induction of mature Mo-DCs activated by tumor necrosis factor-alpha or CD40. Furthermore, we showed that 11C4 could effectively suppress the maturation of Mo-DCs in vitro as evidenced by the low expression of CD80, CD86, CD83, and human leukocyte antigen-DR, which suggested that ICOS-L may be involved in the maturation of Mo-DCs. Using immunohistochemistry staining with mAb 11C4, the expression of ICOS-L was found in B lymphoma tissues and thyroid tissues from the Grave's disease but not in thyroid adenoma and normal thyroid tissues.

Germline-specific MATH-BTB substrate adaptor MAB1 regulates spindle length and nuclei identity in maize.

Science.gov (United States)

Juranič, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanic, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-12-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Podocyte changes after induction of acute albuminuria in mice by anti-aminopeptidase A mAb.

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Dijkman, Henry B P M; Gerlofs-Nijland, Miriam E; van der Laak, Jeroen A W M; Wetzels, Jack F M; Groenen, Patricia J T A; Assmann, Karel J M

2003-01-01

Administration of a specific combination of anti-aminopeptidase A (APA) mAb (ASD-37/41) in mice induces an acute albuminuria which is independent of angiotensin II, a well-known substrate of APA. In the present experiments, we examined whether binding of the mAb initiated changes in the podocytic expression of cytoskeleton (-associated), adhesion and slit-diaphragm proteins in relation to the time course of albuminuria. In addition, we measured ultrastructurally the extent of foot process retraction (the number of foot processes per microm GBM) and the width of the slit pore between the podocytes by morphometric methods. An injection of the mAb combination ASD-37/41 induced a massive but transient albuminuria that started at 6 h, and peaked at 8 h, after which it declined. However, even at day 7 after injection of the mAbs some albuminuria was present. Injection of the combination ASD-3/41 or saline did not induce an albuminuria. Notably, we observed changes in the staining of CD2AP and podocin, two slit-pore-associated proteins that coincided with the start of the albuminuria. Nephrin staining was reduced and podocytic actin staining became more granular only at a time albuminuria was declining (24 h). The number of foot processes per microm GBM was already decreased at 4 h with a further reduction thereafter. The width of the slit pore was unchanged at the time of peak albuminuria and gradually decreased thereafter. At day 7, podocytic foot process effacement was even more prominent although albuminuria was only slightly abnormal. Expression of CD2AP was still granular. We observed however a change toward normal in the expression of podocin. Injection of saline or ASD-3/41 had no effect on the expression of podocytic proteins, the number of foot processes or width of the slit pore. Our data show that the onset of albuminuria in the anti-APA model is related to alterations in CD2AP and podocin, proteins that are important for maintaining slit-diaphragm structure

Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

Science.gov (United States)

Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

2017-02-01

The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG 2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

SPECT scintigraphy with HDP and Mab BW 250/183 of loosened hip endoprothesis

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Predic, P [Hospital Celje, Celje (Sierra Leone); Gregoric, E [Hospital Izola, Izola (Sierra Leone); Dodig, D [Clinical Hospital Centre, Zagreb (Croatia). Dept. of Nuclear Medicine and Radiation Protection

1994-10-01

Main problem of the loosened hip endoprothesis is in distinguishing between the aseptic and septic loosening of endoprothesis. The study involved 27 pts with a loosened hip; 15 pts with aseptic and 12 pts with septic loosening. The patients were injected 550-770 MBq Tc-99m-HDP and underwent SPECT scintigraphy of the hips to repeat then the examination with only 370 MBq Tc-99m-Mab Bw 230/183. HDP application evidenced positive accumulation at the endoprothesis in all patients with a loosened hip while Mab Bw 250/183 only in the patients with septic loosening. Conclusion: SPECT scintigraphy of hip endoprothesis with HDP and Mab BW 250/183 allows differential diagnosing between septic and aseptic hip loosening and hereby a correct therapeutical approach. (author).

Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

Science.gov (United States)

Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

2017-04-01

Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize[W

Science.gov (United States)

Juranić, Martina; Srilunchang, Kanok-orn; Krohn, Nádia Graciele; Leljak-Levanić, Dunja; Sprunck, Stefanie; Dresselhaus, Thomas

2012-01-01

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle–dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s). PMID:23250449

Conservation Compromises: The MAB and the Legacy of the International Biological Program, 1964-1974.

Science.gov (United States)

Schleper, Simone

2017-02-01

This article looks at the International Biological Program (IBP) as the predecessor of UNESCO's well-known and highly successful Man and the Biosphere Programme (MAB). It argues that international conservation efforts of the 1970s, such as the MAB, must in fact be understood as a compound of two opposing attempts to reform international conservation in the 1960s. The scientific framework of the MAB has its origins in disputes between high-level conservationists affiliated with the International Union for the Conservation of Nature and Natural Resources (IUCN) about what the IBP meant for the future of conservation. Their respective visions entailed different ecological philosophies as much as diverging sets of political ideologies regarding the global implementation of conservation. Within the IBP's Conservation Section, one group propagated a universal systems approach to conservation with a centralized, technocratic management of nature and society by an elite group of independent scientific experts. Within IUCN, a second group based their notion of environmental expert roles on a more descriptive and local ecology of resource mapping as practiced by UNESCO. When the IBP came to an end in 1974, both groups' ecological philosophies played into the scientific framework underlying the MAB's World Network or Biosphere Reserves. The article argues that it is impossible to understand the course of conservation within the MAB without studying the dynamics and discourses between the two underlying expert groups and their respective visions for reforming conservation.

Novel sensitive monoclonal antibody based competitive enzyme-linked immunosorbent assay for the detection of raw and processed bovine beta-casein.

Science.gov (United States)

Castillo, Daniela S; Cassola, Alejandro

2017-01-01

Cow milk protein allergy (CMPA) is the most common childhood food allergy, which can sometimes persist or can newly develop in adulthood with severe symptoms. CMPA's treatment is complete dietary avoidance of milk proteins. To achieve this task, patients have to be aware of milk proteins found as "hidden allergens" in food commodities. In regard to milk proteins, it has been reported that allergenicity of caseins remains unaffected upon heat treatment. For these reasons, we aimed to obtain monoclonal antibodies (mAbs) against native and denatured β-casein, one of the most abundant and antigenic caseins, in order to develop an indirect competitive ELISA (icELISA) to detect and quantify traces of this milk allergen in raw and processed foodstuffs. We developed two specific hybridoma clones, 1H3 and 6A12, which recognized β-casein in its denatured and native conformations by indirect ELISA (iELISA). Cross-reaction analysis by Western blot and iELISA indicated that these mAbs specifically recognized β-casein from bovine and goat milk extracts, while they did not cross-react with proteins present in other food matrixes. These highly specific mAbs enabled the development of sensitive, reliable and reproducible icELISAs to detect and quantify this milk protein allergen in food commodities. The extraction of β-casein from foodstuff was efficiently carried out at 60°C for 15 minutes, using an extraction buffer containing 1% SDS. The present study establishes a valid 1H3 based-icELISA, which allows the detection and quantification -0.29 ppm and 0.80 ppm, respectively- of small amounts of β-casein in raw and processed foods. Furthermore, we were able to detect milk contamination in incurred food samples with the same sensitivity as a commercial sandwich ELISA thus showing that this icELISA constitutes a reliable analytical method for control strategies in food industry and allergy prevention.

Asp f6, an Aspergillus allergen specifically recognized by IgE from patients with allergic bronchopulmonary aspergillosis, is differentially expressed during germination.

Science.gov (United States)

Schwienbacher, M; Israel, L; Heesemann, J; Ebel, F

2005-11-01

Aspergillus fumigatus is a pathogenic mould causing allergic and invasive respiratory diseases. Allergic bronchopulmonary Aspergillosis (ABPA) is a severe pulmonary complication resulting from hypersensitivity to A. fumigatus proteins. Aspergillus allergen Asp f6 is recognized by IgE from ABPA patients, but not from sensitized individuals, a fact that can be used to differentiate between these two groups of allergic patients. Proteins from hyphae, resting and germinating conidia of A. fumigatus were compared by SDS-PAGE. Protein identification was performed using MALDI-TOF mass spectrometry. Recombinant A. fumigatus allergens were used to isolate specific monoclonal antibodies (mab) from a hybridoma bank generated against Aspergillus proteins. A hyphae-specific 23 kDa A. fumigatus protein was identified as the allergen Asp f6/manganese-dependent superoxide dismutase (MnSOD). Differential expression of MnSOD was confirmed by immunoblot using a specific mab. In contrast, Asp f8 another intracellular, but not ABPA-specific allergen, was detected in hyphae and conidia. Aspergillus fumigatus is able to colonize its environment by the formation of hyphae. Hyphae are found in the lung of ABPA patients, but not in patients suffering from atopic asthma. Our finding that Asp f6 is specifically expressed in hyphae might explain why an IgE response to Asp f6 is specific for ABPA patients.

Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

Science.gov (United States)

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance.

Science.gov (United States)

Li, W; Carper, K; Liang, Y; Zheng, X X; Kuhr, C S; Reyes, J D; Perkins, D L; Thomson, A W; Perkins, J D

2006-12-01

Liver allografts are accepted spontaneously in all mouse strain combinations without immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we examined the effect of CD4+ CD25+ T regulatory cells (Treg) on the induction of mouse liver transplant tolerance. Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice. Depleting rat anti-mouse CD25 mAb (PC61) was given to the donors or recipients (250 microg/d IP) pretransplant or to the recipients postoperatively. At day 5 posttransplantation, both effector T cells (mainly CD8) and CD4+ CD25+ Treg were increased in the liver allografts and host spleens compared to naïve mice. Anti-CD25 mAb administration, either pretransplantation or posttransplantation, reduced the ratio of CD4+ CD25+ Treg to the CD3 T cells of liver grafts and recipient spleens and induced liver allograft acute rejection compared to IgG treatment. Anti-CD25 mAb administration elevated anti-donor T-cell proliferative responses and CTL and NK activities of graft infiltrates and host splenocytes; reduced CTLA4, Foxp3, and IDO mRNA levels; increased IL-10 and IFN-gamma; and decreased IL-4 mRNA levels in the livers or host spleens. The number of apoptotic T cells was reduced significantly in the liver grafts and treated host spleens. Therefore, anti-CD25 mAb administration changed the balance of CD4+ CD25+ Treg to activated T cells of liver graft recipients, preventing liver transplant tolerance. This was associated with enhanced anti-donor immune reactivity, downregulated Treg gene expression, and reduced T cell apoptosis in the grafts and host spleens.

The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans

DEFF Research Database (Denmark)

Jafari, Gholamali; Appleford, Peter J; Seago, Julian

2011-01-01

, an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat...

Identification and quantification of predominant metabolites of synthetic cannabinoid MAB-CHMINACA in an authentic human urine specimen.

Science.gov (United States)

Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Watanabe, Kanako; Suzuki, Osamu

2018-02-01

An autopsy case in which the cause of death was judged as drug poisoning by two synthetic cannabinoids, including MAB-CHMINACA, was investigated. Although unchanged MAB-CHMINACA could be detected from solid tissues, blood and stomach contents in the case, the compound could not be detected from a urine specimen. We obtained six kinds of reference standards of MAB-CHMINACA metabolites from a commercial source. The MAB-CHMINACA metabolites from the urine specimen of the abuser were extracted using a QuEChERS method including dispersive solid-phase extraction, and analyzed by liquid chromatography-tandem mass spectrometry with or without hydrolysis with β-glucuronidase. Among the six MAB-CHMINACA metabolites tested, two predominant metabolites could be identified and quantified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, an increase of the two metabolites was not observed. The metabolites detected were a 4-monohydroxycyclohexylmethyl metabolite M1 (N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide) and a dihydroxyl (4-hydroxycyclohexylmethyl and tert-butylhydroxyl) metabolite M11 (N-(1-amino-4-hydroxy-3,3-dimethyl-1-oxobutan-2-yl)-1-((4-hydroxycyclohexyl)methyl)-1H-indazole-3-carboxamide). Their concentrations were 2.17 ± 0.15 and 10.2 ± 0.3 ng/mL (n = 3, each) for M1 and M11, respectively. Although there is one previous in vitro study showing the estimation of metabolism of MAB-CHMINACA using human hepatocytes, this is the first report dealing with in vivo identification and quantification of MAB-CHMINACA metabolites in an authentic human urine specimen. Copyright © 2017 John Wiley & Sons, Ltd.

Co-delivery of doxorubicin and arsenite with reduction and pH dual-sensitive vesicle for synergistic cancer therapy

Science.gov (United States)

Zhang, Lu; Xiao, Hong; Li, Jingguo; Cheng, Du; Shuai, Xintao

2016-06-01

Drug resistance is the underlying cause for therapeutic failure in clinical cancer chemotherapy. A prodrug copolymer mPEG-PAsp(DIP-co-BZA-co-DOX) (PDBD) was synthesized and assembled into a nanoscale vesicle comprising a PEG corona, a reduction and pH dual-sensitive hydrophobic membrane and an aqueous lumen encapsulating doxorubicin hydrochloride (DOX.HCl) and arsenite (As). The dual stimulation-sensitive design of the vesicle gave rise to rapid release of the physically entrapped DOX.HCl and arsenite inside acidic lysosomes, and chemically conjugated DOX inside the cytosol with high glutathione (GSH) concentration. In the optimized concentration range, arsenite previously recognized as a promising anticancer agent from traditional Chinese medicine can down-regulate the expressions of anti-apoptotic and multidrug resistance proteins to sensitize cancer cells to chemotherapy. Consequently, the DOX-As-co-loaded vesicle demonstrated potent anticancer activity. Compared to the only DOX-loaded vesicle, the DOX-As-co-loaded one induced more than twice the apoptotic ratio of MCF-7/ADR breast cancer cells at a low As concentration (0.5 μM), due to the synergistic effects of DOX and As. The drug loading strategy integrating chemical conjugation and physical encapsulation in stimulation-sensitive carriers enabled efficient drug loading in the formulation.Drug resistance is the underlying cause for therapeutic failure in clinical cancer chemotherapy. A prodrug copolymer mPEG-PAsp(DIP-co-BZA-co-DOX) (PDBD) was synthesized and assembled into a nanoscale vesicle comprising a PEG corona, a reduction and pH dual-sensitive hydrophobic membrane and an aqueous lumen encapsulating doxorubicin hydrochloride (DOX.HCl) and arsenite (As). The dual stimulation-sensitive design of the vesicle gave rise to rapid release of the physically entrapped DOX.HCl and arsenite inside acidic lysosomes, and chemically conjugated DOX inside the cytosol with high glutathione (GSH) concentration. In the

Physico-chemical Stability of MabThera Drug-product Solution for Subcutaneous Injection under in-use Conditions with Different Administration Materials.

Science.gov (United States)

Mueller, Claudia; Dietel, Elke; Heynen, Severin R; Nalenz, Heiko; Goldbach, Pierre; Mahler, Hanns-Christian; Schmidt, Johannes; Grauschopf, Ulla; Schoenhamnmer, Karin

2015-01-01

MabThera is an essential component of the standard-of-care regimens in the treatment of non-Hodgkin lymphoma and Chronic Lymphatic Leukemia. MabThera for subcutaneous injection is a novel line extension that has been approved by the European Medicines Agency for the treatment of patients with follicular lymphoma and diffuse large B-cell lymphoma. This study aimed to evaluate in-use stability data of MabThera subcutaneous drug-product solution in single-use syringes for subcutaneous administration according to the European Medicines Agency guideline. The drug-product solution was exposed to material contact surfaces of five different administration setups commonly used in subcutaneous drug delivery. MabThera subcutaneous was transferred under aseptic conditions into polypropylene and polycarbonate syringes and stored for 1, 2, and 4 weeks at 2°C to 8°C followed by 24 hours at 30°C. After storage, subcutaneous administration was simulated and MabThera subcutaneous drug-product solution quality attributes were evaluated by using compendial physico-chemical tests, as well as suitable and validated molecule- and formulation-specific analytical methods. MabThera subcutaneous vials were treated and analyzed in parallel. The physico-chemical results of MabThera subcutaneous in the different setups were comparable to the control for all timepoints. No change in drug-product quality after storage and simulated administration was found compared to the control. However, since single-dose products do not contain preservatives, microbial contamination and growth needs to be avoided and product sterility needs to be ensured. The results showed that MabThera subcutaneous remains compatible and stable, from a physico-chemical perspective, for up to 4 weeks at 2°C to 8°C followed by 24 hours at 30°C with the contact materials tested in this study. In order to avoid and minimize microbial growth, MabThera subcutaneous should be used immediately after removal from the original

IL-8 as antibody therapeutic target in inflammatory diseases: Reduction of clinical activity in palmoplantar pustulosis

DEFF Research Database (Denmark)

Skov, L.; Beurskens, F.J.; Reitamo, S.

2008-01-01

IL-8 is a chemokine that has been implicated in a number of inflammatory diseases involving neutrophil activation. HuMab 10F8 is a novel fully human mAb against IL-8, which binds a discontinuous epitope on IL-8 overlapping the receptor binding site, and which effectively neutralizes IL-8-dependent...... human neutrophil activation and migration. We investigated whether interference in the cytokine network by HuMab 10F8 might benefit patients suffering from palmoplantar pustulosis, a chronic inflammatory skin disease. Treatment of patients with HuMab 10F8 was well tolerated and significantly reduced...... clinical disease activity at all five endpoints, which included a >= 50% reduction in the formation of fresh pustules. IL-8 neutralization was monitored at the site of inflammation by assessing exudates of palmoplantar pustulosis lesions. HuMab 10F8 sequestered IL-8 in situ, as observed by rapid dose...

RECOGNIZING INFANTS' EMOTIONAL EXPRESSIONS: ARE ADOLESCENTS LESS SENSITIVE TO INFANTS' CUES?

Science.gov (United States)

Niessen, Anke; Konrad, Kerstin; Dahmen, Brigitte; Herpertz-Dahlmann, Beate; Firk, Christine

2017-07-01

Previous studies have shown that adolescent mothers interact less sensitively with their infants than do adult mothers. This difference might be due to developmental difficulties in the recognition of infants' emotional states in adolescents. Therefore, the aim of the current study was to explore differences in the recognition of infant signals between nonparous adolescent girls and boys as compared to female and male adults. To this end, we examined 54 childless adolescents and 54 childless adults (50% female). Participants were shown a series of 20 short videos of infants aged 3 to 6 months presenting different emotional states ranging from very distressed to very happy. In addition, participants were asked to report their own parental experiences using the German version, Fragebogen zum erinnerten elterlichen Erziehungsverhalten (J. Schumacher, M. Eisemann, & E. Brähler, ), of the Egna Minnen Befräffande Uppfostran (Own Memories of Parental Rearing Experiences in Childhood; C. Perris, L. Jacobsson, H. Lindstrom, L. von Knorring, & H. Perris, ). Adolescents rated distressed infants as more distressed than did the adults. Furthermore, female participants rated the very distressed infants as more distressed than did male participants. These data suggest that adolescents, in general, are not impaired in recognizing infant emotional states, as compared to adults. Thus, we suggest that more extreme ratings of infant signals of discomfort together with immature sociocognitive regulation processes during adolescence might contribute to reduced sensitivity observed in adolescent mothers. © 2017 Michigan Association for Infant Mental Health.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

Science.gov (United States)

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

TGF-beta Sma/Mab signaling mutations uncouple reproductive aging from somatic aging.

Directory of Open Access Journals (Sweden)

Shijing Luo

2009-12-01

Full Text Available Female reproductive cessation is one of the earliest age-related declines humans experience, occurring in mid-adulthood. Similarly, Caenorhabditis elegans' reproductive span is short relative to its total life span, with reproduction ceasing about a third into its 15-20 day adulthood. All of the known mutations and treatments that extend C. elegans' reproductive period also regulate longevity, suggesting that reproductive span is normally linked to life span. C. elegans has two canonical TGF-beta signaling pathways. We recently found that the TGF-beta Dauer pathway regulates longevity through the Insulin/IGF-1 Signaling (IIS pathway; here we show that this pathway has a moderate effect on reproductive span. By contrast, TGF-beta Sma/Mab signaling mutants exhibit a substantially extended reproductive period, more than doubling reproductive span in some cases. Sma/Mab mutations extend reproductive span disproportionately to life span and act independently of known regulators of somatic aging, such as Insulin/IGF-1 Signaling and Dietary Restriction. This is the first discovery of a pathway that regulates reproductive span independently of longevity and the first identification of the TGF-beta Sma/Mab pathway as a regulator of reproductive aging. Our results suggest that longevity and reproductive span regulation can be uncoupled, although they appear to normally be linked through regulatory pathways.

Structures and Stability of Metal Amidoboranes (MAB): Density Functional Calculations

International Nuclear Information System (INIS)

Li Cailin; Wu Chaoling; Chen Yungui; Zhou Jingjing; Zheng Xin; Pang Lijuan; Deng Gang

2010-01-01

Molecule geometry structures, frequencies, and energetic stabilities of ammonia borane (AB, NH 3 BH 3 ) and metal amidoboranes (MAB, MNH 2 BH 3 ), formed by substituting H atom in AB with one of main group metal atoms, have been investigated by density-functional theory and optimized at the B3LYP levels with 6-311G++ (3df, 3pd) basic set. Their structural parameters and infrared spectrum characteristic peaks have been predicted, which should be the criterion of a successfully synthesized material. Several parameters such as binding energies, vibrational frequencies, and the energy gaps between the HOMO and the LUMO have been adopted to characterize and evaluate their structure stabilities. It is also found that the binding energies and HOMO-LUMO energy gaps of the MAB obviously change with the substitution of the atoms. MgAB has the lowest binding energy and is easier to decompose than any other substitutional structures under same conditions, while CaAB has the highest chemical activity. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

Life‐cycle and cost of goods assessment of fed‐batch and perfusion‐based manufacturing processes for mAbs

Science.gov (United States)

Bunnak, Phumthep; Allmendinger, Richard; Ramasamy, Sri V.; Lettieri, Paola

2016-01-01

Life‐cycle assessment (LCA) is an environmental assessment tool that quantifies the environmental impact associated with a product or a process (e.g., water consumption, energy requirements, and solid waste generation). While LCA is a standard approach in many commercial industries, its application has not been exploited widely in the bioprocessing sector. To contribute toward the design of more cost‐efficient, robust and environmentally‐friendly manufacturing process for monoclonal antibodies (mAbs), a framework consisting of an LCA and economic analysis combined with a sensitivity analysis of manufacturing process parameters and a production scale‐up study is presented. The efficiency of the framework is demonstrated using a comparative study of the two most commonly used upstream configurations for mAb manufacture, namely fed‐batch (FB) and perfusion‐based processes. Results obtained by the framework are presented using a range of visualization tools, and indicate that a standard perfusion process (with a pooling duration of 4 days) has similar cost of goods than a FB process but a larger environmental footprint because it consumed 35% more water, demanded 17% more energy, and emitted 17% more CO2 than the FB process. Water consumption was the most important impact category, especially when scaling‐up the processes, as energy was required to produce process water and water‐for‐injection, while CO2 was emitted from energy generation. The sensitivity analysis revealed that the perfusion process can be made more environmentally‐friendly than the FB process if the pooling duration is extended to 8 days. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1324–1335, 2016 PMID:27390260

Immuno-PET of undifferentiated thyroid carcinoma with radioiodine-labelled antibody cMAb U36: application to antibody tumour uptake studies

Energy Technology Data Exchange (ETDEWEB)

Fortin, Marc-Andre [Centre Hospitalier Universitaire de Quebec and Laval University, Laboratory for Biomaterials and Bioengineering, Quebec City (Canada); Uppsala University, Biomedical Radiation Sciences, Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Salnikov, Alexei V. [Uppsala University, BMC, Department of Medical Biochemistry and Microbiology, Uppsala (Sweden); German Cancer Research Center, Division of Molecular Immunology, Heidelberg (Germany); Nestor, Marika [Uppsala University, Division of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala (Sweden); Heldin, Nils-Erik [Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala (Sweden); Rubin, Kristofer [Uppsala University, BMC, Department of Medical Biochemistry and Microbiology, Uppsala (Sweden); Lundqvist, Hans [Uppsala University, Biomedical Radiation Sciences, Department of Oncology, Radiology, and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden)

2007-09-15

We tested the suitability of the chimeric monoclonal anti-human CD44 splice version 6 antibody (cMAb U36) for targeting and visualising human anaplastic thyroid carcinoma with PET. We also performed experiments aimed at elucidating the relation between tumour interstitial fluid pressure (TIFP) and the tumour uptake of antibodies. The affinity and specificity of the cMAb U36 for KAT-4 cells were evaluated in vitro, as was the Na{sup +}/I{sup -} symporter (NIS) expression. Biodistribution studies were performed on KAT-4 carcinoma-bearing mice injected with {sup 124}I-cMAb U36 or free iodine. Biodistribution studies were also performed in animals treated with the specific TGF-{beta}1 and -{beta}3 inhibitor Fc:T{beta}RII, which lowers TIFP. Treated and non-treated animals were scanned by microPET. Cultured human undifferentiated/anaplastic thyroid carcinoma KAT-4 cells expressed low levels of NIS and uptake of free iodine was insignificant. The cMAb U36 expressed an affinity (K{sub D}) of 11 {+-} 2 nM. Tumour radioactivity uptake reached maximum values 48 h after injection of {sup 124}I-cMAb U36 ({proportional_to}22%IA/g). KAT-4 carcinomas were readily identified in all {sup 124}I-immuno-PET images. Radioactivity tumour uptake in Fc:T{beta}RII-treated animals was significantly lower at 24 and 48 h after injection, and five times higher thyroid uptake was also noted. We successfully used {sup 124}I-cMAb U36 to visualise CD44v6-expressing human anaplastic thyroid carcinoma. Given the lack of NIS expression in KAT-4, tumour visualisation is not due to free iodine uptake. Lowering the TIFP in KAT-4 carcinomas did not increase the uptake of mAbs into tumour tissue. (orig.)

Preparation for gluing of Carbon prototype MAB at INEGI, Porto

CERN Multimedia

Miguel Moreira, Porto, INEGI

2001-01-01

MAB's will assure the alignment of the CMS detector. It is equipped with muon cameras, measuring the position of the barrel muon stations and at the same time linking via the link elements, connecting the barrel muon detectors with the Tracker. In addition there is a connection with the endcap. More details can be found on the muon/alignment homepage on the web

A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies.

Science.gov (United States)

Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas

2017-03-01

Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.

A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity

DEFF Research Database (Denmark)

Krogh-Madsen, Rikke; Thyfault, John P; Broholm, Christa

2010-01-01

decreased after step reduction, with a post hoc analysis revealing the most pronounced effect after 4 h of insulin infusion. In addition, the 2-wk period induced a 7% decline in VO2 max (ml/min; cardiovascular fitness). Lean mass of legs, but not arms and trunk, decreased concurrently. Taken together, one...... possible biological cause for the public health problem of Type 2 diabetes has been identified. Reduced ambulatory activity for 2 wk in healthy, nonexercising young men significantly reduced peripheral insulin sensitivity, cardiovascular fitness, and lean leg mass........ A reduced number of daily steps induced a significant reduction of 17% in the glucose infusion rate (GIR) during the clamp. This reduction was due to a decline in peripheral insulin sensitivity with no effect on hepatic endogenous glucose production. The insulin-stimulated ratio of pAktthr308/total Akt...

A two-week reduction of ambulatory activity attenuates peripheral insulin sensitivity

DEFF Research Database (Denmark)

Krogh-Madsen, Rikke; Thyfault, John P; Broholm, Christa

2009-01-01

after step reduction, with a post hoc analysis revealing the most pronounced effect after 4 h of insulin infusion. In addition, the two-week period induced a 7% decline in VO2max (ml/min; cardiovascular fitness). Lean mass of legs, but not arms and truck, decreased concurrently. Taken together, one...... possible biological cause for the public health problem of type 2 diabetes has been identified. Reduced ambulatory activity for two weeks in healthy, non-exercising young men significantly reduced peripheral insulin sensitivity, cardiovascular fitness, and lean leg mass. Key words: Inactivity, Insulin...... number of daily steps induced a significant reduction of 17% in the glucose infusion rate (GIR) during the clamp. This reduction was due to a decline in peripheral insulin sensitivity with no effect on hepatic endogenous glucose production. The insulin-stimulated ratio of pAkt(thr308)/total Akt decreased...

Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors

Directory of Open Access Journals (Sweden)

Simmonds Peter

2008-01-01

Full Text Available Abstract Background HIV-1 R5 viruses cause most of the AIDS cases worldwide and are preferentially transmitted compared to CXCR4-using viruses. Furthermore, R5 viruses vary extensively in capacity to infect macrophages and highly macrophage-tropic variants are frequently identified in the brains of patients with dementia. Here, we investigated the sensitivity of R5 envelopes to a range of inhibitors and antibodies that block HIV entry. We studied a large panel of R5 envelopes, derived by PCR amplification without culture from brain, lymph node, blood and semen. These R5 envelopes conferred a wide range of macrophage tropism and included highly macrophage-tropic variants from brain and non-macrophage-tropic variants from lymph node. Results R5 macrophage-tropism correlated with sensitivity to inhibition by reagents that inhibited gp120:CD4 interactions. Thus, increasing macrophage-tropism was associated with increased sensitivity to soluble CD4 and to IgG-CD4 (PRO 542, but with increased resistance to the anti-CD4 monoclonal antibody (mab, Q4120. These observations were highly significant and are consistent with an increased affinity of envelope for CD4 for macrophage-tropic envelopes. No overall correlations were noted between R5 macrophage-tropism and sensitivity to CCR5 antagonists or to gp41 specific reagents. Intriguingly, there was a relationship between increasing macrophage-tropism and increased sensitivity to the CD4 binding site mab, b12, but decreased sensitivity to 2G12, a mab that binds a glycan complex on gp120. Conclusion Variation in R5 macrophage-tropism is caused by envelope variation that predominantly influences sensitivity to reagents that block gp120:CD4 interactions. Such variation has important implications for therapy using viral entry inhibitors and for the design of envelope antigens for vaccines.

Study of the epitope structure of purified Dac G I and Lol p I, the major allergens of Dactylis glomerata and Lolium perenne pollens, using monoclonal antibodies.

Science.gov (United States)

Mourad, W; Mécheri, S; Peltre, G; David, B; Hébert, J

1988-11-15

The use of mAb allowed us to further analyze the cross-reactivity between purified Dac g I and Lol p I, the major allergens of Dactylis glomerata (cocksfoot) and Lolium perenne (Rye grass), respectively. It was first shown, using IEF, followed by immunoprinting, that serum IgE antibodies from most grass-sensitive patients recognize both Dac g I and Lol p I. Second, three different anti-Lol p I mAb, 290A-167, 348A-6, and 539A-6, and one anti-Dac g I mAb, P3B2 were all shown to react with Dac g I and Lol p I, indicating that the two molecules share common epitopes. Epitope specificity of the mAb was determined by competitive binding inhibition of a given labeled mAb to solid phase fixed Dac g I or Lol p I by the mAb. The results indicated that the four mAb are directed against four different and non-overlapping epitopes present on both allergens. Using double-binding RIA, our data strongly suggest that the common epitopes are not repetitive on both molecules. In addition to their similar physicochemical characteristics, such as isolectric points and m.w., Dac g I and Lol p I share four identical epitopes. Binding inhibition of human IgE to Lol p I and Dac g I by the mAb was also assessed. The results indicated that each mAb was able to inhibit such reactions to variable degree but no additive inhibition was observed when two mAb of different specificities were used in combination, suggesting that the human IgE binding site is partially shared by each epitope recognized by the four mAb.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

Science.gov (United States)

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin.

Science.gov (United States)

Takahashi, Hitoshi; Nagata, Shiho; Odagiri, Takato; Kageyama, Tsutomu

2018-04-15

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of the β-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus.

Science.gov (United States)

Lefebvre, Anne-Laure; Le Moigne, Vincent; Bernut, Audrey; Veckerlé, Carole; Compain, Fabrice; Herrmann, Jean-Louis; Kremer, Laurent; Arthur, Michel; Mainardi, Jean-Luc

2017-04-01

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a β-lactam (cefoxitin or imipenem). The triple combination is used without any β-lactamase inhibitor, even though M abscessus produces the broad-spectrum β-lactamase Bla Mab We determine whether inhibition of Bla Mab by avibactam improves the activity of imipenem against M. abscessus The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of Bla Mab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 μg/ml), amikacin (32 μg/ml), and avibactam (4 μg/ml) was bactericidal in broth (imipenem was used at 8 and 32 μg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 μg/ml) and high (32 μg/ml) dosages of imipenem, respectively. In vivo inhibition of Bla Mab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding Bla Mab was induced (20-fold) in the infected macrophages. Inhibition of Bla Mab by avibactam improved the efficacy of imipenem against M. abscessus in vitro , in macrophages, and in zebrafish embryos, indicating that this β-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of Bla Mab , since the production of the β-lactamase is inducible in macrophages. Copyright © 2017 American Society for Microbiology.

Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies.

Science.gov (United States)

Mourad, W; Bernier, D; Jobin, M; Hébert, J

1989-11-01

Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.

Aree Protette del Po e della Collina Torinese: studi propedeutici alla Candidatura MAB - Man and the Biosphere

Directory of Open Access Journals (Sweden)

Cimnaghi Elisabetta

2014-03-01

Full Text Available Il programma MAB fu lanciato dall’UNESCO negli anni ’70 al fine di migliorare il rapporto tra uomo e ambiente. Negli anni il programma ha portato al riconoscimento di Riserve della Biosfera che gli Stati Membri si impegnano a gestire nella prospettiva della conservazione delle risorse. L’Organismo Gestore dell’Area Protetta, partendo, da un lato, dalla consapevolezza della ricchezza ambientale che caratterizza il suo territorio e, dall’altro, del contesto di sviluppo antropico elevato in cui essa è situata, ha riconosciuto nel programma MAB un’opportunità per incrementare le sue attività di protezione e potenziamento delle risorse. Questo articolo descrive i primi risultati ottenuti negli studi preliminari per la Designazione al MAB, con particolare attenzione sia alle opportunità di implementazione di progetti legati al marchio “Collina Po”, creato dall’Organo di Gestione del Parco, si ain termini di scambio di ‘best practices’ con altre Riserve di Biosfera.

The effect of ethics training on students recognizing ethical violations and developing moral sensitivity.

Science.gov (United States)

Baykara, Zehra Gocmen; Demir, Sevil Guler; Yaman, Sengul

2015-09-01

Moral sensitivity is a life-long cognitive ability. It is expected that nurses who work in a professional purpose at "curing human beings" should have a highly developed moral sensitivity. The general opinion is that ethics education plays a significant role in this sense to enhance the moral sensitivity in terms of nurses' professional behaviors and distinguish ethical violations. This study was conducted as intervention research for the purpose of determining the effect of the ethics training on fourth-year students of the nursing department recognizing ethical violations experienced in the hospital and developing ethical sensitivity. The study was conducted with 50 students, with 25 students each in the experiment and control groups. Students in the experiment group were provided ethics training and consultancy services. The data were collected through the data collection form, which consists of questions on the socio-demographic characteristics and ethical sensitivity of the students, Moral Sensitivity Questionnaire, and the observation form on ethical principle violations/protection in the clinic environment. The data were digitized on the computer with the SPSS for Windows 13.0 program. The data were evaluated utilizing number, percentile calculation, paired samples t-test, Wilcoxon test, and the McNemar test. The total Moral Sensitivity Questionnaire pre-test score averages of students in the experiment group were determined to be 93.88 ± 13.57, and their total post-test score averages were determined to be 89.24 ± 15.90. The total pre-test score averages of students in the control group were determined to be 91.48 ± 17.59, and their total post-test score averages were determined to be 97.72 ± 19.91. In the study, it was determined that the post-training ethical sensitivity of students in the experiment group increased; however, this was statistically not significant. Furthermore, it was determined that the number of ethical principle protection

The Mycoplasma hominis P120 membrane protein contains a 216 amino acid hypervariable domain that is recognized by the human humoral immune response

DEFF Research Database (Denmark)

Nyvold, Charlotte Guldborg; Birkelund, Svend; Christiansen, Gunna

1997-01-01

In the antigenically heterogeneous species Mycoplasma hominis a monoclonal antibody, mAb 26.7D, was previously found to recognize a 120 kDa polypeptide from M. hominis 7488. This antibody did not react with the type strain PG21. The homologous gene from M. hominis PG21 was cloned and sequenced an...... response. Such a variable domain may be important in evasion of the host's immune response, and thus aid survival of the micro-organism....

Anti-GD2 mAb and Vorinostat synergize in the treatment of neuroblastoma

NARCIS (Netherlands)

Kroesen, M.; Bull, C.; Gielen, P.R.; Brok, I.C.; Armandari, I.; Wassink, M.; Looman, M.W.G.; Boon, L.; Brok, M.H.M.G.M. den; Hoogerbrugge, P.M.; Adema, G.J.

2016-01-01

Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical

Experimental Treatment of Bladder Cancer with Bi-213-anti-EGFR MAb

International Nuclear Information System (INIS)

Seidl, Christof; Pfost, Birgit; Müller, Felix

2013-01-01

Therapy of non-muscle-invasive bladder cancer (carcinoma in situ) comprises transurethral resection of the tumour and subsequent instillation of the chemotherapeutic drug mitomycin C in order to eradicate remaining tumour cells. Yet 15 – 40% of treated patients relapse within 5 years. Therefore, new therapeutic strategies to combat tumour recurrence are needed. Alpha-particle emitting radionuclides efficiently kill single tumour cells or small tumour cell clusters. Because the epidermal growth factor receptor (EGFR) is overexpressed on bladder cancer cells, conjugates composed of the alpha-emitter Bi-213 and the anti-EGFR antibody matuzumab should provide a powerful drug to eliminate disseminated bladder cancer cells. Therefore, the aims of our study were (i) to analyse the cytotoxic effects of Bi-213-anti-EGFR radioimmunoconjugates at the cellular level, (ii) to evaluate therapeutic efficacy of intravesically applied Bi-213- anti-EGFR-Mab in a nude mouse model with intravesical human bladder cancer xenografts, (iii) to compare Bi- 213-anti-EGFR-Mab efficacy with chemotherapy using mitomycin C and (iv) to demonstrate that radioimmunotherapy is not toxic to cells of the bladder wall and of the kidneys

Analysis of the epitope structure of Plum pox virus coat protein.

Science.gov (United States)

Candresse, Thierry; Saenz, Pilar; García, Juan Antonio; Boscia, Donato; Navratil, Milan; Gorris, Maria Teresa; Cambra, Mariano

2011-05-01

Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

Science.gov (United States)

Mehrotra nee Tandon, P; Lind, D S; Bear, H D; Susskind, B M

1992-08-01

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

Directory of Open Access Journals (Sweden)

Luisa W. Cheng

2015-11-01

Full Text Available Botulinum neurotoxins (BoNT are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL immunoassay for BoNT/B, using monoclonal antibodies (mAbs MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

Directory of Open Access Journals (Sweden)

Li H

2015-07-01

Full Text Available Hua-Fei Li,1–3,* Cong Wu,4,* Ting Chen,5,* Ge Zhang,1 He Zhao,1 Chang-Hong Ke,1 Zheng Xu21International Joint Cancer Institute, Translation Medicine Institute, 2Planning Division, Scientific Research Department, 3Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, 4Department of Laboratory Diagnosis, Changhai Hospital, 5Department of Cardiology, Changhai Hospital, the Second Military Medical University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The CD20-directed monoclonal antibody rituximab (RTX established a new era in the treatment of non-Hodgkin lymphoma (NHL; however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD, they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer–RTX–tositumomab [PPRT nanocomb] was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced “off-rate” to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, “cross-cell link”-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With

Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

International Nuclear Information System (INIS)

Zaffino, Paolo; Spadea, Maria Francesca; Raudaschl, Patrik; Fritscher, Karl; Sharp, Gregory C.

2016-01-01

Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, where a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against

Technical Note: PLASTIMATCH MABS, an open source tool for automatic image segmentation

Energy Technology Data Exchange (ETDEWEB)

Zaffino, Paolo; Spadea, Maria Francesca [Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Catanzaro 88100 (Italy); Raudaschl, Patrik; Fritscher, Karl [Institute for Biomedical Image Analysis, Private University of Health Sciences, Medical Informatics and Technology, Hall in Tirol 6060 (Austria); Sharp, Gregory C. [Department for Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts 02114 (United States)

2016-09-15

Purpose: Multiatlas based segmentation is largely used in many clinical and research applications. Due to its good performances, it has recently been included in some commercial platforms for radiotherapy planning and surgery guidance. Anyway, to date, a software with no restrictions about the anatomical district and image modality is still missing. In this paper we introduce PLASTIMATCH MABS, an open source software that can be used with any image modality for automatic segmentation. Methods: PLASTIMATCH MABS workflow consists of two main parts: (1) an offline phase, where optimal registration and voting parameters are tuned and (2) an online phase, where a new patient is labeled from scratch by using the same parameters as identified in the former phase. Several registration strategies, as well as different voting criteria can be selected. A flexible atlas selection scheme is also available. To prove the effectiveness of the proposed software across anatomical districts and image modalities, it was tested on two very different scenarios: head and neck (H&N) CT segmentation for radiotherapy application, and magnetic resonance image brain labeling for neuroscience investigation. Results: For the neurological study, minimum dice was equal to 0.76 (investigated structures: left and right caudate, putamen, thalamus, and hippocampus). For head and neck case, minimum dice was 0.42 for the most challenging structures (optic nerves and submandibular glands) and 0.62 for the other ones (mandible, brainstem, and parotid glands). Time required to obtain the labels was compatible with a real clinical workflow (35 and 120 min). Conclusions: The proposed software fills a gap in the multiatlas based segmentation field, since all currently available tools (both for commercial and for research purposes) are restricted to a well specified application. Furthermore, it can be adopted as a platform for exploring MABS parameters and as a reference implementation for comparing against

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Primary Lymphoid Tumors and Lymph Node Metastasis

Science.gov (United States)

Blanco, Rancés; Blanco, Damián; Quintana, Yisel; Escobar, Xiomara; Rengifo, Charles E.; Osorio, Marta; Gutiérrez, Zailí; Lamadrid, Janet; Cedeño, Mercedes; Frómeta, Milagros; Carr, Adriana; Rengifo, Enrique

2013-01-01

The reactivity of the 14F7 Mab, a highly specific IgG1 against N-glycolyl GM3 ganglioside (NeuGcGM3) in normal tissues, lymphomas, lymph node metastasis, and other metastatic sites was assessed by immunohistochemistry. In addition, the effect of chemical fixation on the 14F7 Mab staining using monolayers of P3X63Ag.653 cells was also evaluated. Moreover, the ability of 14F7 to bind NeuGcGM3 ganglioside inducing complement-independent cytotoxicity by a flow cytometry-based assay was measured. The 14F7 Mab was reactive in unfixed, 4% paraformaldehyde, 4% formaldehyde, and acetone fixed cells. Postfixation with acetone did not alter the localization of NeuGcGM3, while the staining with 14F7 Mab was significantly eliminated in both cells fixed and postfixed with methanol but only partially reduced with ethanol. The staining with 14F7 Mab was evidenced in the 89.2%, 89.4%, and 88.9% of lymphomas, lymph node metastasis, and other metastatic sites, respectively, but not in normal tissues. The treatment with 14F7 Mab affected both morphology and membrane integrity of P3X63Ag.653 cells. This cytotoxic activity was dose-dependent and ranged from 24.0 to 84.7% (10–1000 μg/mL) as compared to the negative control. Our data could support the possible use of NeuGcGM3 as target for both active and passive immunotherapy against malignancies expressing this molecule. PMID:24381785

Do You Recognize This Parent?

Science.gov (United States)

Wallace, Edna

1997-01-01

Suggests effective ways to work with parents who may be permissive, busy, detached, overprotective, or negative. Recommends that child care professionals be sensitive and understanding, recognize other demands on parents' time and communicate competitively with them, use terms parents understand, accept various levels of parental involvement, be…

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

International Nuclear Information System (INIS)

Nourshargh, S.; Rampart, M.; Hellewell, P.G.; Jose, P.J.; Harlan, J.M.; Edwards, A.J.; Williams, T.J.

1989-01-01

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: (1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; (2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and (3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules

Folate-containing reduction-sensitive lipid-polymer hybrid nanoparticles for targeted delivery of doxorubicin.

Science.gov (United States)

Wu, Bo; Yu, Ping; Cui, Can; Wu, Ming; Zhang, Yang; Liu, Lei; Wang, Cai-Xia; Zhuo, Ren-Xi; Huang, Shi-Wen

2015-04-01

The development and evaluation of folate-targeted and reduction-triggered biodegradable nanoparticles are introduced to the research on targeted delivery of doxorubicin (DOX). This type of folate-targeted lipid-polymer hybrid nanoparticles (FLPNPs) is comprised of a poly(D,L-lactide-co-glycolide) (PLGA) core, a soybean lecithin monolayer, a monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) reduction-sensitive shell, and a folic acid-targeted ligand. FLPNPs exhibited high size stability but fast disassembly in a simulated cancer cell reductive environment. The experiments on the release process in vitro revealed that as a reduction-sensitive drug delivery system, FLPNPs released DOX faster in the presence of 10 mM dithiothreitol (DTT). Results from flow cytometry, confocal image and in vitro cytotoxicity assays revealed that FLPNPs further enhanced cell uptake and generated higher cytotoxicity against human epidermoid carcinoma in the oral cavity than non-targeted redox-sensitive and targeted redox-insensitive controls. Furthermore, in vivo animal experiments demonstrated that systemic administration of DOX-loaded FLPNPs remarkably reduced tumor growth. Experiments on biodistribution of DOX-loaded FLPNPs showed that an increasing amount of DOX accumulated in the tumor. Therefore, FLPNPs formulations have proved to be a stable, controllable and targeted anticancer drug delivery system.

Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies

International Nuclear Information System (INIS)

Kim, Chan-Lan; Umetani, Atsushi; Matsui, Toshio; Ishiguro, Naotaka; Shinagawa, Morikazu; Horiuchi, Motohiro

2004-01-01

We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrP Sc ) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56-90, 119-127, 137-143, 143-149, 147-151, 163-169, and 219-229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrP Sc . However, no mAbs reacted with protease-treated PrP Sc purified from scrapie-affected mice, even when PrP Sc was dispersed into a detergent-lipid protein complex, to reduce the size of PrP Sc aggregates. In contrast, denaturation of PrP Sc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrP Sc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrP Sc , the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrP Sc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

OpenAIRE

Norihisa Nishimichi; Nagako Kawashima; Yasuyuki Yokosaki

2015-01-01

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face o...

A comparison of targetting of neuroblastoma with MIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients

International Nuclear Information System (INIS)

Hoefnagel, C.A.; Rutgers, M.; Buitenhuis, C.K.M.; Smets, L.A.; Kraker, J. de; Meli, M.; Carrel, F.; Schubiger, P.A.; Novak-Hofer, I.; Amstutz, H.

2001-01-01

Modine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131 I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targetting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of 131 I-MIBG (110 MBq) and with 131 I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with 131 I-chCE7, the subcutaneous tumours nearly disappeared; treatment with 131 I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with 131 I-mAb chCE7 and of 24 days with 131 I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by 131 I-chCE7 compared with 131 I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with 131 I-MIBG and 131 I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. 131 I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not

Effect of weight reduction on insulin sensitivity, sex hormone-binding globulin, sex hormones and gonadotrophins in obese children

DEFF Research Database (Denmark)

Birkebaek, N H; Lange, Aksel; Holland-Fischer, P

2010-01-01

Obesity in men is associated with reduced insulin sensitivity and hypoandrogenism, while obesity in women is associated with reduced insulin sensitivity and hyperandrogenism. In children, the effect of obesity and weight reduction on the hypothalamo-pituitary-gonadal axis is rarely investigated. ....... The aim of the present study was to investigate the effect of weight reduction in obese Caucasian children on insulin sensitivity, sex hormone-binding globulin (SHBG), DHEAS and the hypothalamo-pituitary-gonadal axis.......Obesity in men is associated with reduced insulin sensitivity and hypoandrogenism, while obesity in women is associated with reduced insulin sensitivity and hyperandrogenism. In children, the effect of obesity and weight reduction on the hypothalamo-pituitary-gonadal axis is rarely investigated...

Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies

International Nuclear Information System (INIS)

Piechaczyk, M.; Baldet, L.; Pau, B.; Bastide, J.M.

1989-01-01

A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs

Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus.

Science.gov (United States)

Yu, Cui; Yang, Cuiyun; Song, Shaoyi; Yu, Zixiang; Zhou, Xueping; Wu, Jianxiang

2018-04-04

Iris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. We report the development of a high-sensitive Luminex xMAP-based microsphere immunoassay (MIA) for specific detection of IYSV. The nucleocapsid (N) gene of IYSV was cloned and expressed in Escherichia coli to produce the His-tagged recombinant N protein. A panel of monoclonal antibodies (MAbs) against IYSV was generated by immunizing the mice with recombinant N protein. Five specific MAbs (16D9, 11C6, 7F4, 12C10, and 14H12) were identified and used for developing the Luminex xMAP-based MIA systems along with a polyclonal antibody against IYSV. Comparative analyses of their sensitivity and specificity in detecting IYSV from infected tobacco leaves identified 7F4 as the best-performed MAb in MIA. We then optimized the working conditions of Luminex xMAP-based MIA in specific detection of IYSV from infected tobacco leaves by using appropriate blocking buffer and proper concentration of biotin-labeled antibodies as well as the suitable ratio between the antibodies and the streptavidin R-phycoerythrin (SA-RPE). Under the optimized conditions the Luminex xMAP-based MIA was able to specifically detect IYSV with much higher sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). Importantly, the Luminex xMAP-based MIA is time-saving and the whole procedure could be completed within 2.5 h. We generated five specific MAbs against IYSV and developed the Luminex xMAP-based MIA method for specific detection of IYSV in plants. This assay provides a sensitive, high-specific, easy to perform and likely cost-effective approach for IYSV detection from infected plants, implicating potential broad usefulness of MIA in plant virus diagnosis.

Reduction-sensitive micelles self-assembled from amphiphilic chondroitin sulfate A-deoxycholic acid conjugate for triggered release of doxorubicin.

Science.gov (United States)

Liu, Hongxia; Wu, Shuqin; Yu, Jingmou; Fan, Dun; Ren, Jin; Zhang, Lei; Zhao, Jianguo

2017-06-01

Reduction-sensitive chondroitin sulfate A (CSA)-based micelles were developed. CSA was conjugated with deoxycholic acid (DOCA) via a disulfide linkage. The bioreducible conjugate (CSA-ss-DOCA) can form self-assembled micelles in aqueous medium. The critical micelle concentration (CMC) of CSA-ss-DOCA conjugate is 0.047mg/mL, and its mean diameter is 387nm. The anticancer drug doxorubicin (DOX) was chosen as a model drug, and was effectively encapsulated into the micelles with high loading efficiency. Reduction-sensitive micelles and reduction-insensitive control micelles displayed similar DOX release behavior in phosphate buffered saline (PBS, pH7.4). Notably, DOX release from the reduction-sensitive micelles in vitro was accelerated in the presence of 20mM glutathione-containing PBS environment. Moreover, DOX-loaded CSA-ss-DOCA (CSA-ss-DOCA/DOX) micelles exhibited intracellular reduction-responsive characteristics in human gastric cancer HGC-27 cells determined by confocal laser scanning microscopy (CLSM). Furthermore, CSA-ss-DOCA/DOX micelles demonstrated higher antitumor efficacy than reduction-insensitive control micelles in HGC-27 cells. These results suggested that reduction-sensitive CSA-ss-DOCA micelles had the potential as intracellular targeted carriers of anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

Science.gov (United States)

Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

2014-11-14

We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb(P) CO17-1A × BR55-treated. The mAb(P) CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb(P) CO17-1A × BR55. In addition, the mAb(P) CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation

Directory of Open Access Journals (Sweden)

Devesh Radhakrishnan

2017-12-01

Full Text Available In order to meet desired drug product quality targets, the glycosylation profile of biotherapeutics such as monoclonal antibodies (mAbs must be maintained consistently during manufacturing. Achieving consistent glycan distribution profiles requires identifying factors that influence glycosylation, and manipulating them appropriately via well-designed control strategies. Now, the cell culture media supplement, MnCl2, is known to alter the glycosylation profile in mAbs generally, but its effect, particularly when introduced at different stages during cell growth, has yet to be investigated and quantified. In this study, we evaluate the effect of time-dependent addition of MnCl2 on the glycan profile quantitatively, using factorial design experiments. Our results show that MnCl2 addition during the lag and exponential phases affects the glycan profile significantly more than stationary phase supplementation does. Also, using a novel computational technique, we identify various combinations of glycan species that are affected by this dynamic media supplementation scheme, and quantify the effects mathematically. Our experiments demonstrate the importance of taking into consideration the time of addition of these trace supplements, not just their concentrations, and our computational analysis provides insight into what supplements to add, when, and how much, in order to induce desired changes.

Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus

DEFF Research Database (Denmark)

Martinez-Torrecuadrada, J.L.; Langeveld, J.P.M.; Venteo, A.

1999-01-01

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological...... in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coil using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most....... Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques...

Development of a reduction-sensitive diselenide-conjugated oligoethylenimine nanoparticulate system as a gene carrier

Directory of Open Access Journals (Sweden)

Cheng G

2012-07-01

Full Text Available Gang Cheng,1,2 Yiyan He,1 Li Xie,1 Yu Nie,1 Bin He,1 Zhirong Zhang,2 Zhongwei Gu11National Engineering Research Center for Biomaterials, 2Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, People's Republic of ChinaBackground: The reduction-sensitive cationic polymer is a promising nonviral carrier for gene delivery. Until now, disulfide bonds have been the only golden standard for its design. The aim of this research was to develop a novel reduction-responsive cationic polymer as a gene carrier.Methods: Polycationic carriers were synthesized by addition of branched oligoethylenimine 800 Da (OEI800 via an active ester containing diselenide bonds. Disulfide bonds cross-linked with OEI800-SSx and monoselenide bonds linked with OEI800-Sex were synthesized and compared. Their molecular weights and degradation properties were determined using gel permeation chromatography. Changes in particle size, morphology, and DNA binding were investigated by dynamic light scattering, transmission electron microscopy, and electrophoresis assay in a reduction environment. Cytotoxicity and transfection in vitro were evaluated in a murine melanoma cell line (B16F10 and a human cervical epithelial carcinoma cell line (HeLa, while intracellular degradation and dissociation with DNA were studied by confocal laser scanning microscopy with FITC-labeled OEI800 derivatives and Cy5-labeled DNA.Results: Diselenide-conjugated OEI800 (OEI800-Se Sex polymer carriers of high molecular weight were successfully synthesized. After compacting with DNA, the OEI800-SeSex polymers formed nanoparticles with an average size of 140 nm at an adequate C/P ratio. OEI800-Se Sex showed reduction-responsive degradation properties similar to those of the OEI800-SSx via gel permeation chromatography, dynamic light scattering, and transmission electron microscopy. OEI800-SeSex showed much lower cytotoxicity than PEI25k

Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

Science.gov (United States)

Seepiban, Channarong; Charoenvilaisiri, Saengsoon; Warin, Nuchnard; Bhunchoth, Anjana; Phironrit, Namthip; Phuangrat, Bencharong; Chatchawankanphanich, Orawan; Attathom, Supat; Gajanandana, Oraprapai

2017-05-30

Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars. The recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene. Two MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72-81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78-96% identity with each other and 72-91% identity with those that were not detected by MAb D2. TAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of

64Cu-DOTA-Anti-CTLA-4 mAb Enabled PET Visualization of CTLA-4 on the T-Cell Infiltrating Tumor Tissues

Science.gov (United States)

Higashikawa, Kei; Yagi, Katsuharu; Watanabe, Keiko; Kamino, Shinichiro; Ueda, Masashi; Hiromura, Makoto; Enomoto, Shuichi

2014-01-01

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) targeted therapy by anti-CTLA-4 monoclonal antibody (mAb) is highly effective in cancer patients. However, it is extremely expensive and potentially produces autoimmune-related adverse effects. Therefore, the development of a method to evaluate CTLA-4 expression prior to CTLA-4-targeted therapy is expected to open doors to evidence-based and cost-efficient medical care and to avoid adverse effects brought about by ineffective therapy. In this study, we aimed to develop a molecular imaging probe for CTLA-4 visualization in tumor. First, we examined CTLA-4 expression in normal colon tissues, cultured CT26 cells, and CT26 tumor tissues from tumor-bearing BALB/c mice and BALB/c nude mice by reverse transcription polymerase chain reaction (RT-PCR) analysis and confirmed whether CTLA-4 is strongly expressed in CT26 tumor tissues. Second, we newly synthesized 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-anti-mouse CTLA-4 mAb (64Cu-DOTA-anti-CTLA-4 mAb) and evaluated its usefulness in positron emission tomography (PET) and ex-vivo biodistribution analysis in CT26-bearing BALB/c mice. High CTLA-4 expression was confirmed in the CT26 tumor tissues of tumor-bearing BALB/c mice. However, CTLA-4 expression was extremely low in the cultured CT26 cells and the CT26 tumor tissues of tumor-bearing BALB/c nude mice. The results suggested that T cells were responsible for the high CTLA-4 expression. Furthermore, 64Cu-DOTA-anti-CTLA-4 mAb displayed significantly high accumulation in the CT26 tumor, thereby realizing non-invasive CTLA-4 visualization in the tumor. Together, the results indicate that 64Cu-DOTA-anti-CTLA-4 mAb would be useful for the evaluation of CTLA-4 expression in tumor. PMID:25365349

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV).

Science.gov (United States)

Satoh, Ryoichi; Kaku, Ayumi; Satomura, Megumi; Kohori, Michiyo; Noura, Kanako; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Hohdatsu, Tsutomu

2011-06-01

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats. Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Sensitivity Enhancement in Low Cutoff Wavelength Long-Period Fiber Gratings by Cladding Diameter Reduction

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Ignacio Del Villar

2017-09-01

Full Text Available The diameter of long-period fiber gratings (LPFGs fabricated in optical fibers with a low cutoff wavelength was be reduced by hydrofluoric acid etching, enhancing the sensitivity to refractive index by more than a factor of 3, to 2611 nm/refractive index unit in the range from 1.333 to 1.4278. The grating period selected for the LPFGs allowed access to the dispersion turning point at wavelengths close to the visible range of the optical spectrum, where optical equipment is less expensive. As an example of an application, a pH sensor based on the deposition of a polymeric coating was analyzed in two situations: with an LPFG without diameter reduction and with an LPFG with diameter reduction. Again, a sensitivity increase of a factor of near 3 was obtained, demonstrating the ability of this method to enhance the sensitivity of thin-film-coated LPFG chemical sensors.

Sensitivity Enhancement in Low Cutoff Wavelength Long-Period Fiber Gratings by Cladding Diameter Reduction.

Science.gov (United States)

Del Villar, Ignacio; Partridge, Matthew; Rodriguez, Wenceslao Eduardo; Fuentes, Omar; Socorro, Abian Bentor; Diaz, Silvia; Corres, Jesus Maria; James, Stephen Wayne; Tatam, Ralph Peter

2017-09-13

The diameter of long-period fiber gratings (LPFGs) fabricated in optical fibers with a low cutoff wavelength was be reduced by hydrofluoric acid etching, enhancing the sensitivity to refractive index by more than a factor of 3, to 2611 nm/refractive index unit in the range from 1.333 to 1.4278. The grating period selected for the LPFGs allowed access to the dispersion turning point at wavelengths close to the visible range of the optical spectrum, where optical equipment is less expensive. As an example of an application, a pH sensor based on the deposition of a polymeric coating was analyzed in two situations: with an LPFG without diameter reduction and with an LPFG with diameter reduction. Again, a sensitivity increase of a factor of near 3 was obtained, demonstrating the ability of this method to enhance the sensitivity of thin-film-coated LPFG chemical sensors.

Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.

Science.gov (United States)

Kaneko, Mika K; Nakamura, Takuro; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Abe, Shinji; Takagi, Michiaki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

OpenAIRE

Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

2016-01-01

Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab cou...

Strategies for cost-effective carbon reductions: A sensitivity analysis of alternative scenarios

International Nuclear Information System (INIS)

Gumerman, Etan; Koomey, Jonathan G.; Brown, Marilyn

2001-01-01

Analyses of alternative futures often present results for a limited set of scenarios, with little if any sensitivity analysis to identify the factors affecting the scenario results. This approach creates an artificial impression of certainty associated with the scenarios considered, and inhibits understanding of the underlying forces. This paper summarizes the economic and carbon savings sensitivity analysis completed for the Scenarios for a Clean Energy Future study (IWG, 2000). Its 19 sensitivity cases provide insight into the costs and carbon-reduction impacts of a carbon permit trading system, demand-side efficiency programs, and supply-side policies. Impacts under different natural gas and oil price trajectories are also examined. The results provide compelling evidence that policy opportunities exist to reduce carbon emissions and save society money

Immunoreactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Epithelial Malignant Tumors from Digestive System

OpenAIRE

Blanco, Rancés; Rengifo, Enrique; Cedeño, Mercedes; Rengifo, Charles E.; Alonso, Daniel F.; Carr, Adriana

2011-01-01

The limited expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in human normal tissues, as well as its presence in melanoma and breast carcinoma using 14F7 Mab (anti-NeuGcGM3), has been previously reported. In this work we evaluated for the first time the 14F7 Mab immunorecognition in some digestive system tumors. Immunohistochemical assays were made with 14F7, followed by anti-mouse biotinylated antibody and ABC/HRP system in normal and pathological human tissues were made. No immunoreactio...

Multiple epitopes in a dodecapeptide of myelin basic protein determined bymonoclonal antibodies

International Nuclear Information System (INIS)

Price, J.O.; Whitaker, J.N.; Vasu, R.I.; Metzger, D.W.

1986-01-01

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

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Andrabi Raiees

2012-09-01

Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

Aeroallergen analyses and their clinical relevance. I. Immunochemical quantification of allergens by RAST-inhibition, Mab-ELISA, basophil histamine release, and counter current immuno electrophoresis

DEFF Research Database (Denmark)

Johnsen, C R; Abrahamsen, L; Stahl Skov, P

1991-01-01

of indoor aeroallergens, cat, dog, and Derm. pter. allergen extracts were selected for the experiments. To evaluate unspecific interference, these allergens were compared mutually and with Cladosporium herbarum. Allergen extracts in varying dilutions were mixed with crushed glass fibre filter materials......, eluted, recovered by centrifugation, and allergen concentration quantified by the assays. Equal sensitivity was found for both IgE- and IgG4-RI assaying cat allergen (in the range 5-50 SQ-U/ml) and dog allergen (in the range 10(2)-10(3) SQ-U/ml). The IgG4-RI assaying Derm. pter. was more sensitive (50 SQ......-U/ml) than IgE-RI (2*10(3) SQ-U/ml). The ranges of allergen detection limits for the Mab-ELISA were equal for cat and Derm. pter. (10-10(2) SQ-U/ml). The range of allergen detection limits for CCIE, assaying dog were 10(4)-10(5) SQ-U/ml. The ranges of allergen detection limits for HR were equal for cat...

Mapping of epitopes on Poa p I and Lol p I allergens with monoclonal antibodies.

Science.gov (United States)

Lin, Z W; Ekramoddoullah, A K; Jaggi, K S; Dzuba-Fischer, J; Rector, E; Kisil, F T

1990-01-01

Allergen Poa p I isolated from the dialysed aqueous extract of Kentucky blue grass pollen by affinity chromatography with an anti-Lol p I murine monoclonal antibody (MAb) 290A-167 was previously shown to consist of a 35.8-kilodalton (kD) component with a pI of 6.4, designated as Poa p Ia, and a 33-kD component with a pI of 9.1, designated as Poa p Ib. The present study reports on the comparative antigenic analyses of these two components, using MAbs produced separately against Poa p I and Lol p I. Thus, anti-Poa p I MAbs 60 and 61 and anti-Lol p I MAb 290A-167 recognized Poa p Ia and Poa p Ib whereas anti-Poa p I MAbs 62, 63 and 64 and anti-Lol p I MAb 348A-6 recognized only Poa p Ia. The specificities of the MAbs were further resolved by comparing their respective abilities to inhibit the binding of 125I-Poa p I or 125I-Lol p I to the different MAbs prepared in the form of solid phase. These studies revealed that at least 4 distinct epitopes (designated as E1, E2, E3 and E4) were shared by both Poa p I and Lol p I. All 4 epitopes were present on Poa p Ia whereas only E1 and E3 were detected on Poa p Ib. E1 was recognized by MAbs 60 and 61, E2 by MAbs 62, 63 and 64, E3 by MAb 290A-167 and E4 by MAb 348A-6.(ABSTRACT TRUNCATED AT 250 WORDS)

CHARACTERIZATION OF TWO MONOCLONAL ANTIBODIES WHICH RECOGNIZE DIFFERENT SUBPOPULATIONS OF CHICKEN T LYMPHOCYTES

OpenAIRE

KONDO, Takashi; HATTORI, Masakazu; KODAMA, Hiroshi; ONUMA, Misao; MIKAMI, Takeshi

1990-01-01

Distribution among peripheral T lymphocyte subpopulations and biochemical properties of the chicken lymphocyte surface antigens defined by monoclonal antibodies (mAbs) Lc-4 and Lc-6 were examined. Two-color immunofluorescence analysis revealed that Lc-4 and Lc-6 antigens were expressed on mutually exclusive subpopulations of peripheral T lymphocytes but not on B lymphocytes. Lc-4 mAb precipitated a polypeptide with apparent molecular mass of 35 and 65 kilodalton under reducing and non-reducin...

Tissue Reactivity of the 14F7 Mab Raised against N-Glycolyl GM3 Ganglioside in Tumors of Neuroectodermal, Mesodermal, and Epithelial Origin

Science.gov (United States)

Blanco, Rancés; Quintana, Yisel; Blanco, Damián; Cedeño, Mercedes; Rengifo, Charles E.; Frómeta, Milagros; Ríos, Martha; Rengifo, Enrique; Carr, Adriana

2013-01-01

The expression of N-glycolylneuraminic acid forming the structure of gangliosides and/or other glycoconjugates (Hanganutziu-Deicher antigen) in human has been considered as a tumor-associated antigen. Specifically, some reports of 14F7 Mab (a highly specific Mab raised against N-glycolyl GM3 ganglioside) reactivity in human tumors have been recently published. Nevertheless, tumors of epithelial origin have been mostly evaluated. The goal of the present paper was to evaluate the immunohistochemical recognition of 14F7 Mab in different human tumors of neuroectodermal, mesodermal, and epithelial origins using an immunoperoxidase staining method. Samples of fetal, normal, and reactive astrocytosis of the brain were also included in the study. In general, nontumoral tissues, as well as, low-grade brain tumors showed no or a limited immunoreaction with 14F7 Mab. Nevertheless, high-grade astrocytomas (III-IV) and neuroblastomas, as well as, sarcomas and thyroid carcinomas were mostly reactive with 14F7. No reaction was evidenced in medulloblastomas and ependymoblastomas. Our data suggest that the expression of N-glycolyl GM3 ganglioside could be related to the aggressive behavior of malignant cells, without depending on the tumor origin. Our data could also support the possible use of N-glycolyl GM3 as a target for both active and passive immunotherapies of malignancies expressing this molecule. PMID:26317019

Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

Science.gov (United States)

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Chang, Yao-Wen; Harada, Hiroyuki; Kato, Yukinari

2017-12-01

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti-NGF monoclonal antibodies.

Science.gov (United States)

Medina-Cucurella, Angélica V; Zhu, Yaqi; Bowen, Scott J; Bergeron, Lisa M; Whitehead, Timothy A

2018-04-12

Nerve growth factor (NGF) plays a central role in multiple chronic pain conditions. As such, anti-NGF monoclonal antibodies (mAbs) that function by antagonizing NGF downstream signaling are leading drug candidates for non-opioid pain relief. To evaluate anti-canine NGF (cNGF) mAbs we sought a yeast surface display platform of cNGF. Both mature cNGF and pro-cNGF displayed on the yeast surface but bound conformationally sensitive mAbs at most 2.5-fold in mean fluorescence intensity above background, suggesting that cNGF was mostly misfolded. To improve the amount of folded, displayed cNGF, we used comprehensive mutagenesis, FACS, and deep sequencing to identify point mutants in the pro-region of canine NGF that properly enhance the folded protein displayed on the yeast surface. Out of 1,737 tested single point mutants in the pro region, 49 increased the amount of NGF recognized by conformationally sensitive mAbs. These gain-of-function mutations cluster around residues A-61-P-26. Gain-of-function mutants were additive, and a construct containing three mutations increased amount of folded cNGF to 23- fold above background. Using this new cNGF construct, fine conformational epitopes for tanezumab and three anti-cNGF mAbs were evaluated. The epitope revealed by the yeast experiments largely overlapped with the tanezumab epitope previously determined by X-ray crystallography. The other mAbs showed site-specific differences with tanezumab. As the number of binding epitopes of functionally neutralizing anti-NGF mAbs on NGF are limited, subtle differences in the individual interacting residues on NGF that bind each mAb contribute to the understanding of each antibody and variations in its neutralizing activity. These results demonstrate the potential of deep sequencing-guided protein engineering to improve the production of folded surface-displayed protein, and the resulting cNGF construct provides a platform to map conformational epitopes for other anti-neurotrophin mAbs

Russian biosphere reserves at the youth MAB-2017 Forum in Italy

Directory of Open Access Journals (Sweden)

Elena A. Shuyskaya

2018-02-01

Full Text Available Russia was represented by 9 participants from Biosphere Reserves at the MAB Youth Forum in Italy. The main question of the Forum was «How to involve the young in the work of biosphere reserves?» The debate resulted in a Declaration, elaborated by participants from around the world (282 delegates from 85 countries. Measures to improve scientific cooperation, data exchange in the sphere of educational tourism and administrative management, development of joint projects in environmental education were formulated. The article contains a number of recommendations for the network of biosphere reserves in Russia, based on Seville strategy and Lima Action Plan.

Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

Science.gov (United States)

Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

2015-01-01

Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

Science.gov (United States)

Yang, Baolin; Wang, Mingxia; Liu, Wenming; Xu, Zhiqiang; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

2017-12-01

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8 TLLEDRILT 16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr 8 and Asp 12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.

A literatura como resistência política: traços neorrealistas na produção literária do Movimento dos Atingidos por Barragens (MAB

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Carolina Alves Pereira

2018-04-01

Full Text Available Este trabalho busca analisar as relações existentes entre as produções literárias do inicio do século XX, com o surgimento da corrente neo-realista, e a literatura produzida pelo Movimento dos Atingidos por Barragens (MAB, como manifestação de resistência à cultura elitista, no que tange ao espaço das criações artísticas. Para tanto, discute-se a característica do neo-realismo como expressão literária definidora dos aspectos sociais e políticos representados nas obras da época. Analisa-se, também, o conceito de resistência como determinante para a definição da literatura marginalizada do movimento campesino (MAB. Por fim, a seguinte pesquisa ratifica a importância da literatura e das artes produzida para além das universidades e dos cânones habituais. Palavras-chave: Literatura de resistência, Neorrealismo, Movimento dos Atingidos por Barragens (MAB. _________________________ This paper analyzes the relationship between the literary productions of the early twentieth century, with the rise of neo-realist current, and the literature produced by the Brazilian social group called Movimento do Atingidos por Barragens (MAB as a manifestation of resistance to elitist culture in terms the space of artistic creations. To this aim, I discuss the characteristic of neorealism as the defining literary expression of social and political represented in the works of the time aspects. In addition, I analyze the concept of resistance as a determinant for defining the literature of marginalized campesino movement (MAB. Finally, the following research confirms the importance of literature and the arts produced in excess of the usual canons and universities. Keywords: Literature of Resistance, Neorealism, Movimento dos Atingidos por Barragens (MAB.

Monoclonal antibodies (MAb) made against insect-derived metacyclic trypomastigotes (IMT) of Trypanosoma cruzi (TC) cross-react with other parasite forms

International Nuclear Information System (INIS)

Kirchhoff, L.V.; Gilliam, F.C.

1986-01-01

Considerable information has been generated in recent years about stage-specific surface membrane antigens of a number of protozoa, and this phenomenon has been observed among several stages of TC as well. However, little is known about the surface antigens of IMT, the true infective stage of TC, because of the difficulty of obtaining sufficient numbers of these organisms for analysis. The Tulahuen strain of TC was maintained in the reduviid vector Dipetalogaster maximus by repeated feeding on mice with high parasitemias. IMT collected with insect urine were irradiated (150 krad) and used to immunize a BALB/c mouse for hybridoma production. Supernatants were screened by immunofluorescence assay for the presence of IgG MAb that react with methanol-fixed IMT, epimastogotes (EPI) and culture-derived metacyclic trypomastigoes (CMT). Of 41 MAb obtained, 40 reacted with IMT, 37 with EPI and 38 with CMT. Four MAb immunoprecipitated radioiodinated proteins or protein conjugates of M/sub r/ 80, 72, 45 and 45 from lysates of 125 I surface-labeled EPI. These results indicate that, at least at the epitopic level, there is considerable overlap among IMT, EPI and CMT surface antigens. This finding suggests that analysis of surface proteins of the latter 2 parasite forms may lead to identification of molecules useful for vaccine development

IL-2/anti-IL-2 mAb immunocomplexes: A renascence of IL-2 in cancer immunotherapy?

Czech Academy of Sciences Publication Activity Database

Tomala, Jakub; Kovář, Marek

2016-01-01

Roč. 5, č. 3 (2016), e1102829 ISSN 2162-402X R&D Projects: GA ČR GA13-12885S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Anti-IL-2 mAb * cancer immunotherapy * IL-2 Subject RIV: EE - Microbiology, Virology Impact factor: 7.719, year: 2016

Differential expression of Meis2, Mab21l2 and Tbx3 during limb development associated with diversification of limb morphology in mammals.

Science.gov (United States)

Dai, Mengyao; Wang, Yao; Fang, Lu; Irwin, David M; Zhu, Tengteng; Zhang, Junpeng; Zhang, Shuyi; Wang, Zhe

2014-01-01

Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5' end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.

Antibodies recognizing both IgM isotypes in Atlantic salmon

DEFF Research Database (Denmark)

Hedfors, Ida Aagård; Bakke, Hege; Skjødt, Karsten

2012-01-01

these molecules. The present study aimed at identifying tools to separate IgM positive (IgM(+)) B cells from IgM negative (IgM(-)) non-B cell populations using flow cytometry. Several monoclonal antibodies (mAbs), and one polyclonal antibody (pAb) to both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon...... (Salmo salar) IgM, either commercially available or locally produced were tested for their recognition of Atlantic salmon IgM(+) cells. Leukocytes were isolated from peripheral blood (PB), spleen (S) and head kidney (HK) and stained with all mAbs and the pAb, to possibly verify the approximate number...... of IgM(+) cells in the respective tissues in salmon. To our surprise, this seemingly simple task did not reveal similar staining patterns for all antibodies as expected, but rather large differences in the number of positively stained cells were discovered. In short, positively stained cells by each...

Comparison between anti-CEA and anti-HER2 212Pb-labeled mAbs during α-RIT of small volume peritoneal carcinomatosis - Role of activity distribution on therapeutic efficacy and toxicity?

International Nuclear Information System (INIS)

Elgqvist, J.; Boudousq, V.; Bobyk, L.; Busson, M.; Lozza, C.; Navarro-Teulon, I.; Pouget, J.P.; Maquaire, P.; Torgue, J.

2015-01-01

Full text of publication follows. Objectives: we investigated the role of internalizing/non-internalizing monoclonal antibodies (mAbs) on the final outcome (efficacy/toxicity) of mice treated with alpha radioimmunotherapy (α- RIT) using 212 Pb-labeled mAbs. The relationship between distribution of radioactivity at the tissue level and biological parameters was also assessed. Methods: nude mice bearing 2-3 mm peritoneal nodules obtained by xenograft of A-431 tumor cells, expressing low and high level of HER2 and CEA receptors, respectively, were intraperitoneally (i.p.) injected with increasing activities (370-1480 kBq; 37 MBq/mg) of either 35A7 (non-internalising anti-CEA), Trastuzumab (internalizing anti-HER2) or PX (non-specific) 212 Pb-labeled mAbs. Control groups were injected with corresponding amount of unlabeled mAbs or with NaCl. Tumor growth was followed by bioluminescence and median survival (MS) of control and treated mice was determined. 212 Pb-35A7 and 212 Pb-Trastuzumab biodistribution was used to determine the cumulative uptake of radioactivity (UOR) in organs and tumors. Mean absorbed doses were calculated using the MIRD formalism. Haematological, liver and kidney toxicities were also assessed. Distribution of radioactivity at the tissue level was determined by digital micro-autoradiography and the relationship with biological markers of tissue damage was investigated using immunohistochemistry. Results: a mild and transient haematological toxicity in groups treated with the highest amount of activity was observed. MS of the groups treated either with internalizing or non-internalizing 212 Pb-labeled mAbs was significantly improved compared to those treated with non-specific 212 Pb-PX or those only given unlabeled mAbs or just NaCl. MS ranged from 42 days to 94 days using various activity levels of anti-CEA 212 Pb-35A7 while MS was not reached over the follow-up period of 130 days for mice treated with anti-HER2 212 Pb-Trastuzumab. However, UOR and

Monoclonal antibodies for the identification and purification of vNAR domains and IgNAR immunoglobulins from the horn shark Heterodontus francisci.

Science.gov (United States)

Juarez, Karla; Dubberke, Gudrun; Lugo, Pavel; Koch-Nolte, Friedrich; Buck, Friedrich; Haag, Friedrich; Licea, Alexei

2011-08-01

In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.

Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

Science.gov (United States)

Valliere-Douglass, John F; Lewis, Patsy; Salas-Solano, Oscar; Jiang, Shan

2015-02-01

We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling.

Science.gov (United States)

Grom, Matic; Kozorog, Mirijam; Caserman, Simon; Pohar, Andrej; Likozar, Blaž

2018-04-15

Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes. Copyright © 2018 Elsevier B.V. All rights reserved.

Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by SN1 (indirect-acting) and SN2 (direct-acting) alkylating agents: breaking a "salt-link".

Science.gov (United States)

Oh, H K; Teo, A K; Ali, R B; Lim, A; Ayi, T C; Yarosh, D B; Li, B F

1996-09-24

Human O6-methylguanine-DNA methyltransferase (MGMT) repairs DNA by transferring alkyl (R-) adducts from O6-alkylguanine (6RG) in DNA to its own cysteine residue at codon 145 (formation of R-MGMT). We show here that R-MGMT in cell extracts, which is sensitive to protease V8 cleavage at the glutamic acid residues at codons 30 (E30) and 172 (E172), can be specifically immunoprecipitated with an MGMT monoclonal antibody, Mab.3C7. This Mab recognizes an epitope of human MGMT including the lysine 107 (K107) which is within the most basic region that is highly conserved among mammalian MGMTs. Surprisingly, the K107L mutant protein is repair-deficient and readily cleaved by protease V8 similar to R-MGMT. We propose that R-MGMT adopted an altered conformation which exposed the Mab.3C7 epitope and rendered that protein sensitive to protease V8 attack. This proposal could be explained by the disruption of a structural "salt-link" within the molecule based on the available structural and biochemical data. The specific binding of Mab.3C7 to R-MGMT has been compared with the protease V8 method in the detection of R-MGMT in extracts of cells treated with low dosages of methyliodide (SN2) and O6-benzylguanine. Their identical behaviors in producing protease V8 sensitive R-MGMT and Mab.3C7 immunoprecipitates suggest that probably methyl iodide (an ineffective agent in producing 6RG in DNA) can directly alkylate the active site of cellular MGMT similar to O6-benzylguanine. The effectiveness of MeI in producing R-MGMT, i.e., inactivation of cellular MGMT, indicates that this agent can increase the effectiveness of environmental and endogenously produced alkylating carcinogens in producing the mutagenic O6-alkylguanine residues in DNA in vivo.

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

Science.gov (United States)

Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

2002-10-01

We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

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Floyd Hassenrück

2018-01-01

Full Text Available The antibody-dependent cell-mediated cytotoxicity (ADCC of the anti-CD20 monoclonal antibodies (mAbs rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.

Polynomic nonlinear dynamical systems - A residual sensitivity method for model reduction

Science.gov (United States)

Yurkovich, S.; Bugajski, D.; Sain, M.

1985-01-01

The motivation for using polynomic combinations of system states and inputs to model nonlinear dynamics systems is founded upon the classical theories of analysis and function representation. A feature of such representations is the need to make available all possible monomials in these variables, up to the degree specified, so as to provide for the description of widely varying functions within a broad class. For a particular application, however, certain monomials may be quite superfluous. This paper examines the possibility of removing monomials from the model in accordance with the level of sensitivity displayed by the residuals to their absence. Critical in these studies is the effect of system input excitation, and the effect of discarding monomial terms, upon the model parameter set. Therefore, model reduction is approached iteratively, with inputs redesigned at each iteration to ensure sufficient excitation of remaining monomials for parameter approximation. Examples are reported to illustrate the performance of such model reduction approaches.

Myeloablative radioimmunotherapy with {sup 188}Re-CD66mAb before stem cell transplantation. No increase of proinflammatory cytokine levels of TNF-{alpha}; Myeloablative Radioimmuntherapie mit {sup 188}Re-CD66mAb vor Stammzelltransplantation. Kein Anstieg proinflammatorischer Zytokinspiegel von TNF-{alpha}

Energy Technology Data Exchange (ETDEWEB)

Mutschler, J.; Reske, S.N. [Universitaetsklinik Ulm (Germany). Klinik fuer Nuklearmedizin; Steinbach, G. [Universitaetsklinik Ulm (Germany). Abt. Klinische Chemie; Bunjes, D. [Universitaetsklinik Ulm (Germany). Medizinische Klinik III; Buchmann, I. [Universitaetsklinik Heidelberg (Germany). Abt. fuer Nuklearmedizin

2009-07-01

Tumour necrosis factor-{alpha} (TNF-{alpha}) serum levels may increase due to intensive conditioning regimes with high-dose chemotherapy and total body irradiation (TBI) before stem cell transplantation. This increases the risk for developing acute graft versus host disease (aGvHD) after stem cell transplantation. In this prospective study we investigated the influence of radioimmunotherapy with {sup 188}Re-CD-66-mAb on changes on TNF-{alpha} serum levels. Patients, methods: In 18 patients we measured TNF-{alpha} before and up to 96 hours after radioimmunotherapy, in 2 patients in addition following TBI, in 9 patients also following chemotherapy. For measuring TNF-{alpha} we used an automated immunochemiluminescence assay (Immulite 1000 DPC Biermann, Bad Nauheim). The mean follow up period to record incidence of aGVHD was 100 days after stem cell transplantation. Compared to the basal levels before, the levels of TNF-{alpha} after conditioning with {sup 188}Re-CD-66-mAb did not increase significantly and remained in the physiological range. In contrast, these initial physiological cytokine levels increased and became pathological following 48 h after total body irradiation (13.2 {+-} 6.6 pg/ml) and chemotherapy (10.8 {+-} 15.7 pg/ml). In our study we found a low incidence of aGvHD (22.2%, n = 4/18). Conclusion: These results demonstrate that additional conditioning therapy with {sup 188}Re-CD-66-mAb does not increase proinflammatory cytokine levels of TNF-{alpha}. This finding may indicate that additive radioimmunotherapy may not be a significant factor for increasing the rate of conditioning- associated aGvHD. (orig.)

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system

International Nuclear Information System (INIS)

Kuroki, Masahide; Arakawa, Fumiko; Matsunaga, Akira; Okamoto, Naomi; Takakura, Kyoko; Matsuoka, Yuji; Higuchi, Hiroshi.

1987-01-01

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radio-immunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels. (author)

Effects of anti-CD40 mAb on inducing malignant B cells proliferation arrest and apoptosis and its mechanism

International Nuclear Information System (INIS)

Tang Lin; Zhuang Yumei; Zhou Zhaohua; Yu Gehua; Pan Jianzhong; Zhang Xueguang

2002-01-01

Objective: To study the expression of CD 40 molecule and the biological effects mediated by CD 40 molecules on malignant B cells. Methods: Agonistic anti-human CD 40 monoclonal antibody (clone 5C11) was added to cell culture system. Cell counting, PI staining, Annexin-V staining and flow cytometric analysis were used to study the behavior of malignant B cell lines after treatment with mAb clone 5C11. Results: 5C11 induced homotypic aggregation and proliferation arrest and mediated apoptosis in multiple myeloma cell line XG2 that expressed CD 40 strongly; 5C11 induced B lymphoma cell line Daudi homotypic aggregation and proliferation arrest and apoptosis, the apoptosis of XG2 and Daudi by CD40 activation was not mediated by TNF. Conclusion: Agonistic anti-CD 40 mAb 5C11 can inhibit the proliferation of malignant B cells by inducing them to die apoplectically

Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin

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Sultana Akter

2017-09-01

Full Text Available Nodularin (NOD is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E,6(E-dienoic acid monoclonal antibody (Mab. One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR. It was expressed in Escherichia coli as a single chain antibody fragment (scFv fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

Science.gov (United States)

Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

2016-06-01

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monoclonal antibody identification of subpopulations of cerebral cortical neurons affected in Alzheimer's disease

International Nuclear Information System (INIS)

Miller, C.A.; Rudnicka, M.; Hinton, D.R.; Blanks, J.C.; Kozlowski, M.

1987-01-01

Neuronal degeneration is one of the hallmarks of Alzheimer's disease (AD). Given the paucity of molecular markers available for the identification of neuronal subtypes, the specificity of neuronal loss within the cerebral cortex has been difficult to evaluate. With a panel of four monoclonal antibodies (mAbs) applied to central nervous system tissues from AD patients, the authors have immunocytochemically identified a population of vulnerable cortical neurons; a subpopulation of pyramidal neurons is recognized by mAbs 3F12 and 44.1 in the hippocampus and neocortex, and clusters of multipolar neurons in the entorhinal cortex reactive with mAb 44.1 show selective degeneration. Closely adjacent stellate-like neurons in these regions, identified by mAb 6A2, show striking preservation in AD. The neurons recognized by mAbs 3F12 and 44.1 do not comprise a single known neurotransmitter system. mAb 3A4 identifies a phosphorylated antigen that is undetectable in normal brain but accumulates early in the course of AD in somas of vulnerable neurons. Antigen 3A4 is distinct from material reactive with thioflavin S or antibody generated against paired helical filaments. Initially, antigen 3A4 is localized to neurons in the entorhinal cortex and subiculum, later in the association neocortex, and, ultimately in cases of long duration, in primary sensory cortical regions. mAb 3F12 recognizes multiple bands of immunoblots of homogenates of normal and AD cortical tissues, whereas mAb 3A4 does not bind to immunoblots containing neurofilament proteins or brain homogenates from AD patients. Ultrastructurally, antigen 3A4 is localized to paired-helical filaments. Using these mAbs, further molecular characterization of the affected cortical neurons is now possible

Recognizing Facial Slivers.

Science.gov (United States)

Gilad-Gutnick, Sharon; Harmatz, Elia Samuel; Tsourides, Kleovoulos; Yovel, Galit; Sinha, Pawan

2018-07-01

We report here an unexpectedly robust ability of healthy human individuals ( n = 40) to recognize extremely distorted needle-like facial images, challenging the well-entrenched notion that veridical spatial configuration is necessary for extracting facial identity. In face identification tasks of parametrically compressed internal and external features, we found that the sum of performances on each cue falls significantly short of performance on full faces, despite the equal visual information available from both measures (with full faces essentially being a superposition of internal and external features). We hypothesize that this large deficit stems from the use of positional information about how the internal features are positioned relative to the external features. To test this, we systematically changed the relations between internal and external features and found preferential encoding of vertical but not horizontal spatial relationships in facial representations ( n = 20). Finally, we employ magnetoencephalography imaging ( n = 20) to demonstrate a close mapping between the behavioral psychometric curve and the amplitude of the M250 face familiarity, but not M170 face-sensitive evoked response field component, providing evidence that the M250 can be modulated by faces that are perceptually identifiable, irrespective of extreme distortions to the face's veridical configuration. We theorize that the tolerance to compressive distortions has evolved from the need to recognize faces across varying viewpoints. Our findings help clarify the important, but poorly defined, concept of facial configuration and also enable an association between behavioral performance and previously reported neural correlates of face perception.

Prevention and reversal of experimental autoimmune thyroiditis (EAT) in mice by administration of anti-L3T4 monoclonal antibody at different stages of disease development.

Science.gov (United States)

Stull, S J; Kyriakos, M; Sharp, G C; Braley-Mullen, H

1988-11-01

Experimental autoimmune thyroiditis (EAT) can be induced in CBA/J mice following the transfer of spleen cells from mouse thyroglobulin (MTg)-sensitized donors that have been activated in vitro with MTg. Since L3T4+ T cells are required to transfer EAT in this model, the present study was undertaken to assess the effectiveness of the anti-L3T4 monoclonal antibody (mAb) GK1.5 in preventing or arresting the development of EAT. Spleen cells from mice given mAb GK1.5 prior to sensitization with MTg and adjuvant could not transfer EAT to normal recipients and cells from these mice did not proliferate in vitro to MTg. Donor mice given GK1.5 before immunization did not develop anti-MTg autoantibody and recipients of cells from such mice also produced little anti-MTg. GK1.5 could also prevent the proliferation and activation of sensitized effector cell precursors when added to in vitro cultures. When a single injection of mAb GK1.5 was given to recipients of in vitro-activated spleen cells, EAT was reduced whether the mAb was given prior to cell transfer or as late as 19 days after cell transfer. Whereas the incidence and severity of EAT was consistently reduced by injecting recipient mice with GK1.5, the same mice generally had no reduction in anti-MTg autoantibody. Since EAT is consistently induced in control recipients by 14-19 days after cell transfer, the ability of mAb GK1.5 to inhibit EAT when injected 14 or 19 days after cell transfer indicates that a single injection of the mAb GK1.5 can cause reversal of the histopathologic lesions of EAT in mice. These studies further establish the important role of L3T4+ T cells in the pathogenesis of EAT in mice and also suggest that therapy with an appropriate mAb may be an effective treatment for certain autoimmune diseases even when the therapy is initiated late in the course of the disease.

Aeroallergen analyses and their clinical relevance. I. Immunochemical quantification of allergens by RAST-inhibition, Mab-ELISA, basophil histamine release, and counter current immuno electrophoresis

DEFF Research Database (Denmark)

Johnsen, C R; Abrahamsen, L; Stahl Skov, P

1991-01-01

The aim was to compare IgE and IgG4 RAST-inhibition assay (RI), monoclonal antibody ELISA (Mab-ELISA), counter current immuno electrophoresis (CCIE) and histamine release from basophil leukocytes (HR) for allergen quantification with special reference to aeroallergen detection. As components......-U/ml) than IgE-RI (2*10(3) SQ-U/ml). The ranges of allergen detection limits for the Mab-ELISA were equal for cat and Derm. pter. (10-10(2) SQ-U/ml). The range of allergen detection limits for CCIE, assaying dog were 10(4)-10(5) SQ-U/ml. The ranges of allergen detection limits for HR were equal for cat...

Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

International Nuclear Information System (INIS)

Gromowski, Gregory D.; Barrett, Alan D.T.

2007-01-01

The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity (∼ 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant

Upconverting nanophosphors as reporters in a highly sensitive heterogeneous immunoassay for cardiac troponin I

Energy Technology Data Exchange (ETDEWEB)

Sirkka, Nina, E-mail: nkelon@utu.fi; Lyytikäinen, Annika; Savukoski, Tanja; Soukka, Tero

2016-06-21

Photon upconverting nanophosphors (UCNPs) have a unique capability to produce anti-Stokes emission at visible wavelengths via sequential multiphoton absorption upon infrared excitation. Since the anti-Stokes emission can be easily spectrally resolved from the Stokes' shifted autofluorescence, the upconversion luminescence (UCL) is a highly attractive reporter technology for optical biosensors and biomolecular binding assays – potentially enabling unprecedented sensitivity in separation-based solid-phase immunoassays. UCL technology has not previously been applied in sensitive detection of cardiac troponin I (cTnI), which requires highly sensitive detection to enable accurate and timely diagnosis of myocardial infarction. We have developed an UCL-based immunoassay for cTnI using NaYF{sub 4}: Yb{sup 3+}, Er{sup 3+} UCNPs as reporters. Biotinylated anti-cTnI monoclonal antibody (Mab) and Fab fragment immobilized to streptavidin-coated wells were used to capture cTnI. Captured cTnI was detected from dry well surface after a 15 min incubation with poly(acrylic acid) coated UCNPs conjugated to second anti-cTnI Mab. UCL was measured with a dedicated UCL microplate reader. The UCL-based immunoassay allowed sensitive detection of cTnI. The limit of detection was 3.14 ng L{sup −1}. The calibration curve was linear up to cTnI concentration 50,000 ng L{sup −1}. Plasma recoveries of added cTnI were 92–117%. Obtained cTnI concentrations from five normal plasma samples were 4.13–10.7 ng L{sup −1} (median 5.06 ng L{sup −1}). There is yet significant potential for even further improved limit of detection by reducing non-specifically bound fraction of the Mab-conjugated UCNPs. The assay background with zero calibrator was over 40-fold compared to the background obtained from wells where the reporter conjugate had been excluded. - Highlights: • Detection of attomole analyte quantity in microwell using upconversion luminescence. • Upconverting

Evaluating two model reduction approaches for large scale hedonic models sensitive to omitted variables and multicollinearity

DEFF Research Database (Denmark)

Panduro, Toke Emil; Thorsen, Bo Jellesmark

2014-01-01

Hedonic models in environmental valuation studies have grown in terms of number of transactions and number of explanatory variables. We focus on the practical challenge of model reduction, when aiming for reliable parsimonious models, sensitive to omitted variable bias and multicollinearity. We...

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

Science.gov (United States)

A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.

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Tetsuji Kamata

Full Text Available Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.

Does exercise aid smoking cessation through reductions in anxiety sensitivity and dysphoria?

Science.gov (United States)

Zvolensky, Michael J; Rosenfield, David; Garey, Lorra; Kauffman, Brooke Y; Langdon, Kirsten J; Powers, Mark B; Otto, Michael W; Davis, Michelle L; Marcus, Bess H; Church, Timothy S; Frierson, Georita M; Hopkins, Lindsey B; Paulus, Daniel J; Baird, Scarlett O; Smits, Jasper A J

2018-07-01

Research shows that high anxiety sensitivity (AS) and dysphoria are related to poor smoking cessation outcomes. Engaging in exercise may contribute to improvement in smoking cessation outcomes through reductions in AS and dysphoria. In the current study, we examined whether exercise can aid smoking cessation through reductions in AS and dysphoria. Participants were sedentary and low activity adult daily smokers (N = 136) with elevated AS who participated in a randomized controlled trial comparing smoking cessation treatment (ST) plus an exercise intervention (ST + EX) to ST plus wellness education (ST + CTRL). Self-reported smoking status was assessed in-person weekly from baseline through week 16 (end of-treatment; EOT), at week 22 (4 months postquit day), and at week 30 (6 months postquit day), and verified biochemically. Results indicated that both AS and dysphoria at 6-month follow-up were significantly lower in the ST + EX group compared to the ST + CTRL group (controlling for baseline levels). Moreover, reductions in AS and dysphoria emerged as independent mechanisms of action explaining success in quitting. These novel findings offer clinically significant evidence suggesting that vigorous-intensity exercise can effectively engage affective constructs in the context of smoking cessation. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Depletion GPT-free sensitivity analysis for reactor eigenvalue problems

International Nuclear Information System (INIS)

Kennedy, C.; Abdel-Khalik, H.

2013-01-01

This manuscript introduces a novel approach to solving depletion perturbation theory problems without the need to set up or solve the generalized perturbation theory (GPT) equations. The approach, hereinafter denoted generalized perturbation theory free (GPT-Free), constructs a reduced order model (ROM) using methods based in perturbation theory and computes response sensitivity profiles in a manner that is independent of the number or type of responses, allowing for an efficient computation of sensitivities when many responses are required. Moreover, the reduction error from using the ROM is quantified in the GPT-Free approach by means of a Wilks' order statistics error metric denoted the K-metric. Traditional GPT has been recognized as the most computationally efficient approach for performing sensitivity analyses of models with many input parameters, e.g. when forward sensitivity analyses are computationally intractable. However, most neutronics codes that can solve the fundamental (homogenous) adjoint eigenvalue problem do not have GPT capabilities unless envisioned during code development. The GPT-Free approach addresses this limitation by requiring only the ability to compute the fundamental adjoint. This manuscript demonstrates the GPT-Free approach for depletion reactor calculations performed in SCALE6 using the 7x7 UAM assembly model. A ROM is developed for the assembly over a time horizon of 990 days. The approach both calculates the reduction error over the lifetime of the simulation using the K-metric and benchmarks the obtained sensitivities using sample calculations. (authors)

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

Science.gov (United States)

Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

2015-09-09

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

Generation and Application of Monoclonal Antibody Against Lycopene.

Science.gov (United States)

Tsibezov, Valeriy V; Bashmakov, Yuriy K; Pristenskiy, Dmitry V; Zigangirova, Naylia A; Kostina, Ludmila V; Chalyk, Natalya E; Kozlov, Alexey Y; Morgunova, Elena Y; Chernyshova, Marina P; Lozbiakova, Marina V; Kyle, Nigel H; Petyaev, Ivan M

2017-04-01

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

Reduction of pain sensitivity after somatosensory therapy in adults with cerebral palsy

Directory of Open Access Journals (Sweden)

Inmaculada eRiquelme

2013-06-01

Full Text Available Objective. Pain and deficits in somatosensory processing seem to play a relevant role in cerebral palsy (CP. Rehabilitation techniques based on neuroplasticity mechanisms may induce powerful changes in the organization of the primary somatosensory cortex and have been proved to reduce levels of pain and discomfort in neurological pathologies. However, little is known about the efficacy of such interventions for pain sensitivity in CP individuals. Methods. Adults with cerebral palsy participated in the study and were randomly assigned to the intervention (n=17 or the control group (n=20. The intervention group received a somatosensory therapy including 4 types of exercises (touch, proprioception, vibration, and stereognosis. All participants were asked to continue their standardized motor therapy during the study period. Several somatosensory (pain and touch thresholds, stereognosis, propioception, texture recognition and motor parameters (fine motor skills were assessed before, immediately after and three months after the therapy (follow-up. Results. Participants of the intervention group showed a significant reduction on pain sensitivity after treatment and at follow-up after three months, whereas participants in the control group displayed increasing pain sensitivity over time. No improvements were found on touch sensitivity, proprioception, texture recognition or fine motor skills. Conclusions. Data suggest the possibility that somatosensory therapy was effective in eliciting changes in central somatosensory processing. This hypothesis may have implications for future neuromodulatory treatment of pain complaints in children and adults with cerebral palsy.

Reduction and Uncertainty Analysis of Chemical Mechanisms Based on Local and Global Sensitivities

Science.gov (United States)

Esposito, Gaetano

Numerical simulations of critical reacting flow phenomena in hypersonic propulsion devices require accurate representation of finite-rate chemical kinetics. The chemical kinetic models available for hydrocarbon fuel combustion are rather large, involving hundreds of species and thousands of reactions. As a consequence, they cannot be used in multi-dimensional computational fluid dynamic calculations in the foreseeable future due to the prohibitive computational cost. In addition to the computational difficulties, it is also known that some fundamental chemical kinetic parameters of detailed models have significant level of uncertainty due to limited experimental data available and to poor understanding of interactions among kinetic parameters. In the present investigation, local and global sensitivity analysis techniques are employed to develop a systematic approach of reducing and analyzing detailed chemical kinetic models. Unlike previous studies in which skeletal model reduction was based on the separate analysis of simple cases, in this work a novel strategy based on Principal Component Analysis of local sensitivity values is presented. This new approach is capable of simultaneously taking into account all the relevant canonical combustion configurations over different composition, temperature and pressure conditions. Moreover, the procedure developed in this work represents the first documented inclusion of non-premixed extinction phenomena, which is of great relevance in hypersonic combustors, in an automated reduction algorithm. The application of the skeletal reduction to a detailed kinetic model consisting of 111 species in 784 reactions is demonstrated. The resulting reduced skeletal model of 37--38 species showed that the global ignition/propagation/extinction phenomena of ethylene-air mixtures can be predicted within an accuracy of 2% of the full detailed model. The problems of both understanding non-linear interactions between kinetic parameters and

Monoclonal antibodies specific to heat-treated porcine blood.

Science.gov (United States)

Raja Nhari, Raja Mohd Hafidz; Hamid, Muhajir; Rasli, Nurmunirah Mohamad; Omar, Abdul Rahman; El Sheikha, Aly Farag; Mustafa, Shuhaimi

2016-05-01

Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Chimpanzee-Human Monoclonal Antibodies for Treatment of Chronic Poliovirus Excretors and Emergency Postexposure Prophylaxis▿‡

Science.gov (United States)

Chen, Zhaochun; Chumakov, Konstantin; Dragunsky, Eugenia; Kouiavskaia, Diana; Makiya, Michelle; Neverov, Alexander; Rezapkin, Gennady; Sebrell, Andrew; Purcell, Robert

2011-01-01

Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case. PMID:21345966

Measuring value sensitivity in medicine.

Science.gov (United States)

Ineichen, Christian; Christen, Markus; Tanner, Carmen

2017-01-28

Value sensitivity - the ability to recognize value-related issues when they arise in practice - is an indispensable competence for medical practitioners to enter decision-making processes related to ethical questions. However, the psychological competence of value sensitivity is seldom an explicit subject in the training of medical professionals. In this contribution, we outline the traditional concept of moral sensitivity in medicine and its revised form conceptualized as value sensitivity and we propose an instrument that measures value sensitivity. We developed an instrument for assessing the sensitivity for three value groups (moral-related values, values related to the principles of biomedical ethics, strategy-related values) in a four step procedure: 1) value identification (n = 317); 2) value representation (n = 317); 3) vignette construction and quality evaluation (n = 37); and 4) instrument validation by comparing nursing professionals with hospital managers (n = 48). We find that nursing professionals recognize and ascribe importance to principle-related issues more than professionals from hospital management. The latter are more likely to recognize and ascribe importance to strategy-related issues. These hypothesis-driven results demonstrate the discriminatory power of our newly developed instrument, which makes it useful not only for health care professionals in practice but for students and people working in the clinical context as well.

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

Science.gov (United States)

Weins, G.; Chien, M.S.; Winton, J.R.; Kaatari, S.L.

1999-01-01

Renibacterium salmoninarum, the causative agent of bacterial kidney disease, produces large quantities of a 57-58 kDa protein (p57) during growth in broth culture and during infection of salmonid fish. Biological activities of secreted p57 include agglutination of salrnonid leucocytes and rabbit erythrocytes. We define the location of epitopes on p57 recognized by agglutination-blocking monoclonal antibodies (MAbs) 4Cl1, 4H8 and 4D3, and demonstrate that the majority of secreted p57 is a nlonomer that retains salrnonid leucocyte agglutinat~ng activity. The 3 MAbs bound a recombinant, amino-terminal fragment of p57 (211 aa) but not a carboxy-terminal fragment (315 aa) demonstrating that the neutralizing epitopes are located within the amino-terminal portion of p57. When combinations of the MAbs were used in an antigen capture ELISA. the epitopes recognized by the 3 MAbs were shown to be sterically separate. However, when the same MAb was used as both the coating and detection MAb, binding of the biotinylated detection MAb was not observed. These data indicate that the epitopes recognized by the 3 agglutination-blocking antibodies are functionally available only once per molecule and that native p57 exists as a monomer Similar ELISA results were obtained when kidney tissues from 3 naturally infected chinook salmon were assayed. Finally, a p57 monomer was purified using anion exchange and size exclusion chromatography that retained in vitro agglutinating activity. A model in which p57 is released from R. salmoninarum as a biologically active monomer during infection of salmonid fish is proposed.

The shape of change in perceived stress, negative affect, and stress sensitivity during mindfulness based stress reduction

NARCIS (Netherlands)

Snippe, E.; Dziak, J.J.; Lanza, S.T.; Nyklicek, I.; Wichers, M.

2017-01-01

Both daily stress and the tendency to react to stress with heightened levels of negative affect (i.e., stress sensitivity) are important vulnerability factors for adverse mental health outcomes. Mindfulness-based stress reduction (MBSR) may help to reduce perceived daily stress and stress

The Shape of Change in Perceived Stress, Negative Affect, and Stress Sensitivity During Mindfulness-Based Stress Reduction

NARCIS (Netherlands)

Snippe, Evelien; Dziak, John J.; Lanza, Stephanie T.; Nykliek, Ivan; Wichers, Marieke

Both daily stress and the tendency to react to stress with heightened levels of negative affect (i.e., stress sensitivity) are important vulnerability factors for adverse mental health outcomes. Mindfulness-based stress reduction (MBSR) may help to reduce perceived daily stress and stress

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

Science.gov (United States)

Itai, Shunsuke; Fujii, Yuki; Nakamura, Takuro; Chang, Yao-Wen; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Suzuki, Hiroyoshi; Harada, Hiroyuki; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-10-01

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG 2a , kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

Science.gov (United States)

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

2013-12-01

Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

Myeloablative radioimmunotherapy with 188Re-CD66mAb before stem cell transplantation. No increase of proinflammatory cytokine levels of TNF-α

International Nuclear Information System (INIS)

Mutschler, J.; Reske, S.N.; Steinbach, G.; Bunjes, D.; Buchmann, I.

2009-01-01

Tumour necrosis factor-α (TNF-α) serum levels may increase due to intensive conditioning regimes with high-dose chemotherapy and total body irradiation (TBI) before stem cell transplantation. This increases the risk for developing acute graft versus host disease (aGvHD) after stem cell transplantation. In this prospective study we investigated the influence of radioimmunotherapy with 188 Re-CD-66-mAb on changes on TNF-α serum levels. Patients, methods: In 18 patients we measured TNF-α before and up to 96 hours after radioimmunotherapy, in 2 patients in addition following TBI, in 9 patients also following chemotherapy. For measuring TNF-α we used an automated immunochemiluminescence assay (Immulite 1000 DPC Biermann, Bad Nauheim). The mean follow up period to record incidence of aGVHD was 100 days after stem cell transplantation. Compared to the basal levels before, the levels of TNF-α after conditioning with 188 Re-CD-66-mAb did not increase significantly and remained in the physiological range. In contrast, these initial physiological cytokine levels increased and became pathological following 48 h after total body irradiation (13.2 ± 6.6 pg/ml) and chemotherapy (10.8 ± 15.7 pg/ml). In our study we found a low incidence of aGvHD (22.2%, n = 4/18). Conclusion: These results demonstrate that additional conditioning therapy with 188 Re-CD-66-mAb does not increase proinflammatory cytokine levels of TNF-α. This finding may indicate that additive radioimmunotherapy may not be a significant factor for increasing the rate of conditioning- associated aGvHD. (orig.)

Production, Characterization, and Epitope Mapping of Monoclonal Antibodies Against Different Subtypes of Rabbit Hemorrhagic Disease Virus (RHDV).

Science.gov (United States)

Kong, Desheng; Liu, Jiasen; Jiang, Qian; Yu, Zuo; Hu, Xiaoliang; Guo, Dongchun; Huang, Qianqian; Jiao, Meihui; Qu, Liandong

2016-02-16

In 2010, a new rabbit hemorrhagic disease virus (RHDV) variant, designated RHDV2, was identified for the first time in Italy. Studies have shown that RHDV2 differs from RHDV1 (traditional RHDV) in terms of its antigenic profile and genetic characteristics. The VP60 protein of RHDV is a structural protein that plays important roles in viral replication, assembly, and immunogenicity. In this study, we immunized BALB/c mice with recombinant VP60 proteins from different RHDV subtypes. After three rounds of subcloning, type-specific positive hybridoma clones of RHDV1 and RHDV2 were further identified by an enzyme-linked immunosorbent assay, Western blotting, and an indirect immunofluorescence assay. Finally, three monoclonal antibodies (MAbs) (1D6, 1H2, and 3F2) that only recognize RHDV1, and four MAbs (1G2, 2C1, 3B7, and 5D6) that only recognize RHDV2 were identified. The epitopes recognized by these MAbs were mapped by Western blotting. Sequence analysis showed that the epitope sequences recognized by 1D6, 1H2, and 3F2 are highly conserved (98%) among RHDV1 strains, whereas the epitope sequences recognized by 1G2, 2C1, 3B7, and 5D6 are 100% conserved among RHDV2 strains. The high conservation of the epitope sequence showed that the screened MAbs were type-specific, and that they could distinguish different RHDV subtypes.

Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

Science.gov (United States)

Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

2010-01-01

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

Recognizing emotions from EEG subbands using wavelet analysis.

Science.gov (United States)

Candra, Henry; Yuwono, Mitchell; Handojoseno, Ardi; Chai, Rifai; Su, Steven; Nguyen, Hung T

2015-01-01

Objectively recognizing emotions is a particularly important task to ensure that patients with emotional symptoms are given the appropriate treatments. The aim of this study was to develop an emotion recognition system using Electroencephalogram (EEG) signals to identify four emotions including happy, sad, angry, and relaxed. We approached this objective by firstly investigating the relevant EEG frequency band followed by deciding the appropriate feature extraction method. Two features were considered namely: 1. Wavelet Energy, and 2. Wavelet Entropy. EEG Channels reduction was then implemented to reduce the complexity of the features. The ground truth emotional states of each subject were inferred using Russel's circumplex model of emotion, that is, by mapping the subjectively reported degrees of valence (pleasure) and arousal to the appropriate emotions - for example, an emotion with high valence and high arousal is equivalent to a `happy' emotional state, while low valence and low arousal is equivalent to a `sad' emotional state. The Support Vector Machine (SVM) classifier was then used for mapping each feature vector into corresponding discrete emotions. The results presented in this study indicated thatWavelet features extracted from alpha, beta and gamma bands seem to provide the necessary information for describing the aforementioned emotions. Using the DEAP (Dataset for Emotion Analysis using electroencephalogram, Physiological and Video Signals), our proposed method achieved an average sensitivity and specificity of 77.4% ± 14.1% and 69.1% ± 12.8%, respectively.

A highly sensitive monoclonal antibody based biosensor for quantifying 3–5 ring polycyclic aromatic hydrocarbons (PAHs in aqueous environmental samples

Directory of Open Access Journals (Sweden)

Xin Li

2016-03-01

Full Text Available Immunoassays based on monoclonal antibodies (mAbs are highly sensitive for the detection of polycyclic aromatic hydrocarbons (PAHs and can be employed to determine concentrations in near real-time. A sensitive generic mAb against PAHs, named as 2G8, was developed by a three-step screening procedure. It exhibited nearly uniformly high sensitivity against 3-ring to 5-ring unsubstituted PAHs and their common environmental methylated PAHs, with IC50 values between 1.68 and 31 μg/L (ppb. 2G8 has been successfully applied on the KinExA Inline Biosensor system for quantifying 3–5 ring PAHs in aqueous environmental samples. PAHs were detected at a concentration as low as 0.2 μg/L. Furthermore, the analyses only required 10 min for each sample. To evaluate the accuracy of the 2G8-based biosensor, the total PAH concentrations in a series of environmental samples analyzed by biosensor and GC–MS were compared. In most cases, the results yielded a good correlation between methods. This indicates that generic antibody 2G8 based biosensor possesses significant promise for a low cost, rapid method for PAH determination in aqueous samples. Keywords: Monoclonal antibody, PAH, Pore water, Biosensor, Pyrene

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

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Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

2016-01-01

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

Science.gov (United States)

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid–polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment

Directory of Open Access Journals (Sweden)

Wu B

2017-03-01

Full Text Available Bo Wu,1,2,* Shu-Ting Lu,1,* Liu-Jie Zhang,2 Ren-Xi Zhuo,2 Hai-Bo Xu,1 Shi-Wen Huang2 1Department of Radiology, Zhongnan Hospital of Wuhan University, 2Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan, People’s Republic of China *These authors contributed equally to this work Abstract: Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid–polymer hybrid nanoparticles (LPNPs drug delivery system composed of monomethoxy-poly(ethylene glycol-S-S-hexadecyl (mPEG-S-S-C16, soybean lecithin, and poly(d,l-lactide-co-glycolide (PLGA was used for codelivery of doxorubicin (DOX and a Chinese herb extract triptolide (TPL. Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs exhibited a high level of synergistic activation with low combination index (CI in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells. Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment. Keywords: triptolide, codelivery, reduction sensitive, synergistic effect

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

DEFF Research Database (Denmark)

Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

2012-01-01

, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human m......Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

International Nuclear Information System (INIS)

Chou, Te-hui W.; Wang, Shixia; Sakhatskyy, Pavlo V.; Mboudoudjeck, Innocent; Lawrence, John M.; Huang Song; Coley, Scott; Yang Baoan; Li Jiaming; Zhu Qingyu; Lu Shan

2005-01-01

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV

Identification of two distinct allergenic sites on ryegrass-pollen allergen, Lol p IV.

Science.gov (United States)

Jaggi, K S; Ekramoddoullah, A K; Kisil, F T; Dzuba-Fischer, J M; Rector, E S; Sehon, A H

1989-04-01

Lol p IV is an important allergen of ryegrass pollen. For the immunochemical identification of antigenic and/or allergenic site(s), murine monoclonal antibodies (MAbs) were prepared against Lol p IV. The hybridoma cell-culture supernatants were screened for anti-Lol p IV antibodies by a combination of ELISA and Western immunoblot analyses. The MAbs were finally purified from ascites on a Mono Q ion-exchange column. In a competitive radioimmunoassay with Lol p IV as the solid phase and 125I-labeled MAbs, it was established that MAbs 90, 91, 92, 93, and 94, although they differed in their relative affinities, recognized in common with one another an epitope designated as antigenic site A, whereas MAb 12 recognized a different epitope referred to as site B. Sites A and B were also demonstrated to constitute allergenic determinants of Lol p IV. Differences in the repertoire of specificities of the human IgE antibodies directed to Lol p IV were also demonstrated. Interestingly, it was found that sera from both allergic as well as from nonatopic individuals had IgG antibodies to sites A and/or B.

Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

Science.gov (United States)

Burastero, Samuele E; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo

2011-01-01

To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

Broad-spectrum inhibition of HIV-1 by a monoclonal antibody directed against a gp120-induced epitope of CD4.

Directory of Open Access Journals (Sweden)

Samuele E Burastero

Full Text Available To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.

Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

International Nuclear Information System (INIS)

Zhao Hongwei; Xue Changguo; Nan Tiegui; Tan Guiyu; Li Zhaohu; Li, Qing X.; Zhang Qingchuan; Wang Baomin

2010-01-01

A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL -1 of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL -1 , respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

Science.gov (United States)

Lusso, Paolo; Earl, Patricia L; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A; Burastero, Samuele E

2005-06-01

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on

Proteins of bovine viral diarrhea virus: characterization, biotype-specific differences, and immunological properties

International Nuclear Information System (INIS)

Donis, R.O.

1987-01-01

Virus-specific polypeptides in bovine viral diarrhea-mucosal disease (BVD) virus-infected bovine cells were studied by radiolabeling. A total of 12 polypeptides with apparent Mr of 165, 135, 118, 80, 75, 62, 56-58, 48, 37, 32, 25 and 19 kilodaltons (k) were identified in infected cells. Five glycoproteins were detected in infected cells. Two abundant species had apparent Mr of 48 k and 56-58 k while the minor species had masses of 118, 75 and 65 k. When cells were radiolabeled with L-[ 35 S]-methionine in the presence of tunicamycin the 56-58 k migrated with apparent masses of 54 k and 48-50 K in PAGE. Endoglycosidase F digestion of virus-induced polypeptides caused a 4-6 K reduction in the apparent molecular mass of the 56-58 k yielding a 52 k digested product. Tunicamycin caused a drastic reduction in the yield of infectious virus indicating that the carbohydrate moieties serve a vital role in the infection cycle of BVD virus. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be consistently differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of unique polypeptide profiles they induce in the infected cell: the most abundant polypeptide in CB-BVD infected cells is the 80 kD polypeptide while NCB-BVD lack this polypeptide and induce a predominant 118 k polypeptide. A panel of 25 murine monoclonal antibodies (Mabs) against the two major glycoproteins of BVD virus was produced. Based on their viral polypeptide specificity and on their ability to neutralize viral infectivity the Mabs in the panel were divided into 3 classes: Class 1 Mabs reacted with the 56-58 k glycoprotein and neutralized the virus, Class 2 Mabs recognized the 56-58 k glycoprotein but were not neutralizing and Class 3 Mabs reacted with the 48 k glycoprotein and did not neutralize the virus. These results identify the 56-58 k as one of the envelope glycoproteins of BVD virus

Stability of monoclonal antibodies at high-concentration

DEFF Research Database (Denmark)

Neergaard, Martin S; Nielsen, Anders D; Parshad, Henrik

2014-01-01

Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG1 and one IgG4 ) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences betw...

Photodegradation of emamectin benzoate in aqueous solutions

International Nuclear Information System (INIS)

Mushtaq, M.; Chukwudebe, A.C.; Wrzesinski, C.; Allen, L.R.S.; Luffer-Atlas, D.; Arison, B.H.

1998-01-01

The half-life of [ 14 C]4'-deoxy-4'-(epi-methylamino)avermectin B1a (MAB1a) benzoate (1 ppm) photodegradation in buffer (pH 7), natural pond water, and sensitized buffer (1% acetone in pH 7 buffer) determined at Three Bridges, NJ (latitude approximately 40 degrees N) during the fall season under natural sunlight was 22, 7, and 1 days, respectively. The half-life of [ 14 C]MAB1a benzoate (10-12 ppm) photodegradation in buffer (pH 7) containing 1% (v/v) acetonitrile, ethanol, or acetone as cosolvent under continuous exposure with a xenon lamp was 64.5, 8.5, or 0.5 days, respectively. The photoisomer 8,9-Z-MAB1a, 8a-hydroxy-MAB1a, and unknown polar residues were found in light-exposed samples of MAB1a in buffer and natural pond water. In light-exposed sensitized buffer samples, 8a-oxo-MAB1a and MAB1a-10,11-14,15-diepoxide were additional products. Very polar residues found in the organic and aqueous phases after extraction increased with time, and their formation followed the order sensitized buffer natural pond water buffer. (author)

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid–polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment

Science.gov (United States)

Wu, Bo; Lu, Shu-Ting; Zhang, Liu-Jie; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

2017-01-01

Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid–polymer hybrid nanoparticles (LPNPs) drug delivery system composed of monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16), soybean lecithin, and poly(D,L-lactide-co-glycolide) (PLGA) was used for codelivery of doxorubicin (DOX) and a Chinese herb extract triptolide (TPL). Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs) exhibited a high level of synergistic activation with low combination index (CI) in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells). Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment. PMID:28331310

Codelivery of doxorubicin and triptolide with reduction-sensitive lipid-polymer hybrid nanoparticles for in vitro and in vivo synergistic cancer treatment.

Science.gov (United States)

Wu, Bo; Lu, Shu-Ting; Zhang, Liu-Jie; Zhuo, Ren-Xi; Xu, Hai-Bo; Huang, Shi-Wen

2017-01-01

Codelivery is a promising strategy to overcome the limitations of single chemotherapeutic agents in cancer treatment. Despite progress, codelivery of two or more different functional drugs to increase anticancer efficiency still remains a challenge. Here, reduction-sensitive lipid-polymer hybrid nanoparticles (LPNPs) drug delivery system composed of monomethoxy-poly(ethylene glycol)- S - S -hexadecyl (mPEG- S - S -C 16 ), soybean lecithin, and poly(D,L-lactide-co-glycolide) (PLGA) was used for codelivery of doxorubicin (DOX) and a Chinese herb extract triptolide (TPL). Hydrophobic DOX and TPL could be successfully loaded in LPNPs by self-assembly. More importantly, drug release and cellular uptake experiments demonstrated that the two drugs were reduction sensitive, released simultaneously from LPNPs, and taken up effectively by the tumor cells. DOX/TPL-coloaded LPNPs (DOX/TPL-LPNPs) exhibited a high level of synergistic activation with low combination index (CI) in vitro and in vivo. Moreover, the highest synergistic therapeutic effect was achieved at the ratio of 1:0.2 DOX/TPL. Further experiments showed that TPL enhanced the uptake of DOX by human oral cavity squamous cell carcinoma cells (KB cells). Overall, DOX/TPL-coencapsulated reduction-sensitive nanoparticles will be a promising strategy for cancer treatment.

ICE on the road to auditory sensitivity reduction and sound localization in the frog.

Science.gov (United States)

Narins, Peter M

2016-10-01

Frogs and toads are capable of producing calls at potentially damaging levels that exceed 110 dB SPL at 50 cm. Most frog species have internally coupled ears (ICE) in which the tympanic membranes (TyMs) communicate directly via the large, permanently open Eustachian tubes, resulting in an inherently directional asymmetrical pressure-difference receiver. One active mechanism for auditory sensitivity reduction involves the pressure increase during vocalization that distends the TyM, reducing its low-frequency airborne sound sensitivity. Moreover, if sounds generated by the vocal folds arrive at both surfaces of the TyM with nearly equal amplitudes and phases, the net motion of the eardrum would be greatly attenuated. Both of these processes appear to reduce the motion of the frog's TyM during vocalizations. The implications of ICE in amphibians with respect to sound localizations are discussed, and the particularly interesting case of frogs that use ultrasound for communication yet exhibit exquisitely small localization jump errors is brought to light.

Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection.

Science.gov (United States)

Wan, Shu-Wen; Chen, Pei-Wei; Chen, Chin-Yu; Lai, Yen-Chung; Chu, Ya-Ting; Hung, Chia-Yi; Lee, Han; Wu, Hsuan Franziska; Chuang, Yung-Chun; Lin, Jessica; Chang, Chih-Peng; Wang, Shuying; Liu, Ching-Chuan; Ho, Tzong-Shiann; Lin, Chiou-Feng; Lee, Chien-Kuo; Wu-Hsieh, Betty A; Anderson, Robert; Yeh, Trai-Ming; Lin, Yee-Shin

2017-10-15

Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease. Copyright © 2017 by The American Association of Immunologists, Inc.

Action Potential Broadening in Capsaicin-Sensitive DRG Neurons from Frequency-Dependent Reduction of Kv3 Current.

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Liu, Pin W; Blair, Nathaniel T; Bean, Bruce P

2017-10-04

Action potential (AP) shape is a key determinant of cellular electrophysiological behavior. We found that in small-diameter, capsaicin-sensitive dorsal root ganglia neurons corresponding to nociceptors (from rats of either sex), stimulation at frequencies as low as 1 Hz produced progressive broadening of the APs. Stimulation at 10 Hz for 3 s resulted in an increase in AP width by an average of 76 ± 7% at 22°C and by 38 ± 3% at 35°C. AP clamp experiments showed that spike broadening results from frequency-dependent reduction of potassium current during spike repolarization. The major current responsible for frequency-dependent reduction of overall spike-repolarizing potassium current was identified as Kv3 current by its sensitivity to low concentrations of 4-aminopyridine (IC 50 action potentials of small-diameter rat DRG neurons showed spike broadening at frequencies as low as 1 Hz and that spike broadening resulted predominantly from frequency-dependent inactivation of Kv3 channels. Spike width helps to control transmitter release, conduction velocity, and firing patterns and understanding the role of particular potassium channels can help to guide new pharmacological strategies for targeting pain-sensing neurons selectively. Copyright © 2017 the authors 0270-6474/17/379705-10$15.00/0.

Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

Science.gov (United States)

Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

2014-12-01

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of a conformational neutralizing epitope on the VP1 protein of type A foot-and-mouth disease virus.

Science.gov (United States)

Liu, Wenming; Yang, Baolin; Wang, Mingxia; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

2017-12-01

Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype A and protect against future pandemics. In this study, we generated a serotype A FMDV-specific potent neutralizing monoclonal antibody (MAb), 6C9, which recognizes a conformation-dependent epitope. MAb 6C9 potently neutralized FMDV A/XJBC/CHA/2010 with a 50% neutralization titer (NT 50 ) of 4096. Screening of a phage-displayed random 12-mer peptide library revealed that MAb 6C9 bound to phages displaying the consensus motif YxxPxGDLG, which is highly homologous to the 135 YxxPxxxxxGDLG 147 motif found in the serotype A FMDV virus-encoded structural protein VP1. To further verify the authentic epitope recognized by MAb 6C9, two FMDV A/XJBC/CHA/2010 mutant viruses, P138A and G144A, were generated using a reverse genetic system. Subsequent micro-neutralization assays and double-antibody sandwich (DAS) ELISA analyses revealed that the Pro 138 and Gly 144 residues of the conformational epitope that are recognized by 6C9 are important for MAb 6C9 binding. Importantly, the epitope 135 YxxPxxxxxGDLG 147 was highly conserved among different topotypes of serotype A FMDV strains in a sequence alignment analysis. Thus, the results of this study could have potential applications in the development of novel epitope-based vaccines and suitable a MAb-based diagnostic method for the detection of serotype A FMDV and the quantitation of antibodies against this serotype. Copyright © 2017 Elsevier Ltd. All rights reserved.

Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance

NARCIS (Netherlands)

Lasonder, E; Schellekens, GA; Koedijk, DGAM; Damhof, RA; WellingWester, S; Feijlbrief, M; Scheffer, AJ; Welling, GW

1996-01-01

The interaction between mAb A16 and glycoprorein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical

Production and characterization of monoclonal antibodies specific to pangasius catfish, basa, and tra.

Science.gov (United States)

Gajewski, K G; Chen, Y-T; Hsieh, Y-H P

2009-04-01

Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

Science.gov (United States)

Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

2015-04-01

Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV. © 2015 The Societies and Wiley Publishing Asia Pty Ltd.

A 44 bp intestine-specific hermaphrodite-specific enhancer from the C. elegans vit-2 vitellogenin gene is directly regulated by ELT-2, MAB-3, FKH-9 and DAF-16 and indirectly regulated by the germline, by daf-2/insulin signaling and by the TGF-β/Sma/Mab pathway.

Science.gov (United States)

Goszczynski, Barbara; Captan, Vasile V; Danielson, Alicia M; Lancaster, Brett R; McGhee, James D

2016-05-01

The Caenorhabditis elegans vitellogenin genes are transcribed in the intestine of adult hermaphrodites but not of males. A 44-bp region from the vit-2 gene promoter is able largely to reconstitute this tissue-, stage- and sex-specific-expression. This "enhancer" contains a binding site for the DM-domain factor MAB-3, the male-specific repressor of vitellogenesis, as well as an activator site that we show is the direct target of the intestinal GATA factor ELT-2. We further show that the enhancer is directly activated by the winged-helix/forkhead-factor FKH-9, (whose gene has been shown by others to be a direct target of DAF-16), by an unknown activator binding to the MAB-3 site, and by the full C. elegans TGF-β/Sma/Mab pathway acting within the intestine. The vit-2 gene has been shown by others to be repressed by the daf-2/daf-16 insulin signaling pathway, which so strongly influences aging and longevity in C. elegans. We show that the activity of the 44 bp vit-2 enhancer is abolished by loss of daf-2 but is restored by simultaneous loss of daf-16. DAF-2 acts from outside of the intestine but DAF-16 acts both from outside of the intestine and from within the intestine where it binds directly to the same non-canonical target site that interacts with FKH-9. Activity of the 44 bp vit-2 enhancer is also inhibited by loss of the germline, in a manner that is only weakly influenced by DAF-16 but that is strongly influenced by KRI-1, a key downstream effector in the pathway by which germline loss increases C. elegans lifespan. The complex behavior of this enhancer presumably allows vitellogenin gene transcription to adjust to demands of body size, germline proliferation and nutritional state but we suggest that the apparent involvement of this enhancer in aging and longevity "pathways" could be incidental. Copyright © 2016 Elsevier Inc. All rights reserved.

Prion Protein-specific antibodies that detect multiple TSE Agents with high sensitivity

NARCIS (Netherlands)

McCutcheon, S.; Langeveld, J.P.M.; Tan, B.C.; Gill, A.C.; Wolf, de C.A.; Martin, S.; Gonzalez, L.; Alibhai, J.; Alejo Blanco, A.R.; Campbell, L.; Hunter, N.; Houston, E.F.

2014-01-01

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning

Identification of IgE-binding proteins from Lepidoglyphus destructor and production of monoclonal antibodies to a major allergen.

Science.gov (United States)

Ventas, P; Carreira, J; Polo, F

1991-08-01

The allergen composition of one of the most important storage mites, Lepidoglyphus destructor, has been studied by immunodetection after SDS-PAGE with individual patient sera. An allergenic polypeptide of 14 kDa was identified with 95% of the sera. This major allergen was isolated in the supernatant of 60% ammonium sulfate salt precipitation of the whole extract, which was subsequently used to immunize BALB/c mice so as to produce monoclonal antibodies. Four mAbs recognizing molecules with IgE-binding ability were obtained. The specificity of the mAbs was assayed against different allergenic extracts, and the molecules recognized by them were characterized by immunoblotting. Two mAbs (Le5B5 and Le9E4) were directed to the 14-kDa allergen; the other two to several proteins of lesser allergenic significance.

Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

Directory of Open Access Journals (Sweden)

Kélen Fabíola Arroteia

Full Text Available The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

New amphiphilic glycopolypeptide conjugate capable of self-assembly in water into reduction-sensitive micelles for triggered drug release

Energy Technology Data Exchange (ETDEWEB)

Yang, Hui-Kang [DSAPM Lab and PCFM Lab, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275 (China); Zhang, Li-Ming, E-mail: ceszhlm@mail.sysu.edu.cn [DSAPM Lab and PCFM Lab, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275 (China); Guangdong Provincial Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006 (China)

2014-08-01

For the development of biomimetic carriers for stimuli-sensitive delivery of anticancer drugs, a novel amphiphilic glycopolypeptide conjugate containing the disulfide bond was prepared for the first time by the ring-opening polymerization of benzyl glutamate N-carboxy anhydride in the presence of (propargyl carbamate)ethyl dithio ethylamine and then click conjugation with α-azido dextran. Its structure was characterized by Fourier-transform infrared spectroscopy and nuclear magnetic resonance analyses. Owing to its amphiphilic nature, such a conjugate could self assemble into nanosize micelles in aqueous medium, as confirmed by fluorometry, transmission electron microscopy and dynamic light scattering. For the resultant micelles, it was found to encapsulate poorly water-soluble anticancer drug (methotrexate, MTX) with the loading efficiency of 45.2%. By the in vitro drug release tests, the release rate of encapsulated MTX was observed to be accelerated significantly in the presence of 10 mM 1,4-dithio-DL-threitol (DTT), analogous to the intracellular redox potential. - Graphical abstract: New amphiphilic glycopolypeptide conjugate containing the disulfide bond could self-assemble in aqueous solution into reduction-sensitive micelles for triggered release of an anticancer drug (methotrexate, MTX) in the presence of 10 mM 1,4-dithio-DL-threitol (DTT). - Highlights: • Amphiphilic glycopolypeptide conjugate containing disulfide bond was prepared. • Such a conjugate self assembled in aqueous solution into nanosize micelles. • The resultant micelles could encapsulate effectively methotrexate drug. • The drug-loaded micelles showed a reduction-sensitive drug release behavior.

New amphiphilic glycopolypeptide conjugate capable of self-assembly in water into reduction-sensitive micelles for triggered drug release

International Nuclear Information System (INIS)

Yang, Hui-Kang; Zhang, Li-Ming

2014-01-01

For the development of biomimetic carriers for stimuli-sensitive delivery of anticancer drugs, a novel amphiphilic glycopolypeptide conjugate containing the disulfide bond was prepared for the first time by the ring-opening polymerization of benzyl glutamate N-carboxy anhydride in the presence of (propargyl carbamate)ethyl dithio ethylamine and then click conjugation with α-azido dextran. Its structure was characterized by Fourier-transform infrared spectroscopy and nuclear magnetic resonance analyses. Owing to its amphiphilic nature, such a conjugate could self assemble into nanosize micelles in aqueous medium, as confirmed by fluorometry, transmission electron microscopy and dynamic light scattering. For the resultant micelles, it was found to encapsulate poorly water-soluble anticancer drug (methotrexate, MTX) with the loading efficiency of 45.2%. By the in vitro drug release tests, the release rate of encapsulated MTX was observed to be accelerated significantly in the presence of 10 mM 1,4-dithio-DL-threitol (DTT), analogous to the intracellular redox potential. - Graphical abstract: New amphiphilic glycopolypeptide conjugate containing the disulfide bond could self-assemble in aqueous solution into reduction-sensitive micelles for triggered release of an anticancer drug (methotrexate, MTX) in the presence of 10 mM 1,4-dithio-DL-threitol (DTT). - Highlights: • Amphiphilic glycopolypeptide conjugate containing disulfide bond was prepared. • Such a conjugate self assembled in aqueous solution into nanosize micelles. • The resultant micelles could encapsulate effectively methotrexate drug. • The drug-loaded micelles showed a reduction-sensitive drug release behavior

A Sensitivity Analysis of Timing and Costs of Greenhouse Gas Emission Reductions

International Nuclear Information System (INIS)

Gerlagh, R.; Van der Zwaan, B.

2004-01-01

This paper analyses the optimal timing and macro-economic costs of carbon emission reductions that mitigate the global average atmospheric temperature increase. We use a macro-economic model in which there are two competing energy sources, fossil-fuelled and non-fossil-fuelled. Technological change is represented endogenously through learning curves, and niche markets exist implying positive demand for the relatively expensive non-fossil-fuelled energy source. Under these conditions, with a temperature increase constraint of 2C, early abatement is found to be optimal, and, compared to the results of many existing top-down models, the costs of this strategy prove to be low. We perform an extensive sensitivity analysis of our results regarding the uncertainties that dominate various economic and technological modeling parameters. Uncertainties in the learning rate and the elasticity of substitution between the two different energy sources most significantly affect the robustness of our findings

Detection of copper ions using microcantilever immunosensors and enzyme-linked immunosorbent assay

Energy Technology Data Exchange (ETDEWEB)

Zhao Hongwei [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Xue Changguo [Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027 (China); Nan Tiegui; Tan Guiyu; Li Zhaohu [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Li, Qing X. [Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822 (United States); Zhang Qingchuan, E-mail: zhangqc@ustc.edu.cn [Key Laboratory of Mechanical Behavior and Design of Material of Chinese Academy of Sciences, University of Science and Technology of China, Hefei 230027 (China); Wang Baomin, E-mail: wbaomin@263.com [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China)

2010-08-31

A sensitive and specific monoclonal antibody (designated as mAb6A9) recognizing a Cu(II)-ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) complex but not metal-free EDTA was obtained by using an 1-(4-aminobenzyl)-EDTA-Cu(II) complex covalently coupled to a carrier protein as an immunogen to immunize the Balb/c mice. A mAb6A9-modified microcantilever sensor (MCS) was developed. A bending response was found to occur at or below 1 ng mL{sup -1} of Cu(II)-EDTA complex. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed with mAb6A9. The icELISA had a half maximum inhibition concentration and working range of approximately 1.8 and 0.2-17 ng mL{sup -1}, respectively. The icELISA showed cross-reactivity of 18.8%, 1.1% and less than 1% with bivalent cobalt, mercury and other metals, respectively. The icELISA and functionalized MCSs were utilized to analyze the content of copper in spiked tap water samples. The assay conditions were optimized. The results of icELISA and MCS correlated well with those obtained by graphite furnace atomic absorption spectrometry.

Antibody-mediated targeting of the urokinase-type plasminogen activator proteolytic function neutralizes fibrinolysis in vivo

DEFF Research Database (Denmark)

Lund, Ida K; Jögi, Annika; Rønø, Birgitte

2008-01-01

highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only...

Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

Directory of Open Access Journals (Sweden)

Fang He

Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

NARCIS (Netherlands)

Marissen, W.E.; Kramer, R.A.; Rice, A.; Weldon, W.C.; Niezgoda, M.; Faber, M.; Slootstra, J.W.; Meloen, R.H.; Clijsters-van der Horst, M.; Visser, T.J.; Jongeneelen, M.; Thijsse, S.; Throsby, M.; Kruif, de J.; Rupprecht, C.E.; Dietzschold, B.; Goudsmit, J.; Bakker, A.B.H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

NARCIS (Netherlands)

Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen

DEFF Research Database (Denmark)

Birkelund, Svend; Mygind, P; Holm, A

1996-01-01

this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding......Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against...... with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized...

A study of the use of radioimmunoscintigraphy (RIA) with the monoclonal antibody MAb-170, and fluorine-18 flurodeoxyglucose positron emission tomography (18FDG-PET) for the preoperative imaging of complex ovarian masses and their ability to identify ovarian cancer

Science.gov (United States)

Lieberman, Gidon

The hypothesis for this study is whether the newer diagnostic techniques of radioimmunoscintigraphy (RIS) utilising radiolabelled monoclonal antibodies and 2-[[18]F] fluoro-2-deoxy-D-glucose ([18]FDG) imaging using a double headed gamma camera offer improvements in preoperative selection for referral of patients to Cancer Centres. Monoclonal antibody radioimmunoscintigraphy (RIS) is hindered by several factors including false positive results due to physiological excretion, concern over production of human anti-mouse antibodies (HAMA) that would prevent repeated doses and difficulty in precisely relating areas of accumulation and anatomy. [18]FDG imaging relies on the accumulation of radiolabelled sugars, and subsequent breakdown products within tumour. [18]FDG imaging with dedicated positron emission tomography has real potential, but its use is limited by large capital outlay. Newer techniques involving "dual headed cameras" (DHC) offer PET capability at a lower cost. Chapter two describes the evaluation of a monoclonal antibody (MAb-170) in 27 women who presented with suspicious pelvic masses. The preoperative clinical, radiological and radioimmunoscintigraphy findings are compared to those at surgery and subsequent histology. All 18 patients with malignant or borderline ovarian cancer were correctly identified using RIS. The overall sensitivity and specificity for all sites were 100% and 38%. RIS was particularly useful in the identification of (intra-abdominal) serosal deposits. Enzyme linked immunosorbant assay (ELISA) was used to quantify the HAMA. A strong HAMA production was seen in at least 3 patients, however HAMA response was independent of clinical parameters. Chapter three describes the immunohistochemical staining of paraffin embedded biopsy specimens from the 27 patients who underwent RIS with MAb-170. The original research into the cellular location of the specific epitope to which the antibody interacts was performed on isopentane frozen biopsies

The Duty to Recognize Culture

DEFF Research Database (Denmark)

Nielsen, Morten Ebbe Juul

2012-01-01

On Taylor and Honneth's theories of recognition and whether one can derive a "duty to recognize Culture" from these......On Taylor and Honneth's theories of recognition and whether one can derive a "duty to recognize Culture" from these...

Temperature effect on the photoinduced reduction of methyl viologen with several sensitizers and the evolution of hydrogen from water

Energy Technology Data Exchange (ETDEWEB)

Nenadovic, M.T.; Micic, O.I.; Rajh, T.; Savic, D.

1983-01-01

Irradiation by visible light of an aqueous solution containing a photosensitizer, methyl viologen (MV/sup 2 +/) and ethylenediaminetetraacetic acid leads to the formation of the reduced form of methyl viologen (MV/sup +/). The quantum yield for the formation of MV/sup +/ depends strongly on the time during which the formation is observed owing to the reaction of MV/sup +/ with oxidative products and its reduction to MV/sup 0/. Proflavin, acridine yellow and ruthenium(II)tris(2,2-bipyridyl) were used as photosensitizers and showed the same ability to promote hydrogen evolution. When CdS was used as a sensitizer a factor of 10 less hydrogen was obtained than when the dyes were used. The redox catalysts platinum, Pt-TiO/sub 2/-RuO/sub 2/ and Pt-CdS in colloidal systems showed approximately the same activity towards the reduction of water. The reduction of MV/sup 2 +/ and the evolution of hydrogen were enhanced at higher temperatures (70/sup 0/C). The optimum conditions for water reduction on redox catalysts in colloidal system under continuous illumination are analysed.

Epitope mapping of functional domains of human factor V with human and mouse monoclonal antibodies

International Nuclear Information System (INIS)

Annamalai, A.E.; Rao, A.K.; Chiu, H.C.; Wang, D.; Dutta-Roy, A.K.; Colman, R.W.

1986-01-01

The authors previously described two human monoclonal antibodies (MAbs) which inactivated factor V. The authors have now purified the predominant antibody (H2) on protein A Sepharose using a pH gradient and typed it as IgG 1 ,. Immunoprecipitation of 125 I-human factor Va with H2 demonstrated specificity for the heavy chain (D), Mr = 105,000. The authors compared using ELISA the competitive binding to factor Va, of H2, H1 and two mouse MAbs, B38 (directed to E) and B10 (to activation peptide, Cl). All four antibodies recognized distinct epitopes in factor V with steric overlap in some cases. Factor Xa showed a concentration dependent competition for binding of H1, H2 and B38 but not B10 to factor V/Va in ELISA. All MAbs bound to factor V/Va in the absence of Ca ++ . However, Ca ++ at 8 mM increased the binding of H1 and H2 to 165% and 360% and did not have any effect on the binding of either mouse MAbs. Prothrombin at a concentration of up to 400 μg/ml did not inhibit binding of any of these antibodies. Thus, both the light (E) and heavy (D) chains of factor Va but not the activation peptide (Cl) interact with factor Xa as defined by the MAbs. In addition, sites on both chains for Ca ++ are recognized by particular MAbs (H1 and H2). These studies increase their knowledge of the interactions of factor V domains in the formation of prothrombinase complex

Toward the elucidation of factors concerning the individual difference of radiation sensitivity, and the reduction of radiation risks

International Nuclear Information System (INIS)

Nenoi, Mitsuru; Nakajima, Tetsuo; Wang, Bing

2013-01-01

This article describes studies aiming at the title subject and contains 2 topics of genetic and non-genetic factors modifying the radiation sensitivity. The ultimate purposes of those studies are the introduction of individual weighting factor to correct the individual differences of the sensitivity (IDS) and the practical control of the sensitivity-concerned factors, in the field of medical exposure. For genetic factors, described are studies on factors modifying the sensitivity at DNA repair and on the control of the sensitivity through the DNA repairing factors. The former, using cultured cells, aims at identifying protein (gene) of possible biomarker for IDS in non-homologous end-joining (NHEJ), an important mechanism in repairing the double strand break of DNA. Ku protein is found as the candidate. The latter has revealed that cells lacking Artemis, XRCC4 or MDC1 gene are highly sensitive, and are planning to suppress Artemis activity artificially, which may lead to the reduction of radiation cancer formation due to the death of highly sensitive cells. For non-genetic factors, described are studies on the life habits modifying the sensitivity, on the control of the sensitivity through the radiation-induced adaptive response and with steroid hormone. In the first, in mice treated with high-calorie diet and X-irradiation, a possible radiation response is suggested in the hepatic DNA-methylation and micro-RNA. Second, the combination of radiation adaptive response in the genome damage and restriction of diet ingestion is shown to lower the sensitivity of mice with use of C, Ne ion or X-ray irradiation. Third, in studies on the radiation-induced formation and condensation of breast cancer stem cells in the presence of progesterone, the hormone is found to produce micro-RNA molecules relating with the suppression of cellular senescence and repressed carcinogenesis with over-expression of apoptosis inhibitory molecules. (T.T.)

Novel monoclonal autoantibody specificity associated with ribonucleoprotein complexes

International Nuclear Information System (INIS)

Winkler, A.; Watson-McKown, R.; Wise, K.

1986-01-01

The authors describe an IgG/sub 2a/, kappa monoclonal autoantibody (mAb) F78 derived from a 6-month old MRL-Mp lpr/lpr mouse that recognizes a novel epitope associated with small nuclear ribonuclear protein complexes (snRNP). Indirect immunofluorescent staining of HEp-2 cells with F78 showed a nonnucleolar speckled nuclear pattern characteristic of anti-RNP and anti-Sm mAbs which could be abrogated by pretreating fixed cells with 0.1M HCl prior to staining. Immunoblots of whole cell extracts (dissociated in SDS, urea and mercaptan at 4 0 C then subjected to SDS-PAGE) showed that F78 selectively bound to a component of M/sub r/ = 100,000 clearly distinct from components recognized by two mAbs described by Billings et al that detected, respectively, proteins of M/sub r/ = 70,000 associated with RNP and M/sub r/ = 13,000 associated with Sm. Incubation of extracts at 100 0 C prior to SDS-PAGE eliminated subsequent binding of F78 but not of the other nAbs. F78 as well as the other mAbs selectively immunoprecipitated characteristic patterns of small nuclear RNAs (U 1 , U 2 , U 4 , U 5 , U 6 ) from extracts of 32 P-phosphate labeled HeLa cells. These results suggest a new specificity associated with snRNP that is recognized in the MRL autoimmune response

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

Science.gov (United States)

Florentino, Pilar T. V.; Real, Fernando; Orikaza, Cristina M.; da Cunha, Julia P. C.; Vitorino, Francisca N. L.; Cordero, Esteban M.; Sobreira, Tiago J. P.; Mortara, Renato A.

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. PMID:29692765

High-throughput screening of monoclonal antibodies against plant cell wall glycans by hierarchical clustering of their carbohydrate microarray binding profiles

DEFF Research Database (Denmark)

Moller, Isabel Eva; Marcus, Susan E.; Haeger, Ash

2008-01-01

Antibody-producing hybridoma cell lines were created following immunisation with a crude extract of cell wall polymers from the plant Arabidopsis thaliana. In order to rapidly screen the specificities of individual monoclonal antibodies (mAbs), their binding to microarrays containing 50 cell wall...... investigated using subsequent immunochemical and biochemical analyses and two novel mAbs are described in detail. mAb LM13 binds to an arabinanase-sensitive pectic epitope and mAb LM14, binds to an epitope occurring on arabinogalactan-proteins. Both mAbs display novel patterns of recognition of cell walls...

Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp*

Science.gov (United States)

Wongtangprasert, Tossapon; Natakuathung, Wirongrong; Pimpitak, Umaporn; Buakeaw, Anumart; Palaga, Tanapat; Komolpis, Kittinan; Khongchareonporn, Nanthika

2014-01-01

A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%–118% for an intra-assay and 96%–113% for an inter-assay. The coefficients of variation of the assays were 3.9%–13.9% and 5.5%–14.9%, respectively. PMID:24510709

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

Science.gov (United States)

Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

2016-01-01

ABSTRACT The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit

Curcumin increases the sensitivity of Paclitaxel-resistant NSCLC cells to Paclitaxel through microRNA-30c-mediated MTA1 reduction.

Science.gov (United States)

Lu, Yimin; Wang, Jun; Liu, Lei; Yu, Lequn; Zhao, Nian; Zhou, Xingju; Lu, Xudong

2017-04-01

Non-small-cell lung cancer is one of the most lethal cancers in the worldwide. Although Paclitaxel-based combinational therapies have long been used as a standard treatment in aggressive non-small-cell lung cancers, Paclitaxel resistance emerges as a major clinical problem. It has been demonstrated that Curcumin from Curcuma longa as a traditional Chinese medicine can inhibit cancer cell proliferation. However, the role of Curcumin in Paclitaxel-resistant non-small-cell lung cancer cells is not clear. In this study, we investigated the effect of Curcumin on the Paclitaxel-resistant non-small-cell lung cancer cells and found that Curcumin treatment markedly increased the sensitivity of Paclitaxel-resistant non-small-cell lung cancer cells to Paclitaxel. Mechanically, the study revealed that Curcumin could reduce the expression of metastasis-associated gene 1 (MTA1) gene through upregulation of microRNA-30c in Paclitaxel-resistant non-small-cell lung cancer cells. During the course, MTA1 reduction sensitized Paclitaxel-resistant non-small-cell lung cancer cells and enhanced the effect of Paclitaxel. Taken together, our studies indicate that Curcumin increases the sensitivity of Paclitaxel-resistant non-small-cell lung cancer cells to Paclitaxel through microRNA-30c-mediated MTA1 reduction. Curcumin might be a potential adjuvant for non-small-cell lung cancer patients during Paclitaxel treatment.

Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms

International Nuclear Information System (INIS)

Shedlock, Devon J.; Bailey, Michael A.; Popernack, Paul M.; Cunningham, James M.; Burton, Dennis R.; Sullivan, Nancy J.

2010-01-01

Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.

Microbially enhanced dissolution and reductive dechlorination of PCE by a mixed culture: Model validation and sensitivity analysis

Science.gov (United States)

Chen, Mingjie; Abriola, Linda M.; Amos, Benjamin K.; Suchomel, Eric J.; Pennell, Kurt D.; Löffler, Frank E.; Christ, John A.

2013-08-01

Reductive dechlorination catalyzed by organohalide-respiring bacteria is often considered for remediation of non-aqueous phase liquid (NAPL) source zones due to cost savings, ease of implementation, regulatory acceptance, and sustainability. Despite knowledge of the key dechlorinators, an understanding of the processes and factors that control NAPL dissolution rates and detoxification (i.e., ethene formation) is lacking. A recent column study demonstrated a 5-fold cumulative enhancement in tetrachloroethene (PCE) dissolution and ethene formation (Amos et al., 2009). Spatial and temporal monitoring of key geochemical and microbial (i.e., Geobacter lovleyi and Dehalococcoides mccartyi strains) parameters in the column generated a data set used herein as the basis for refinement and testing of a multiphase, compositional transport model. The refined model is capable of simulating the reactive transport of multiple chemical constituents produced and consumed by organohalide-respiring bacteria and accounts for substrate limitations and competitive inhibition. Parameter estimation techniques were used to optimize the values of sensitive microbial kinetic parameters, including maximum utilization rates, biomass yield coefficients, and endogenous decay rates. Comparison and calibration of model simulations with the experimental data demonstrate that the model is able to accurately reproduce measured effluent concentrations, while delineating trends in dechlorinator growth and reductive dechlorination kinetics along the column. Sensitivity analyses performed on the optimized model parameters indicate that the rates of PCE and cis-1,2-dichloroethene (cis-DCE) transformation and Dehalococcoides growth govern bioenhanced dissolution, as long as electron donor (i.e., hydrogen flux) is not limiting. Dissolution enhancements were shown to be independent of cis-DCE accumulation; however, accumulation of cis-DCE, as well as column length and flow rate (i.e., column residence time

Elucidating the weak protein-protein interaction mechanisms behind the liquid-liquid phase separation of a mAb solution by different types of additives.

Science.gov (United States)

Wu, Guoliang; Wang, Shujing; Tian, Zhou; Zhang, Ning; Sheng, Han; Dai, Weiguo; Qian, Feng

2017-11-01

Liquid-liquid phase separation (LLPS) has long been observed during the physical stability investigation of therapeutic protein formulations. The buffer conditions and the presence of various excipients are thought to play important roles in the formulation development of monoclonal antibodies (mAbs). In this study, the effects of several small-molecule excipients (histidine, alanine, glycine, sodium phosphate, sodium chloride, sorbitol and sucrose) with diverse physical-chemical properties on LLPS of a model IgG1 (JM2) solutions were investigated by multiple techniques, including UV-vis spectroscopy, circular dichroism, differential scanning calorimetry/fluorimetry, size exclusion chromatography and dynamic light scattering. The LLPS of JM2 was confirmed to be a thermodynamic equilibrium process with no structural changes or irreversible aggregation of proteins. Phase diagrams of various JM2 formulations were constructed, suggesting that the phase behavior of JM2 was dependent on the solution pH, ionic strength and the presence of other excipients such as glycine, alanine, sorbitol and sucrose. Furthermore, we demonstrated that for this mAb, the interaction parameter (k D ) determined at low protein concentration appeared to be a good predictor for the occurrence of LLPS at high concentration. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of a novel monoclonal antibody in diagnosis of Barrett's esophagus.

Science.gov (United States)

Griffel, L H; Amenta, P S; Das, K M

2000-01-01

A novel monoclonal antibody (MAbDAS-1), that specifically reacts with colonic but not small intestinal epithelium, recognizes specialized columnar epithelium (SCE) in the esophagus. The frequency of its reactivity in biopsy specimens of patients with endoscopically suspected Barrett's Esophagus (BE) is examined. Fifty-two biopsy specimens of the distal esophagus from 38 patients were tested by immunoperoxidase method using MAbDAS-1. Fifty-four samples of cardia-type mucosa biopsied from the stomach were used as controls. Results were compared with histology and Alcian blue/high iron diamine (AB/HID). Of the 52 specimens, 29 had glandular epithelium and the rest had only squamous epithelium. Ten were diagnosed to have SCE by histology. All 10 samples reacted with MAbDAS-1 and with Alcian blue. Of the remaining 19 specimens, five also reacted with MAbDAS-1. None of the squamous epithelium and cardia specimens reacted with MAbDAS-1. MAbDAS-1 may detect intestinal metaplasia of the esophagus of colonic phenotype in the absence of histological evidence of SCE.

Monoclonal antibodies based on hybridoma technology.

Science.gov (United States)

Yagami, Hisanori; Kato, Hiroshi; Tsumoto, Kanta; Tomita, Masahiro

2013-03-01

Based on the size and scope of the present global market for medicine, monoclonal antibodies (mAbs) have a very promising future, with applications for cancers through autoimmune ailments to infectious disease. Since mAbs recognize only their target antigens and not other unrelated proteins, pinpoint medical treatment is possible. Global demand is dramatically expanding. Hybridoma technology, which allows production of mAbs directed against antigens of interest is therefore privileged. However, there are some pivotal points for further development to generate therapeutic antibodies. One is selective generation of human mAbs. Employment of transgenic mice producing human antibodies would overcome this problem. Another focus is recognition sites and conformational epitopes in antigens may be just as important as linear epitopes, especially when membrane proteins such as receptors are targeted. Recognition of intact structures is of critical importance for medical purposes. In this review, we describe patent related information for therapeutic mAbs based on hybridoma technology and also discuss new advances in hybridoma technology that facilitate selective production of stereospecific mAbs.

Recognizing teen depression

Science.gov (United States)

... medlineplus.gov/ency/patientinstructions/000648.htm Recognizing teen depression To use the sharing features on this page, ... life. Be Aware of the Risk for Teen Depression Your teen is more at risk for depression ...

Antibody Prophylaxis Against Dengue Virus 2 Infection in Non-Human Primates.

Science.gov (United States)

Simmons, Monika; Putnak, Robert; Sun, Peifang; Burgess, Timothy; Marasco, Wayne A

2016-11-02

Passive immunization with anti-dengue virus (DENV) immune serum globulin (ISG) or monoclonal antibodies (Mabs) may serve to supplement or replace vaccination for short-term dengue immune prophylaxis. In the present study, we sought to establish proof-of-concept by evaluating several DENV-neutralizing antibodies for their ability to protect rhesus macaques against viremia following live virus challenge, including human anti-dengue ISG, and a human Mab (Mab11/wt) and its genetically engineered variant (Mab11/mutFc) that is unable to bind to cells with Fc gamma receptors (FcγR) and potentiate antibody-dependent enhancement (ADE). In the first experiment, groups of animals received ISG or Mab11/wt at low doses (3-10 mg/kg) or a saline control followed by challenge with DENV-2 at day 10 or 30. After passive immunization, only low-titered circulating virus-neutralizing antibody titers were measured in both groups, which were undetectable by day 30. After challenge at day 10, a reduction in viremia duration compared with the control was seen only in the ISG group (75%). However, after a day 30 challenge, no reduction in viremia was observed in both immunized groups. In a second experiment to test the effect of higher antibody doses on short-term protection, groups received either ISG, Mab11/wt, Mab11/mutFc (each at 25 mg/kg) or saline followed by challenge with DENV-2 on day 10. Increased virus-neutralizing antibody titers were detected in all groups at day 5 postinjection, with geometric mean titers (GMTs) of 464 (ISG), 313 (Mab11/wt), and 309 (Mab11/mutFc). After challenge, there was complete protection against viremia in the group that received ISG, and a reduction in viremia duration of 89% and 83% in groups that received Mab11/wt and Mab11/mutFc, respectively. An in vitro ADE assay in Fcγ receptor-bearing K562 cells with sera collected immediately before challenge showed increased DENV-2 infection levels in the presence of both ISG and Mab11/wt, which peaked at a

Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies.

Science.gov (United States)

Liu, G; Gelboin, H V; Myers, M J

1991-02-01

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

International Nuclear Information System (INIS)

Boonsathorn, Naphatsawan; Panthong, Sumolrat; Koksunan, Sarawut; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Yasugi, Mayo; Ono, Ken-ichiro; Arai, Yasuha

2014-01-01

Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Energy Technology Data Exchange (ETDEWEB)

Boonsathorn, Naphatsawan; Panthong, Sumolrat [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Koksunan, Sarawut [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya [National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Prachasupap, Apichai [Medical Life Sciences Institute, Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Sasaki, Tadahiro [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); Yasugi, Mayo [Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); Ono, Ken-ichiro [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano (Japan); Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS), Tokyo (Japan); Arai, Yasuha [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); and others

2014-09-26

Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

A Comprehensive Study of Neutralizing Antigenic Sites on the Hepatitis E Virus (HEV) Capsid by Constructing, Clustering, and Characterizing a Tool Box*

Science.gov (United States)

Zhao, Min; Li, Xiao-Jing; Tang, Zi-Min; Yang, Fan; Wang, Si-Ling; Cai, Wei; Zhang, Ke; Xia, Ning-Shao; Zheng, Zi-Zheng

2015-01-01

The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459–606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process. PMID:26085097

Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin.

Science.gov (United States)

Yamada, Shinji; Kaneko, Mika K; Nakamura, Takuro; Ichii, Osamu; Konnai, Satoru; Kato, Yukinari

2017-04-01

Podoplanin (PDPN), the ligand of C-type lectin-like receptor-2, is used as a lymphatic endothelial marker. We previously established clone PMab-1 of rat IgG 2a as a specific monoclonal antibody (mAb) against mouse PDPN. PMab-1 is also very sensitive in immunohistochemical analysis; however, rat mAbs seem to be unfavorable for pathologists because anti-mouse IgG and anti-rabbit IgG are usually used as secondary antibodies in commercially available kits for immunohistochemical analysis. In this study, we develop a mouse-rat chimeric antibody, mPMab-1 of mouse IgG 2a , which was derived from rat PMab-1 mAb. Immunohistochemical analysis shows that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Importantly, mPMab-1 is more sensitive than PMab-1. This conversion strategy from rat mAb to mouse mAb could be applicable to other mAbs.

Human Monoclonal antibodies - A dual advantaged weapon to tackle cancer and viruses

Directory of Open Access Journals (Sweden)

Kurosawa G

2014-11-01

Full Text Available Human monoclonal antibodies (mAbs are powerful tools as pharmaceutical agents to tackle cancer and infectious diseases. Antibodies (Abs are present in blood at the concentration of 10 mg/ml and play a vital role in humoral immunity. Many therapeutic Abs have been reported since early 1980s. Human mAb technology was not available at that time and only the hybridoma technology for making mouse mAbs had been well established. In order to avoid various potential problems associated with use of mouse proteins, two different technologies to make human/mouse chimeric Ab as well as humanized Ab were developed crossing the various hurdles for almost twenty years and mAb based drugs such as rituximab, anti-CD20 Ab, and trastuzumab, anti-HER2 Ab, have been approved by the US Food and Drug Administration (FDA for treatment of non-Hodgkin's lymphoma and breast cancer in 1997 and 1998, respectively. These drugs are well recognized and accepted by clinicians for treatment of patients. The clinical outcome of the treatment with mAb has strongly encouraged the researchers to develop much more refined mAbs. In addition to chimeric Ab and humanized Ab, now human mAbs can be produced by two technologies. The first is transgenic mice that produce human Abs and the second is human Ab libraries using phage-display system. Until now, several hundreds of mAbs against several tens of antigens (Ags have been developed and subjected to clinical examinations. While many Abs have been approved as therapeutic agents against hematological malignancies, the successful mAbs against solid tumors are still limited. However, many researchers have suggested that developing potential mAbs agents should be possible and incurable cancers may become curable within another decade. Though it is hard to say explicitly that this prediction is correct, a passion for this development should be worth supporting to lead to a successful outcome which will lead to patient benefits. Our institute

Neutralization of White Spot Syndrome Virus by Monoclonal Antibodies against Viral Envelope Proteins

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Hsiu-Hui Shih

2004-09-01

Full Text Available Two monoclonal antibodies (MAbs recognizing envelope proteins of the white spot syndrome virus (WSSV, 6E1 against VP28 and 3E8 against VP19, were applied to demonstrate their neutralizing ability to this virus by using both in vitro and in vivo assays. Mixtures of MAb 6E1 with virus filtrate were inoculated into the primary explant monolayer culture derived from the lymphoid Oka organs of Penaeus monodon. Mab was likely to neutralize the infectivity of virus to monolayer since cytopathic effects were apparently blocked in experiment group. WSSV was titrated using Blue-Cell ELISA and the neutralizing index was calculated to be 6.90 for 6EI and 5.83 for 3E8. Neutralized virus fluids injected intramuscularly into post larvae of P. monodon. The shrimp in the positive control, which were injected with WSSV only showed an increasing mortality and a 100% mortality was reached at day 34, whereas no shrimp died in the negative control. The mortality for 6E1 was 6.7% and for 3E8 was 13.3%. These results suggest that Mabs recognizing the WSSV envelope proteins could neutralize viral infectivity to both cultured cells and shrimp.

A four-step sandwich radioimmunoassay for direct selection of monoclonal antibodies to allergen molecules

International Nuclear Information System (INIS)

Ley, V.; Corbi, A.L.; Sanchez-Madrid, F.; Carreira, J.C.

1985-01-01

A 4-step radioimmunoassay has been devised for direct identification of monoclonal antibodies (MAb) directed to IgE-binding molecules. Polyvinyl chloride wells coated with purified anti-mouse kappa chain MAb (187-1) were successively incubated with: (1) MAb-containing hybridoma supernatants, (2) allergen extract, (3) allergic patients' serum pool, and (4) 125 I-labeled anti-human IgE antiserum, to detect MAb-allergen-IgE complexes. MAb to allergens from Parietaria judaica pollen and Dermatophagoides mites have been selected with this screening procedure. The affinity-purified allergen molecules competed the binding of IgE to allergen extracts coated to paper discs in a RAST inhibition assay, confirming the anti-allergen specificity of the selected MAb. This screening method is sensitive enough to allow detection of MAb directed to poorly represented allergens. (Auth.)

Systematic Sensitivity Analysis of Metabolic Controllers During Reductions in Skeletal Muscle Blood Flow

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Radhakrishnan, Krishnan; Cabrera, Marco

2000-01-01

An acute reduction in oxygen delivery to skeletal muscle is generally associated with profound derangements in substrate metabolism. Given the complexity of the human bioenergetic system and its components, it is difficult to quantify the interaction of cellular metabolic processes to maintain ATP homeostasis during stress (e.g., hypoxia, ischemia, and exercise). Of special interest is the determination of mechanisms relating tissue oxygenation to observed metabolic responses at the tissue, organ, and whole body levels and the quantification of how changes in oxygen availability affect the pathways of ATP synthesis and their regulation. In this study, we apply a previously developed mathematical model of human bioenergetics to study effects of ischemia during periods of increased ATP turnover (e.g., exercise). By using systematic sensitivity analysis the oxidative phosphorylation rate was found to be the most important rate parameter affecting lactate production during ischemia under resting conditions. Here we examine whether mild exercise under ischemic conditions alters the relative importance of pathways and parameters previously obtained.

In vivo effects of monoclonal anti-L3T4 antibody on immune responsiveness of mice infected with Schistosoma mansoni. Reduction of irradiated cercariae-induced resistance

International Nuclear Information System (INIS)

Kelly, E.A.; Colley, D.G.

1988-01-01

Mice can be partially protected against challenge infections of Schistosoma mansoni cercariae by either single or multiple exposure to irradiated cercariae (x-cerc). The participation of L3T4+ lymphocytes on this resistance phenomenon was evaluated by selectively depleting this cell population through in vivo administration of mAb anti-L3T4 at three different times in relationship to the challenge infections. Treatment with anti-L3T4 before challenge such that depletion was effective during the time of cercarial skin penetration and dermal/s.c. residence significantly reduced the level of resistance induced by x-cerc sensitization. When treatment was delayed until after challenge, depletion of L3T4+ cells coincided with either the lung or post-lung/liver phases of schistosomular migration, and normal levels of x-cerc-induced resistance were induced. In contrast to once-immunized mice, mice hyperimmunized by five exposures to x-cerc and then depleted of L3T4+ cells at the time of challenge still expressed resistance to the challenge. These data suggest that when mice are sensitized only once with x-cerc the challenge infection provides a necessary immunologic boost which requires L3T4+ cells for effective expression of resistance. The requirement for this anamnestic effect by the challenge infection can be circumvented by hyperimmunization. Evaluation of the immune response of one-time sensitized or hyperimmunized mice demonstrated that cellular Ag-specific proliferative responses and mitogen-induced lymphokine production were abrogated after any of the various in vivo regimens of anti-L3T4 antibody. In contrast, immunoblot analysis of humoral responsiveness revealed a correlation between the expression of resistance and the ability of sera from immunized and anti-L3T4 treated mice to recognize a 75-kDa parasite antigenic component

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

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Pilar T. V. Florentino

2018-04-01

Full Text Available Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels, it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease.

The Role of Biosphere Reserves in Environmental Education and Training = Le Role des reserves de la biosphere dans l'education et la formation environnementales. Report of the Unesco/MAB Symposium Held During the Unesco/UNEP International Congress on Environmental Education and Training (Moscow, USSR, August 17-21, 1987). Report 20.

Science.gov (United States)

Francis, George, Ed.

Environmental education and training have been key elements of Unesco's Program on Man and the Biosphere (MAB) since its inception in 1971. The MAB Program is an intergovernmental program of research, training, demonstration and distribution of information, aimed at providing the scientific background and the trained personnel to deal with…

"RecognizeCane" : The new concept of a cane which recognizes the most common objects and safety clues.

Science.gov (United States)

Scherlen, Anne-Catherine; Dumas, Jean Claude; Guedj, Benjamin; Vignot, Alexandre

2007-01-01

This paper introduces the new concept of an electronic cane for blind people. While some systems inform the subject only of the presence of the object and its relative distance, RecognizeCane is also able to recognize most common objects and environment clues to increase the safety and confidence of the navigation process. The originality of RecognizeCane is the use of simple sensors, such as infrared, brilliance or water sensors to inform the subject of the presence, for example, of a stairway, a water puddle, a zebra crossing or a trash can. This cane does not use an embedded vision system. RecognizeCane is equipped with several sensors and microprocessors to collect sensor data and extract the desired information about the close environment by means of a dynamic analysis of output signals.

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

Science.gov (United States)

Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

2013-08-01

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

Use what you can: Storage, abstraction processes and perceptual adjustments help listeners recognize reduced forms

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Katja ePoellmann

2014-05-01

Full Text Available Three eye-tracking experiments tested whether native listeners recognized reduced Dutch words better after having heard the same reduced words, or different reduced words of the same reduction type and whether familiarization with one reduction type helps listeners to deal with another reduction type. In the exposure phase, a segmental reduction group was exposed to /b/-reductions (e.g., minderij instead of binderij, 'book binder' and a syllabic reduction group was exposed to full-vowel deletions (e.g., p'raat instead of paraat, 'ready', while a control group did not hear any reductions. In the test phase, all three groups heard the same speaker producing reduced-/b/ and deleted-vowel words that were either repeated (Experiments 1 & 2 or new (Experiment 3, but that now appeared as targets in semantically neutral sentences. Word-specific learning effects were found for vowel-deletions but not for /b/-reductions. Generalization of learning to new words of the same reduction type occurred only if the exposure words showed a phonologically consistent reduction pattern (/b/-reductions. In contrast, generalization of learning to words of another reduction type occurred only if the exposure words showed a phonologically inconsistent reduction pattern (the vowel deletions; learning about them generalized to recognition of the /b/-reductions. In order to deal with reductions, listeners thus use various means. They store reduced variants (e.g., for the inconsistent vowel-deleted words and they abstract over incoming information to build up and apply mapping rules (e.g., for the consistent /b/-reductions. Experience with inconsistent pronunciations leads to greater perceptual flexibility in dealing with other forms of reduction uttered by the same speaker than experience with consistent pronunciations.

Establishment of novel mAb to human ERC/mesothelin useful for study and diagnosis of ERC/mesothelin-expressing cancers.

Science.gov (United States)

Ishikawa, Kiyoshi; Segawa, Tatsuya; Hagiwara, Yoshiaki; Maeda, Masahiro; Abe, Masaaki; Hino, Okio

2009-03-01

Malignant mesothelioma is a highly aggressive tumor of the serosal cavity that arises from the mesothelial cells of the pleura, peritoneum, or pericardium. The immunohistochemical diagnosis of epithelioid mesothelioma from biopsy or surgically resected specimens has been actively pursued, using markers such as mesothelin. Several markers have indeed been helpful for confirming the diagnosis of mesothelioma and distinguishing between mesothelioma and adenocarcinoma. The authors have developed a novel mAb to human C-ERC/mesothelin, which performed well when used in western blotting, fluorescence-activated cell sorting, immunocytochemistry and immunohistochemistry, and which therefore will be useful in studying the molecular biology of mesothelin, in addition to improving the diagnosis and therapy of mesothelin-expressing cancers.

PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Harada, Hiroyuki; Kagawa, Yumiko; Ichii, Osamu; Konnai, Satoru; Kaneko, Mika K; Kato, Yukinari

2017-10-01

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

Rabbit antibodies for hormone receptors and HER2 evaluation in breast cancer Anticorpos de coelho para avaliação de receptores hormonais e HER2 em câncer de mama

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Rafael Malagoli Rocha

2009-01-01

Full Text Available BACKGROUND: Novel rabbit monoclonal antibodies (RabMab for estrogen (ER, progesterone (PR receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab using tissue microarrays (TMA of breast carcinomas. METHODS: Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1% of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines. RESULTS: RabMab against ER have similar staining patterns compared to the 6F11 (Mab, but stronger than 1D5 (Mab from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH. CONCLUSION: The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.OBJETIVOS: Novos anticorpos monoclonais de coelho (RabMab para a avaliação imuno-histoquímica de receptores de estrógeno (RE, progesterona (RP e HER2 foram lançados comercialmente. Comparamos os RabMab anti-RE, anti-RP e anti-HER2 com os anticorpos monoclonais de camundongo (Mab utilizando tissue microarrays (TMA de carcinomas de mama. MÉTODOS: Foram construídos dois TMAs de

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

Energy Technology Data Exchange (ETDEWEB)

Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

2000-02-01

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

2000-01-01

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

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Christine Bee

Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

Self-control in Online Discussions: Disinhibited Online Behavior as a Failure to Recognize Social Cues.

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Voggeser, Birgit J; Singh, Ranjit K; Göritz, Anja S

2017-01-01

In an online experiment we examined the role of self-control in recognizing social cues in the context of disinhibited online behavior (e.g., flaming and trolling). We temporarily lowered participants' self-control capacity with an ego depletion paradigm (i.e., color Stroop task). Next, we measured participants' sensitivity to social cues with an emotional Stroop task containing neutral, negative, and taboo words. Sensitivity to social cues is represented by the increase in reaction time to negative and especially taboo words compared to neutral words. As expected, undepleted participants were slower to process the color of negative and taboo words. By contrast, depleted participants (i.e., those with lowered self-control capacity) did not react differently to taboo or negative words than they did to neutral words. The experiment illustrates that self-control failure may manifest itself in a failure to recognize social cues. The finding underlines the importance of self-control in understanding disinhibited online behavior: Many instances of disinhibited online behavior may occur not because people are unable to control themselves, but because they do not realize that a situation calls for self-control in the first place.

Self-control in Online Discussions: Disinhibited Online Behavior as a Failure to Recognize Social Cues

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Birgit J. Voggeser

2018-01-01

Full Text Available In an online experiment we examined the role of self-control in recognizing social cues in the context of disinhibited online behavior (e.g., flaming and trolling. We temporarily lowered participants' self-control capacity with an ego depletion paradigm (i.e., color Stroop task. Next, we measured participants' sensitivity to social cues with an emotional Stroop task containing neutral, negative, and taboo words. Sensitivity to social cues is represented by the increase in reaction time to negative and especially taboo words compared to neutral words. As expected, undepleted participants were slower to process the color of negative and taboo words. By contrast, depleted participants (i.e., those with lowered self-control capacity did not react differently to taboo or negative words than they did to neutral words. The experiment illustrates that self-control failure may manifest itself in a failure to recognize social cues. The finding underlines the importance of self-control in understanding disinhibited online behavior: Many instances of disinhibited online behavior may occur not because people are unable to control themselves, but because they do not realize that a situation calls for self-control in the first place.

Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men

DEFF Research Database (Denmark)

Knudsen, Sine Haugaard; Hansen, Louise Seier; Pedersen, Maria

2012-01-01

A lifestyle characterized by inactivity and a high-calorie diet is a known risk factor for impaired insulin sensitivity and development of Type 2 diabetes mellitus. To investigate possible links, nine young healthy men (24 ± 3 yr; body mass index of 21.6 ± 2.5 kg/m(2)) completed 14 days of step...... reduction (10,000 to 1,500 steps/day) and overfeeding (+50% kcal). Body composition (dual X-ray absorptiometry, MRI), aerobic fitness (maximal O(2) consumption), systemic inflammation and insulin sensitivity [oral glucose tolerance test (OGTT), hyperinsulinemic euglycemic clamp] were assessed before (day 0...

Peptide-Based Vaccinology: Experimental and Computational Approaches to Target Hypervariable Viruses through the Fine Characterization of Protective Epitopes Recognized by Monoclonal Antibodies and the Identification of T-Cell-Activating Peptides

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Matteo Castelli

2013-01-01

Full Text Available Defining immunogenic domains of viral proteins capable of eliciting a protective immune response is crucial in the development of novel epitope-based prophylactic strategies. This is particularly important for the selective targeting of conserved regions shared among hypervariable viruses. Studying postinfection and postimmunization sera, as well as cloning and characterization of monoclonal antibodies (mAbs, still represents the best approach to identify protective epitopes. In particular, a protective mAb directed against conserved regions can play a key role in immunogen design and in human therapy as well. Experimental approaches aiming to characterize protective mAb epitopes or to identify T-cell-activating peptides are often burdened by technical limitations and can require long time to be correctly addressed. Thus, in the last decade many epitope predictive algorithms have been developed. These algorithms are continually evolving, and their use to address the empirical research is widely increasing. Here, we review several strategies based on experimental techniques alone or addressed by in silico analysis that are frequently used to predict immunogens to be included in novel epitope-based vaccine approaches. We will list the main strategies aiming to design a new vaccine preparation conferring the protection of a neutralizing mAb combined with an effective cell-mediated response.

Global sensitivity analysis of bogie dynamics with respect to suspension components

Energy Technology Data Exchange (ETDEWEB)

Mousavi Bideleh, Seyed Milad, E-mail: milad.mousavi@chalmers.se; Berbyuk, Viktor, E-mail: viktor.berbyuk@chalmers.se [Chalmers University of Technology, Department of Applied Mechanics (Sweden)

2016-06-15

The effects of bogie primary and secondary suspension stiffness and damping components on the dynamics behavior of a high speed train are scrutinized based on the multiplicative dimensional reduction method (M-DRM). A one-car railway vehicle model is chosen for the analysis at two levels of the bogie suspension system: symmetric and asymmetric configurations. Several operational scenarios including straight and circular curved tracks are considered, and measurement data are used as the track irregularities in different directions. Ride comfort, safety, and wear objective functions are specified to evaluate the vehicle’s dynamics performance on the prescribed operational scenarios. In order to have an appropriate cut center for the sensitivity analysis, the genetic algorithm optimization routine is employed to optimize the primary and secondary suspension components in terms of wear and comfort, respectively. The global sensitivity indices are introduced and the Gaussian quadrature integrals are employed to evaluate the simplified sensitivity indices correlated to the objective functions. In each scenario, the most influential suspension components on bogie dynamics are recognized and a thorough analysis of the results is given. The outcomes of the current research provide informative data that can be beneficial in design and optimization of passive and active suspension components for high speed train bogies.

Global sensitivity analysis of bogie dynamics with respect to suspension components

International Nuclear Information System (INIS)

Mousavi Bideleh, Seyed Milad; Berbyuk, Viktor

2016-01-01

The effects of bogie primary and secondary suspension stiffness and damping components on the dynamics behavior of a high speed train are scrutinized based on the multiplicative dimensional reduction method (M-DRM). A one-car railway vehicle model is chosen for the analysis at two levels of the bogie suspension system: symmetric and asymmetric configurations. Several operational scenarios including straight and circular curved tracks are considered, and measurement data are used as the track irregularities in different directions. Ride comfort, safety, and wear objective functions are specified to evaluate the vehicle’s dynamics performance on the prescribed operational scenarios. In order to have an appropriate cut center for the sensitivity analysis, the genetic algorithm optimization routine is employed to optimize the primary and secondary suspension components in terms of wear and comfort, respectively. The global sensitivity indices are introduced and the Gaussian quadrature integrals are employed to evaluate the simplified sensitivity indices correlated to the objective functions. In each scenario, the most influential suspension components on bogie dynamics are recognized and a thorough analysis of the results is given. The outcomes of the current research provide informative data that can be beneficial in design and optimization of passive and active suspension components for high speed train bogies.

Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

Energy Technology Data Exchange (ETDEWEB)

Nguyen thi Man; Morris, G.E. (North East Wales Inst., Clwyd (United Kingdom))

1993-06-01

The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

Science.gov (United States)

Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

1998-06-01

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Recognizing Bedside Events Using Thermal and Ultrasonic Readings

Directory of Open Access Journals (Sweden)

Danielsen Asbjørn

2017-06-01

Full Text Available Falls in homes of the elderly, in residential care facilities and in hospitals commonly occur in close proximity to the bed. Most approaches for recognizing falls use cameras, which challenge privacy, or sensor devices attached to the bed or the body to recognize bedside events and bedside falls. We use data collected from a ceiling mounted 80 × 60 thermal array combined with an ultrasonic sensor device. This approach makes it possible to monitor activity while preserving privacy in a non-intrusive manner. We evaluate three different approaches towards recognizing location and posture of an individual. Bedside events are recognized using a 10-second floating image rule/filter-based approach, recognizing bedside falls with 98.62% accuracy. Bed-entry and exit events are recognized with 98.66% and 96.73% accuracy, respectively.

Pseudo tumors of the lung after lung volume reduction surgery.

Science.gov (United States)

Oey, Inger F; Jeyapalan, Kanagaratnam; Entwisle, James J; Waller, David A

2004-03-01

We describe 2 patients who underwent lung volume reduction surgery, who postoperatively had computed tomographic scans that showed symptomatic mass lesions suggestive of malignancy and an inhaled foreign body. Investigations excluded these conditions with the remaining likely diagnosis of pseudotumor secondary to buttressing material. These potential sequelae of lung volume reduction surgery should be recognized in follow-up investigations.

Dysregulated stress signal sensitivity and inflammatory disinhibition as a pathophysiological mechanism of stress-related chronic fatigue.

Science.gov (United States)

Strahler, Jana; Skoluda, Nadine; Rohleder, Nicolas; Nater, Urs M

2016-09-01

Chronic stress and its subsequent effects on biological stress systems have long been recognized as predisposing and perpetuating factors in chronic fatigue, although the exact mechanisms are far from being completely understood. In this review, we propose that sensitivity of immune cells to glucocorticoids (GCs) and catecholamines (CATs) may be the missing link in elucidating how stress turns into chronic fatigue. We searched for in vitro studies investigating the impact of GCs or CATs on mitogen-stimulated immune cells in chronically stressed or fatigued populations, with 34 original studies fulfilling our inclusion criteria. Besides mixed cross-sectional findings for stress- and fatigue-related changes of GC sensitivity under basal conditions or acute stress, longitudinal studies indicate a decrease with ongoing stress. Research on CATs is still scarce, but initial findings point towards a reduction of CAT sensitivity under chronic stress. In the long run, resistance of immune cells to stress signals under conditions of chronic stress might translate into self-maintaining inflammation and inflammatory disinhibition under acute stress, which in turn lead to fatigue. Copyright © 2016 Elsevier Ltd. All rights reserved.

A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus

International Nuclear Information System (INIS)

Zhang Shuliu; Li Li; Woodson, Sara E.; Huang, Claire Y.-H.; Kinney, Richard M.; Barrett, Alan D.T.; Beasley, David W.C.

2006-01-01

Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop

Chemoselective reductive nucleophilic addition to tertiary amides, secondary amides, and N-methoxyamides.

Science.gov (United States)

Nakajima, Minami; Oda, Yukiko; Wada, Takamasa; Minamikawa, Ryo; Shirokane, Kenji; Sato, Takaaki; Chida, Noritaka

2014-12-22

As the complexity of targeted molecules increases in modern organic synthesis, chemoselectivity is recognized as an important factor in the development of new methodologies. Chemoselective nucleophilic addition to amide carbonyl centers is a challenge because classical methods require harsh reaction conditions to overcome the poor electrophilicity of the amide carbonyl group. We have successfully developed a reductive nucleophilic addition of mild nucleophiles to tertiary amides, secondary amides, and N-methoxyamides that uses the Schwartz reagent [Cp2 ZrHCl]. The reaction took place in a highly chemoselective fashion in the presence of a variety of sensitive functional groups, such as methyl esters, which conventionally require protection prior to nucleophilic addition. The reaction will be applicable to the concise synthesis of complex natural alkaloids from readily available amide groups. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins.

Directory of Open Access Journals (Sweden)

Victoria Martínez-Sernández

Full Text Available MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1 was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.

FDA Recognized Consensus Standards

Data.gov (United States)

U.S. Department of Health & Human Services — This database consists of those national and international standards recognized by FDA which manufacturers can declare conformity to and is part of the information...

Marker of cemento-periodontal ligament junction associated with periodontal regeneration.

Science.gov (United States)

Hara, Ryohko; Wato, Masahiro; Tanaka, Akio

2005-06-01

The purpose of this study was to identify factors promoting formation of the cemento-periodontal ligament junction. Regeneration of the cemento-periodontal ligament junction is an important factor in recovery of the connective tissue attachment to the cementum and it is important to identify all specific substances that promote its formation. To clarify the substances involved in cemento-periodontal ligament junction formation, we produced a monoclonal antibody (mAb) to human cemento-periodontal ligament junction (designated as the anti-TAP mAb) and examined its immunostaining properties and reactive antigen. Hybridomas producing monoclonal antibody against human cemento-periodontal ligament junction antigens were established by fusing P3U1 mouse myeloma cells with spleen cells from BALB/c mice immunized with homogenized human cemento-periodontal ligament junction. The mAb, the anti-TAP mAb for cemento-periodontal ligament junction, was then isolated. The immunoglobulin class and light chain of the mAb were examined using an isotyping kit. Before immunostaining, antigen determination using an enzymatic method or heating was conducted. Human teeth, hard tissue-forming lesions, and animal tissues were immunostained by the anti-TAP mAb. The anti-TAP mAb was positive in human cemento-periodontal ligament junction and predentin but negative in all other human and animal tissues examined. In the cemento-osseous lesions, the anti-TAP mAb was positive in the peripheral area of the cementum and cementum-like hard tissues and not in the bone and bone-like tissues. The anti-TAP mAb showed IgM (kappa) and recognized phosphoprotein. The anti-TAP mAb is potentially useful for developing new agents promoting cementogenesis and periodontal regeneration.

Why sensitive bacteria are resistant to hospital infection control

Science.gov (United States)

van Kleef, Esther; Luangasanatip, Nantasit; Bonten, Marc J; Cooper, Ben S

2017-01-01

Background: Large reductions in the incidence of antibiotic-resistant strains of Staphylococcus aureus and Clostridium difficile have been observed in response to multifaceted hospital-based interventions. Reductions in antibiotic-sensitive strains have been smaller or non-existent. It has been argued that since infection control measures, such as hand hygiene, should affect resistant and sensitive strains equally, observed changes must have largely resulted from other factors, including changes in antibiotic use. We used a mathematical model to test the validity of this reasoning. Methods: We developed a mechanistic model of resistant and sensitive strains in a hospital and its catchment area. We assumed the resistant strain had a competitive advantage in the hospital and the sensitive strain an advantage in the community. We simulated a hospital hand hygiene intervention that directly affected resistant and sensitive strains equally. The annual incidence rate ratio ( IRR) associated with the intervention was calculated for hospital- and community-acquired infections of both strains. Results: For the resistant strain, there were large reductions in hospital-acquired infections (0.1 ≤ IRR ≤ 0.6) and smaller reductions in community-acquired infections (0.2 ≤ IRR ≤  0.9). These reductions increased in line with increasing importance of nosocomial transmission of the strain. For the sensitive strain, reductions in hospital acquisitions were much smaller (0.6 ≤ IRR ≤ 0.9), while communityacquisitions could increase or decrease (0.9 ≤ IRR ≤ 1.2). The greater the importance of the community environment for the transmission of the sensitive strain, the smaller the reductions. Conclusions: Counter-intuitively, infection control interventions, including hand hygiene, can have strikingly discordant effects on resistant and sensitive strains even though they target them equally, following differences in their adaptation to hospital and community

Why sensitive bacteria are resistant to hospital infection control.

Science.gov (United States)

van Kleef, Esther; Luangasanatip, Nantasit; Bonten, Marc J; Cooper, Ben S

2017-01-01

Large reductions in the incidence of antibiotic-resistant strains of Staphylococcus aureus and Clostridium difficile have been observed in response to multifaceted hospital-based interventions. Reductions in antibiotic-sensitive strains have been smaller or non-existent. It has been argued that since infection control measures, such as hand hygiene, should affect resistant and sensitive strains equally, observed changes must have largely resulted from other factors, including changes in antibiotic use. We used a mathematical model to test the validity of this reasoning. We developed a mechanistic model of resistant and sensitive strains in a hospital and its catchment area. We assumed the resistant strain had a competitive advantage in the hospital and the sensitive strain an advantage in the community. We simulated a hospital hand hygiene intervention that directly affected resistant and sensitive strains equally. The annual incidence rate ratio (IRR) associated with the intervention was calculated for hospital- and community-acquired infections of both strains. For the resistant strain, there were large reductions in hospital-acquired infections (0.1 ≤ IRR ≤ 0.6) and smaller reductions in community-acquired infections (0.2 ≤ IRR ≤ 0.9). These reductions increased in line with increasing importance of nosocomial transmission of the strain. For the sensitive strain, reductions in hospital acquisitions were much smaller (0.6 ≤ IRR ≤ 0.9), while community acquisitions could increase or decrease (0.9 ≤ IRR ≤ 1.2). The greater the importance of the community environment for the transmission of the sensitive strain, the smaller the reductions. Counter-intuitively, infection control interventions, including hand hygiene, can have strikingly discordant effects on resistant and sensitive strains even though they target them equally. This follows from differences in their adaptation to hospital- and community-based transmission. Observed lack of

Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

Science.gov (United States)

2013-08-01

Janelia Farm , VA, travel award 2010 -­‐ Translational Medicine Symposium, HHMI Peer Cluster Meeting, travel award 2010 -­‐ AACR Annual Conference...lab. Science Education Alliance, Janelia Farm , VA, 2001 3. Londoño-Joshi AI, Forero-Torres A, Fineberg NS, Oliver PG, Zhou T, LoBuglio AF, Buchsbaum...rabbit mAb and cleaved caspase 3 rabbit mAb were purchased from Cell Signaling (Billerica, MA). Secondary antibodies, Alexa fluor 405 goat anti-rabbit

Development of recombinant antibody technology for application in plant pathogen diagnosis

NARCIS (Netherlands)

Griep, R.

1999-01-01

This thesis describes the applicability of the novel phage display technique to select plant-pathogen-specific monoclonal antibodies (MAbs) from combinatorial antibody libraries. The retrieved MAbs are so specific that they can be used as diagnostic tools in sensitive immunoassays for the

46 CFR 42.05-60 - Recognized classification society.

Science.gov (United States)

2010-10-01

... 46 Shipping 2 2010-10-01 2010-10-01 false Recognized classification society. 42.05-60 Section 42... FOREIGN VOYAGES BY SEA Definition of Terms Used in This Subchapter § 42.05-60 Recognized classification society. The term recognized classification society means the American Bureau of Shipping or other...

46 CFR 90.10-35 - Recognized classification society.

Science.gov (United States)

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Recognized classification society. 90.10-35 Section 90... VESSELS GENERAL PROVISIONS Definition of Terms Used in This Subchapter § 90.10-35 Recognized classification society. The term recognized classification society means the American Bureau of Shipping or other...

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

Science.gov (United States)

Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

2016-04-01

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Synthesis, characterization and in vitro/in vivo evaluation of novel reduction-sensitive hybrid nano-echinus-like nanomedicine.

Science.gov (United States)

Wang, Kaili; Guo, Chunjing; Zou, Shaohua; Yu, Yueming; Fan, Xinxin; Wang, Bingjie; Liu, Mengna; Fang, Lei; Chen, Daquan

2018-04-27

To remedy the problems resulting from the usage of anti-cancer drugs in cancer chemotherapy, such as deficient drug concentration in tumour cells, low water-solubility and non-specific distribution of antitumour drugs, a kind of reduction-sensitive polymer prodrug of curcumin (Cur) containing in the nano-echinus was synthesized and designed. The nano-echinus-like nanomedicine presented synergistic effect with glycyrrhetic acid (GA) and oligomeric hyaluronic (HA) for targeting and combating HepG2 human liver cancer cell. Firstly, a kind of small molecular prodrug of Cur, dithiodipropionic acid-Cur (-SS-Cur), was chemically conjugated onto the side chain of the conjugated glycyrrhetic acid- oligomeric hyaluronic (GA-HA) to generate an amphiphilic polymeric prodrug of Cur, GA-HA-SS-Cur. The obtained GA-HA-SS-Cur prodrug and subsidiary material mPEG-DSPE could self-assemble into a sea urchin-like micelles in aqueous media and release Cur rapidly in response to glutathion (GSH). Then, Cur was loaded into the nano-echinus with a particle size of (118.1 ± 0.2 nm) and drug-loading efficiency of (8.03 ± 2.1%). The structure of GA-HA-SS-Cur was characterized by 1 H-NMR in this report. The morphology of micelles was observed with a transmission electron microscope (TEM). Subsequently, the reduction-sensitivity of the nano-echinus was confirmed by the changes in in-vitro drug release after different concentrations of GSH treatment. Besides, the cellular uptake behaviour and MTT assays of the nano-echinus were investigated, suggesting that the nano-echinus was of desirable safety and could be taken into HepG2 cells in a time-dependent manner. Later, anti-tumour efficacy in vivo revealed the effective inhibition of tumour growth.

Immunoscintigraphy using 111In-DTPA labeled monoclonal antibodies: Comparison between ETC and planar imaging

International Nuclear Information System (INIS)

Happ, J.; Baum, R.P.; Frohn, J.; Weimer, B.; Hoer, G.; Halbsguth, A.; Lochner, B.; Brandhorst, I.

1987-01-01

The present study was done in order to examine if the use of 111 In-DTPA-labeled MAb fragments in place of 131 I-labeled MAb fragments increases the sensitivity of tomographic immunoscintigraphy to reach the level of that of planar imaging techniques. In 11 patients with various primary tumors, local recurrences or metastases [colorectal carcinoma (n=7), ovarian carcinoma (n=2), papillary thyroid carcinoma (n=1), undifferentiated carcinoma of the lung (n=1)], immuniscintigraphy (IS) was carried out using 111 In-DTPA-labeled F(ab') 2 fragments of various MAbs (anti-CEA, OC 125, anti-hTG) and planar and tomographic imaging were compared intraindividually. By conventional diagnostic procedures, the presence of a tumor mass was confirmed (transmission computer tomography, ultrasound) or verified ( 131 I whole-body scintigraphy, histology) in all cases. Immunoscintigraphy was positive in 9 out of 11 cases by ECT and in 10 out of 11 cases by planar imaging. When using 111 In-labeled MAb fragments, intraindividual comparison of ECT and planar imaging resulted in a similar sensitivity. The increased sensitivity of ECT using this tracer in contrast to 131 I-labeled MAb fragments may be attributed to the fact that the physical properties of 111 In are much more suitable for the gamma cameras most commonly used (single detector, 3/8'' crystal); using 111 In-labelled MAb fragments, count rates sufficient for ECT can be obtained within a reasonable acquisition time. This allows to combine IS with the advantages of ECT regarding tumour localization and prevention of artefacts due to superposition of background. (orig.) [de

System visualization of integrated biofuels and high value chemicals developed within the MacroAlgaeBiorefinery (MAB3) project

DEFF Research Database (Denmark)

Seghetta, Michele; Hasler, Berit; Bastianoni, Simone

MacroAlgaeBiorefinery (MAB3) may functions as production platform and raw material supplier for future sustainable production chains of biofuels and high value chemicals. Biofuels are interesting energy source but challenges in terms of the composition of the biomass and resulting energy...... efficiencies has to be compensated for to make the biofuel prices competitive in replacing fossil fuel. Since it is difficult to increase the yield of the single biorefinery, the overall system productivity can be improved integrating different sub-systems. In this study, macroalgae cultivation in Denmark...... is integrated with a biogas biorefinery, a bioethanol biorefinery and a fish feed industry. The modeled system is able to adapt itself to different amount and quality of feedstock and to maximize valuable outputs (e.g. bio-fuels and chemical). Macroalgae are harvested and utilized as feedstock in bioethanol...

Cooperative binding of anti-tetanus toxin monoclonal antibodies: Implications for designing an efficient biclonal preparation to prevent tetanus toxin intoxication.

Science.gov (United States)

Lukic, Ivana; Filipovic, Ana; Inic-Kanada, Aleksandra; Marinkovic, Emilija; Miljkovic, Radmila; Stojanovic, Marijana

2018-05-15

Oligoclonal combinations of several monoclonal antibodies (MAbs) are being considered for the treatment of various infectious pathologies. These combinations are less sensitive to antigen structural changes than individual MAbs; at the same time, their characteristics can be more efficiently controlled than those of polyclonal antibodies. The main goal of this study was to evaluate the binding characteristics of six biclonal equimolar preparations (BEP) of tetanus toxin (TeNT)-specific MAbs and to investigate how the MAb combination influences the BEPs' protective capacity. We show that a combination of TeNT-specific MAbs, which not only bind TeNT but also exert positive cooperative effects, results in a BEP with superior binding characteristics and protective capacity, when compared with the individual component MAbs. Furthermore, we show that a MAb with only partial protective capacity but positive effects on the binding of the other BEP component can be used as a valuable constituent of the BEP. Copyright © 2018 Elsevier Ltd. All rights reserved.

A comparison of targeting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients

NARCIS (Netherlands)

Hoefnagel, C. A.; Rutgers, M.; Buitenhuis, C. K.; Smets, L. A.; de Kraker, J.; Meli, M.; Carrel, F.; Amstutz, H.; Schubiger, P. A.; Novak-Hofer, I.

2001-01-01

Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targeting ability in

Characterisation of chemically-modified proteins by electrospray ionisation mass spectrometry

International Nuclear Information System (INIS)

Bennett, K.L.

1996-09-01

Electrospray mass spectrometry (ESI-MS) has been used to examine a range of intact monoclonal antibodies (MAbs), antibody fragments such as F(ab') 2 , F ab and F c , chemically-modified fragments and a range of other chemically-modified peptides and proteins as part of a broader study aimed at establishing ESI-MS as a method for the characterisation of radioimmunoconjugates (radiolabelled monoclonal antibodies). For example, the addition of up to 10 biotin molecules to the 'papain-sensitive' 50 kDa F ab fragment can be easily detected in ESI mass spectra. For intact MAbs, however, it is only possible to detect average shifts in the mass of intact antibodies following modification. Successful ESI-MS analysis of complexes formed between chelators and other small molecules conjugated to synthetic peptides, hen egg-white Iysozyme (HEL) (M r 14 306) and horse heart myoglobin (M r 16 951) has been demonstrated. ESI-MS offers considerable advantages compared with existing methods for the characterisation of chemically-conjugated proteins including speed and sensitivity of analysis and the capability for obtaining specific structural information. The conditions for ESI-MS of intact MAbs and MAb fragments have been examined in detail and it was found that 150 kDa MAbs generally required lower sample concentration and higher skimmer potentials compared with the 50 kDa F ab fragment and other lower molecular weight proteins. In addition, the m/z range over which ions from MAbs were observed was higher (m/z ∼2000-4500) than for smaller proteins. ESI-MS was also found to be useful for probing the action of the protease papain, that is used to generate MAb fragments (F(ab) '2, F ab and F c ). Further, different sensitivities to papain for different MAb preparations was demonstrated. Finally, the tandem mass spectra of a range of peptides modified by iodine and biotin were examined. In the case of biotinylated peptides, a characteristic fragment ion was identified that could

Interconnected nitrogen and sulfur dual-doped porous carbon as efficient electrocatalyst for triiodide reduction in dye-sensitized solar cells

Science.gov (United States)

Li, Zhao; Yang, Wang; Xu, Xiuwen; Tang, Yushu; Zeng, Ziwei; Yang, Fan; Zhang, Liqiang; Ning, Guoqing; Xu, Chunming; Li, Yongfeng

2016-09-01

Exploiting cost-effective and efficient counter electrodes (CEs) for the reduction of triiodide (I3-) has been a persistent objective for the development of dye-sensitized solar cells (DSSCs). Here, we propose a strategy for the synthesis of nitrogen and sulfur dual-doped porous carbon (N/S-PC) via a thermal annealing approach by using melamine as N source, and basic magnesium sulfate (BMS) whiskers as S source and templates. Benefiting from the high surface area, unique interconnected structural feature and synergistic effects of N/S dual-doping, the N/S-PC shows excellent electrocatalytic activity toward I3- reduction, which has simultaneously been confirmed by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Tafel polarization measurements. The DSSC devices with N/S-PC CEs exhibit a PCE up to 7.41%, which is higher than that of DSSC devices with single heteroatom (N or S) doped CEs and even Pt CEs (7.14%).

Bubble-induced skin-friction drag reduction and the abrupt transition to air-layer drag reduction

Science.gov (United States)

Elbing, Brian R.; Winkel, Eric S.; Lay, Keary A.; Ceccio, Steven L.; Dowling, David R.; Perlin, Marc

To investigate the phenomena of skin-friction drag reduction in a turbulent boundary layer (TBL) at large scales and high Reynolds numbers, a set of experiments has been conducted at the US Navy's William B. Morgan Large Cavitation Channel (LCC). Drag reduction was achieved by injecting gas (air) from a line source through the wall of a nearly zero-pressure-gradient TBL that formed on a flat-plate test model that was either hydraulically smooth or fully rough. Two distinct drag-reduction phenomena were investigated; bubble drag reduction (BDR) and air-layer drag reduction (ALDR).The streamwise distribution of skin-friction drag reduction was monitored with six skin-friction balances at downstream-distance-based Reynolds numbers to 220 million and at test speeds to 20.0msinitial zone1. These results indicated that there are three distinct regions associated with drag reduction with air injection: Region I, BDR; Region II, transition between BDR and ALDR; and Region III, ALDR. In addition, once ALDR was established: friction drag reduction in excess of 80% was observed over the entire smooth model for speeds to 15.3ms1 with the surface fully roughened (though approximately 50% greater volumetric air flux was required); and ALDR was sensitive to the inflow conditions. The sensitivity to the inflow conditions can be mitigated by employing a small faired step (10mm height in the experiment) that helps to create a fixed separation line.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

Science.gov (United States)

Heyworth, M F; Ho, K E; Pappo, J

1989-11-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Sensitive innovation

DEFF Research Database (Denmark)

Søndergaard, Katia Dupret

Present paper discusses sources of innovation as heterogenic and at times intangible processes. Arguing for heterogeneity and intangibility as sources of innovation originates from a theoretical reading in STS and ANT studies (e.g. Callon 1986, Latour 1996, Mol 2002, Pols 2005) and from field work...... in the area of mental health (Dupret Søndergaard 2009, 2010). The concept of sensitive innovation is developed to capture and conceptualise exactly those heterogenic and intangible processes. Sensitive innovation is therefore primarily a way to understand innovative sources that can be......, but are not necessarily, recognized and acknowledged as such in the outer organisational culture or by management. The added value that qualifies these processes to be defined as “innovative” are thus argued for along different lines than in more traditional innovation studies (e.g. studies that build on the classic...

Optical properties of humic substances and CDOM: effects of borohydride reduction.

Science.gov (United States)

Ma, Jiahai; Del Vecchio, Rossana; Golanoski, Kelli S; Boyle, Erin S; Blough, Neil V

2010-07-15

Treatment of Suwanee River humic (SRHA) and fulvic (SRFA) acids, a commercial lignin (LAC), and a series of solid phase extracts (C18) from the Middle Atlantic Bight (MAB extracts) with sodium borohydride (NaBH(4)), a selective reductant of carbonyl-containing compounds including quinones and aromatic ketones, produces a preferential loss of visible absorption (> or = 50% for SRFA) and substantially enhanced, blue-shifted fluorescence emission (2- to 3-fold increase). Comparison of the results with those obtained from a series of model quinones and hydroquinones demonstrates that these spectral changes cannot be assigned directly to the absorption and emission of visible light by quinones/hydroquinones. Instead, these results are consistent with a charge transfer model in which the visible absorption is due primarily to charge transfer transitions arising among hydroxy- (methoxy-) aromatic donors and carbonyl-containing acceptors. Unlike most of the model hydroquinones, the changes in optical properties of the natural samples following NaBH(4) reduction were largely irreversible in the presence of air and following addition of a Cu(2+) catalyst, providing tentative evidence that aromatic ketones (or other similar carbonyl-containing structures) may play a more important role than quinones in the optical properties of these materials.

Time-resolved fluoroimmunoassay of phytoviruses by monoclonal antibodies. Vremenno-razreshennyj fluoroimmunoanaliz fitovirusos pri pomoshchi monoklonal'nykh antitel

Energy Technology Data Exchange (ETDEWEB)

Saarma, M Yu; Yarvekyul' g, L V; Andreeva, E G; Siniyarv, R K

1989-01-01

Monoclonal antibodies (MAbs) to potato viruses were labelled with lanthanides and used for time-resolved fluoroimmunoassay (TRFIA) of these viruses. TRFIA of potato leafroll virus (PLRV) had the sensitivity less than 1 ng/ml and was therefore approx 10 times more sensitive than ELISA. Possibility of simultaneous quantitative detection of several viruses by different MAbs labelled with europium, samarium and terbium provides TRFIA of fundamental advantage. Data on simultaneous sensitive detection of two viruses - PLRV and PVX (0,5 ng/ml of both) and three viruses - PLRV, PVX and PVM are given.

Characterization of human monoclonal antibodies that neutralize multiple poliovirus serotypes.

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Puligedda, Rama Devudu; Kouiavskaia, Diana; Al-Saleem, Fetweh H; Kattala, Chandana Devi; Nabi, Usman; Yaqoob, Hamid; Bhagavathula, V Sandeep; Sharma, Rashmi; Chumakov, Konstantin; Dessain, Scott K

2017-10-04

Following the eradication of wild poliovirus (PV), achieving and maintaining a polio-free status will require eliminating potentially pathogenic PV strains derived from the oral attenuated vaccine. For this purpose, a combination of non-cross-resistant drugs, such as small molecules and neutralizing monoclonal antibodies (mAbs), may be ideal. We previously isolated chimpanzee and human mAbs capable of neutralizing multiple PV types (cross-neutralization). Here, we describe three additional human mAbs that neutralize types 1 and 2 PV and one mAb that neutralizes all three types. Most bind conformational epitopes and have unusually long heavy chain complementarity determining 3 domains (HC CDR3). We assessed the ability of the mAbs to neutralize A12 escape mutant PV strains, and found that the neutralizing activities of the mAbs were disrupted by different amino acid substitutions. Competitive binding studies further suggested that the specific mAb:PV interactions that enable cross-neutralization differ among mAbs and serotypes. All of the cloned mAbs bind PV in the vicinity of the "canyon", a circular depression around the 5-fold axis of symmetry through which PV recognizes its cellular receptor. We were unable to generate escape mutants to two of the mAbs, suggesting that their epitopes are important for the PV life cycle. These data indicate that PV cross-neutralization involves binding to highly conserved structures within the canyon that binds to the cellular receptor. These may be facilitated by the long HC CDR3 domains, which may adopt alternative binding configurations. We propose that the human and chimpanzee mAbs described here could have potential as anti-PV therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Investigations into the use of radiolabeled monoclonal antibodies for selective cell labeling in whole blood]: Progress report, March 1985-May 1988

International Nuclear Information System (INIS)

Thakur, M.L.

1987-01-01

Seventeen monoclonal antibodies (MAbs), 7 specific for human platelets and 10 specific for human polumorphonuclear leukocytes (PMNs) have been evaluated. One MAb has been identified as the antibody most suitable for canine platelets and another has been evaluted as the best among the group, for human neutrophil studies. Indium-111, Tc-99m, and I-125 have been used as the tracers. Six bifunctional chelating agents (BFCAs) were evaluated in order to determine the most efficient agent for maximal cell labeling efficiency. Among these, the DTPA has given us the best results. (4) To botain maximum In-111 chelation and minimum loss of the MAb affinity, the optimal BFCA to MAb ratios for both IgG and IgM type of MAbs were determined. Four different substances, stannous chloride, ascorbic acid, sodium dithionite and sodium borohydride, were evaluated as reducing agents for Tc-99m reduction and its optimal binding to MAbs. Dithionite at the concentration of 200 ug/ml DTPA-MAb solution provides greater than 50% Tc-99m labeling efficiency and maintains its immunospecificity equal to that of In-111-DTPA-MAb. The ability of radiolabeled MAb to interact with blood cells selectively in whole blood and with isolated blood cells was assessed and compared

26 CFR 1.1374-2 - Net recognized built-in gain.

Science.gov (United States)

2010-04-01

... 26 Internal Revenue 11 2010-04-01 2010-04-01 true Net recognized built-in gain. 1.1374-2 Section 1...-in gain. (a) In general. An S corporation's net recognized built-in gain for any taxable year is the... considering only its recognized built-in gain, recognized built-in loss, and recognized built-in gain...

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

Directory of Open Access Journals (Sweden)

Leticia Barboza Rocha

2017-10-01

Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

Science.gov (United States)

Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong

2015-02-01

Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008

NARCIS (Netherlands)

Head, M.W.; Yull, H.M.; Ritchie, D.L.; Langeveld, J.P.M.; Fletcher, N.A.; Knight, R.S.; Ironside, J.W.

2013-01-01

Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified

Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

Science.gov (United States)

Buck, Elizabeth; Gokhale, Prafulla C; Koujak, Susan; Brown, Eric; Eyzaguirre, Alexandra; Tao, Nianjun; Rosenfeld-Franklin, Maryland; Lerner, Lorena; Chiu, M Isabel; Wild, Robert; Epstein, David; Pachter, Jonathan A; Miglarese, Mark R

2010-10-01

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.

Selective changes in expression of HLA class I polymorphic determinants in human solid tumors

International Nuclear Information System (INIS)

Natali, P.G.; Nicotra, M.R.; Bigotti, A.; Venturo, I.; Giacomini, P.; Marcenaro, L.; Russo, C.

1989-01-01

Analysis of surgical biopsies with monoclonal antibodies (mAbs) to framework determinants of major histocompatibility complex class I antigens has shown that malignant transformation is frequently associated with a marked loss of these cell surface molecules. The present study sought to determine whether more selective losses of major histocompatibility complex class I expression occur. Multiple specimens from 13 different types of primary and metastatic tumors were tested utilizing mAb BB7.2, which recognizes a polymorphic HLA-A2 epitope. In each case, expression of HLA-A,B,C molecules was determined by testing with mAb W6/32 directed to a framework HLA class I determinant. The authors have found that in HLA-A2-positive patients, HLA-A2 products are not detectable or are reduced in their expression in 70-80% of endometrial, colorectal, mammary, and renal tumors; in 40-60% of soft-tissue, skin, ovary, urinary bladder, prostate, and stomach tumors; and in 25-30% of melanomas and lung carcinomas tested. All tumors expressed the framework HLA-A,B.C determinant. The HLA-A2 epitope recognized by mAb BB7.2 is located in a portion of the HLA-A2 molecule postulated to react with the T-cell receptor. The selective loss of an HLA class I polymorphic epitope shown in this study may explain the mechanism by which tumor cells escape both T-cell recognition and natural killer cell surveillance

Dissociation of alloantigen recognition from self major histocompatibility complex-restricted recognition of cytolytic T lymphocytes by monoclonal antireceptor antibodies

International Nuclear Information System (INIS)

Kanagawa, O.; Nagasawa, R.

1987-01-01

Two monoclonal antibodies (mAb) directed to the dual reactive cytolytic T lymphocyte clone OH8 (D/sup b/T H-Y and H-2/sup d/) were established. Analysis by cell surface staining and immunoprecipitation of radiolabeled surface molecules of OH8 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveled that both mAb recognized an identical heterodimeric, clonotypic structure on OH8 cells, i.e., T cell receptor. However, although the MR3-2 mAb inhibited the lysis of either D/sup b/ + H-Y or H-2/sup d/ targets by OH8, the MR3-6 mAb inhibited the lysis of H-2/sup d/ target cells, but not that of D/sup b/ + H-Y target cells. Modulation of T cell receptor by either MR3-2 or MR3-6 mAb rendered the OH8 cytolytic T lyrphocyte incapable of killing both D/sup b/ + H-Y and H-2/sup d/ target cells. These finding suggest that different epitopes of OH8 T cell receptor were involved for the recognition of self + antigen and alloantigen

A novel sandwich enzyme-linked immunosorbent assay with covalently bound monoclonal antibody and gold probe for sensitive and rapid detection of bovine β-lactoglobulin.

Science.gov (United States)

He, Shengfa; Li, Xin; Wu, Yong; Wu, Shandong; Wu, Zhihua; Yang, Anshu; Tong, Ping; Yuan, Juanli; Gao, Jinyan; Chen, Hongbing

2018-06-01

Bovine milk is a recognized allergenic food source with β-lactoglobulin (BLG) as its major allergen. Reliable detection of BLG epitopes can, therefore, be a useful marker for the presence of milk in processed food products, and for potential allergenicity. At the present, enzyme-linked immunosorbent assays (ELISA) for the detection of BLG are time-consuming and generally not specific to BLG IgE epitopes. In this study, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-activated anti-BLG IgE epitope monoclonal antibody (mAb 1G9) was covalently bound onto the KOH-treated microtiter plate surface. Using this mAb-bound plate in sandwich combination with biotinylated anti-BLG polyclonal antibody-labeled gold nanoparticles, a linear dynamic range between 31.25 and 64 × 10 3  ng mL -1 with a limit of detection for BLG of 0.49 ng mL -1 was obtained, which is 32 times wider and 16 times more sensitive than conventional sandwich ELISA (sELISA). Total recovery of BLG in spiked food samples was found, without matrix effects. Also in partially hydrolyzed infant formulas, the allergenic BLG residues were detected quantitatively. Compared with conventional and commercial BLG detection sELISAs, our sELISA is reliable, highly BLG epitope-specific, user-friendly, and time-saving and allows accurate detection of potentially allergenic residues in different types of processed foods. This improved sELISA protocol can be easily extended to detect other well-identified and characterized food allergens. Graphical abstract IgE epitope mAb-bound plate in sandwich combination with gold probe for sensitive and rapid detection of bovine β-lactoglobulin and its potentially allergenic residues.

Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1).

Science.gov (United States)

Gelanew, Tesfaye; Hunsperger, Elizabeth

2018-02-06

Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to - 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1-4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1-4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1-3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.

Prevalence of sensitization to Cannabis sativa. Lipid-transfer and thaumatin-like proteins are relevant allergens.

Science.gov (United States)

Larramendi, Carlos H; López-Matas, M Ángeles; Ferrer, Angel; Huertas, Angel Julio; Pagán, Juan Antonio; Navarro, Luis Ángel; García-Abujeta, José Luis; Andreu, Carmen; Carnés, Jerónimo

2013-01-01

Although allergy to Cannabis sativa was first reported over 40 years ago, the allergenicity has scarcely been studied. The objectives of this study were to investigate the frequency of sensitization to this plant, to analyze the clinical characteristics and allergenic profile of sensitized individuals and to identify the allergens involved. Five hundred and forty-five individuals in Spain attending allergy clinics with respiratory or cutaneous symptoms underwent a skin-prick test (SPT) with C. sativa leaf extract. The extract was characterized by SDS-PAGE and 2-dimensional electrophoresis. Specific IgE to C. sativa was measured in positive SPT individuals. The clinical and allergenic profiles of sensitized individuals were investigated and the most-recognized allergens sequenced and characterized by liquid chromatography-mass spectrometry/mass spectrometry. Of this preselected population, 44 individuals had positive SPT to C. sativa (prevalence 8.1%). Prevalence was higher in individuals who were C. sativa smokers (14.6%). Two individuals reported mild symptoms with C. sativa. Twenty-one individuals from 32 available sera (65.6%) had positive specific IgE to C. sativa. Twelve sera recognized at least 6 different bands in a molecular-weight range of between 10 and 60 kDa. Six of them recognized a 10-kDa band, identified as a lipid transfer protein (LTP) and 8 recognized a 38-kDa band, identified as a thaumatin-like protein. There is a high prevalence of sensitization to C. sativa leaves. The clinical symptoms directly attributed to C. sativa were uncommon and mild. The sensitization profile observed suggests that C. sativa sensitization may be mediated by two mechanisms, i.e. cross-reactivity, mainly with LTP and thaumatin-like protein, and exposure-related 'de novo' sensitization. Copyright © 2013 S. Karger AG, Basel.

76 FR 55915 - Agency Forms Undergoing Paperwork Reduction Act Review

Science.gov (United States)

2011-09-09

... type 2 diabetes DPRP Application Form........ 120 1 1 prevention programs. DPRP Evaluation Data... mechanism for recognizing organizations that deliver effective, community-based type 2 diabetes prevention...] Agency Forms Undergoing Paperwork Reduction Act Review The Centers for Disease Control and Prevention...

Global sensitivity analysis of a model related to memory formation in synapses: Model reduction based on epistemic parameter uncertainties and related issues.

Science.gov (United States)

Kulasiri, Don; Liang, Jingyi; He, Yao; Samarasinghe, Sandhya

2017-04-21

We investigate the epistemic uncertainties of parameters of a mathematical model that describes the dynamics of CaMKII-NMDAR complex related to memory formation in synapses using global sensitivity analysis (GSA). The model, which was published in this journal, is nonlinear and complex with Ca 2+ patterns with different level of frequencies as inputs. We explore the effects of parameter on the key outputs of the model to discover the most sensitive ones using GSA and partial ranking correlation coefficient (PRCC) and to understand why they are sensitive and others are not based on the biology of the problem. We also extend the model to add presynaptic neurotransmitter vesicles release to have action potentials as inputs of different frequencies. We perform GSA on this extended model to show that the parameter sensitivities are different for the extended model as shown by PRCC landscapes. Based on the results of GSA and PRCC, we reduce the original model to a less complex model taking the most important biological processes into account. We validate the reduced model against the outputs of the original model. We show that the parameter sensitivities are dependent on the inputs and GSA would make us understand the sensitivities and the importance of the parameters. A thorough phenomenological understanding of the relationships involved is essential to interpret the results of GSA and hence for the possible model reduction. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

Science.gov (United States)

Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

2016-01-01

Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

Preparation of monoclonal antibodies against radiation-induced protein

International Nuclear Information System (INIS)

Nozawa, R.; Tanaka, A.; Watanabe, H.; Kitayama, S.

1992-01-01

We obtained the 6 monoclonal antibodies against gamma-induced proteins of Deinococcus radiodurans, and these antibodies were designated as Mab-3F, 4B, 4D, 4F, 4G and 12G. Using these antibodies, we investigated the relations between gamma-induced proteins and other stress protein in strain R1, and the induction of proteins were compared among strain R1, resistant mutant (rec1) and radiosensitive mutant (rec30). We found new 6 proteins recognized by these monoclonal antibodies which were induced after gamma-irradiation especially in strain R1 and rec 1, but not induced in strain rec30. We suppose that these proteins participate in repair of DNA damages including double strand breaks caused by gamma-irradiation. One of them was around 46kDa protein band recognized by Mab-12G, and this protein was so induced in a large quantity after irradiation that the protein could detect by gold staining. In addition to this observation, we found some proteins which were induced in R1 and rec 1 by gamma-irradiation and other stress, but not in strain rec30, such as 31kDa protein band recognized by Mab-3F, 4B and 4G, and other 11 proteins which were especially induced in irradiated strain R1. The latter proteins might be reinforcement factor to radioresistance such as GroE and DnaK, or participant in repair of damage by gamma-irradiation in strain R1. (author)

Nerium oleander indirect leaf photosynthesis and light harvesting reductions after clipping injury or Spodoptera eridania herbivory: high sensitivity to injury.

Science.gov (United States)

Delaney, Kevin J

2012-04-01

Variable indirect photosynthetic rate (P(n)) responses occur on injured leaves after insect herbivory. It is important to understand factors that influence indirect P(n) reductions after injury. The current study examines the relationship between gas exchange and chlorophyll a fluorescence parameters with injury intensity (% single leaf tissue removal) from clipping or Spodoptera eridania Stoll (Noctuidae) herbivory on Nerium oleander L. (Apocynaceae). Two experiments showed intercellular [CO(2)] increases but P(n) and stomatal conductance reductions with increasing injury intensity, suggesting non-stomatal P(n) limitation. Also, P(n) recovery was incomplete at 3d post-injury. This is the first report of a negative exponential P(n) impairment function with leaf injury intensity to suggest high N. oleander leaf sensitivity to indirect P(n) impairment. Negative linear functions occurred between most other gas exchange and chlorophyll a fluorescence parameters with injury intensity. The degree of light harvesting impairment increased with injury intensity via lower (1) photochemical efficiency indicated lower energy transfer efficiency from reaction centers to PSII, (2) photochemical quenching indicated reaction center closure, and (3) electron transport rates indicated less energy traveling through PSII. Future studies can examine additional mechanisms (mesophyll conductance, carbon fixation, and cardenolide induction) to cause N. oleander indirect leaf P(n) reductions after injury. Published by Elsevier Ireland Ltd.

Simple and ultra-fast recognition and quantitation of compounded monoclonal antibodies: Application to flow injection analysis combined to UV spectroscopy and matching method.

Science.gov (United States)

Jaccoulet, E; Schweitzer-Chaput, A; Toussaint, B; Prognon, P; Caudron, E

2018-09-01

Compounding of monoclonal antibody (mAbs) constantly increases in hospital. Quality control (QC) of the compounded mAbs based on quantification and identification is required to prevent potential errors and fast method is needed to manage outpatient chemotherapy administration. A simple and ultra-fast (less than 30 s) method using flow injection analysis associated to least square matching method issued from the analyzer software was performed and evaluated for the routine hospital QC of three compounded mAbs: bevacizumab, infliximab and rituximab. The method was evaluated through qualitative and quantitative parameters. Preliminary analysis of the UV absorption and second derivative spectra of the mAbs allowed us to adapt analytical conditions according to the therapeutic range of the mAbs. In terms of quantitative QC, linearity, accuracy and precision were assessed as specified in ICH guidelines. Very satisfactory recovery was achieved and the RSD (%) of the intermediate precision were less than 1.1%. Qualitative analytical parameters were also evaluated in terms of specificity, sensitivity and global precision through a matrix of confusion. Results showed to be concentration and mAbs dependant and excellent (100%) specificity and sensitivity were reached within specific concentration range. Finally, routine application on "real life" samples (n = 209) from different batch of the three mAbs complied with the specifications of the quality control i.e. excellent identification (100%) and ± 15% of targeting concentration belonging to the calibration range. The successful use of the combination of second derivative spectroscopy and partial least square matching method demonstrated the interest of FIA for the ultra-fast QC of mAbs after compounding using matching method. Copyright © 2018 Elsevier B.V. All rights reserved.

Recognizing, Confronting, and Eliminating Workplace Bullying.

Science.gov (United States)

Berry, Peggy Ann; Gillespie, Gordon L; Fisher, Bonnie S; Gormley, Denise K

2016-07-01

Workplace bullying (WPB) behaviors negatively affect nurse productivity, satisfaction, and retention, and hinder safe patient care. The purpose of this article is to define WPB, differentiate between incivility and WPB, and recommend actions to prevent WPB behaviors. Informed occupational and environmental health nurses and nurse leaders must recognize, confront, and eliminate WPB in their facilities and organizations. Recognizing, confronting, and eliminating WPB behaviors in health care is a crucial first step toward sustained improvements in patient care quality and the health and safety of health care employees. © 2016 The Author(s).

Recognizing textual entailment models and applications

CERN Document Server

Dagan, Ido; Sammons, Mark

2013-01-01

In the last few years, a number of NLP researchers have developed and participated in the task of Recognizing Textual Entailment (RTE). This task encapsulates Natural Language Understanding capabilities within a very simple interface: recognizing when the meaning of a text snippet is contained in the meaning of a second piece of text. This simple abstraction of an exceedingly complex problem has broad appeal partly because it can be conceived also as a component in other NLP applications, from Machine Translation to Semantic Search to Information Extraction. It also avoids commitment to any sp

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice.

Science.gov (United States)

Gonzalez-Suarez, Maria L; Salinas-Carmona, Mario C; Pérez-Rivera, Isabel

2009-10-01

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti-N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis. IgG mAb lacked this protective effect. IgM mAb showed a dose-response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.

A study on limb reduction defects in six European regions

NARCIS (Netherlands)

Stoll, C; Calzolari, E; Cornel, M; GarciaMinaur, S; Garne, E; Nevin, N

1996-01-01

Limb reduction defects (LRD) gained especial attention after the thalidomide tragedy in 1962, LRD are common congenital malformations which present as obvious congenital anomalies recognized at birth, Therefore it might be assumed that they are well documented, However classification of LRDs is

PrP(Sc-specific antibodies with the ability to immunodetect prion oligomers.

Directory of Open Access Journals (Sweden)

Mourad Tayebi

Full Text Available The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0 cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.

An oncolytic adenovirus regulated by a radiation-inducible promoter selectively mediates hSulf-1 gene expression and mutually reinforces antitumor activity of I131-metuximab in hepatocellular carcinoma.

Science.gov (United States)

Zhang, Yan; Fang, Lin; Zhang, Quan'an; Zheng, Qin; Tong, Jinlong; Fu, Xiaohui; Jiang, Xiaoqing; Su, Changqing; Zheng, Junnian

2013-06-01

Gene therapy and antibody approaches are crucial auxiliary strategies for hepatocellular carcinoma (HCC) treatment. Previously, we established a survivin promoter-regulated oncolytic adenovirus that has inhibitory effect on HCC growth. The human sulfatase-1 (hSulf-1) gene can suppress the growth factor signaling pathways, then inhibit the proliferation of cancer cells and enhance cellular sensitivity to radiotherapy and chemotherapy. I(131)-metuximab (I(131)-mab) is a monoclonal anti-HCC antibody that conjugated to I(131) and specifically recognizes the HAb18G/CD147 antigen on HCC cells. To integrate the oncolytic adenovirus-based gene therapy and the I(131)-mab-based radioimmunotherapy, this study combined the CArG element of early growth response-l (Egr-l) gene with the survivin promoter to construct a radiation-inducible enhanced promoter, which was used to recombine a radiation-inducible oncolytic adenovirus as hSulf-1 gene vector. When I(131)-mab was incorporated into the treatment regimen, not only could the antibody produce radioimmunotherapeutic effect, but the I(131) radiation was able to further boost adenoviral proliferation. We demonstrated that the CArG-enhanced survivin promoter markedly improved the proliferative activity of the oncolytic adenovirus in HCC cells, thereby augmenting hSulf-1 expression and inducing cancer cell apoptosis. This novel strategy that involved multiple, synergistic mechanisms, including oncolytic therapy, gene therapy and radioimmunotherapy, was demonstrated to exert an excellent anti-cancer outcome, which will be a promising approach in HCC treatment. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A cocatalyst-free Eosin Y-sensitized p-type of Co₃O₄ quantum dot for highly efficient and stable visible-light-driven water reduction and hydrogen production.

Science.gov (United States)

Zhang, Ning; Shi, Jinwen; Niu, Fujun; Wang, Jian; Guo, Liejin

2015-09-07

Owing to the effect of energy band bending, p-type Co3O4 quantum dots sensitized by Eosin Y showed a high and stable photocatalytic activity (∼13,440 μmol h(-1) g(-1)(cat)) for water reduction and hydrogen production under visible-light irradiation without any cocatalyst.

Surface Sensitive Bolometer for the CUORE background reduction

International Nuclear Information System (INIS)

Pedretti, M.; Foggetta, L.; Giuliani, A.; Nones, C.; Sangiorgio, S.

2005-01-01

The most critical point of the CUORE Project [CUORE Proposal, see the web page: http://crio.mib.infn.it/wig] is the background level (BKGL) in the neutrinoless double beta decay (0νββ) region that is dominated by degraded particles coming from materials that face the detectors. Surface Sensitive Bolometers (SSBs) have been developed in order to reduce the BKGL by means of an active background discrimination. The principle of this technique and the first results obtained are briefly described in the following paper

76 FR 33419 - Nationally Recognized Statistical Rating Organizations

Science.gov (United States)

2011-06-08

... 232, 240, 249, et al. Nationally Recognized Statistical Rating Organizations; Proposed Rule #0;#0...-11] RIN 3235-AL15 Nationally Recognized Statistical Rating Organizations AGENCY: Securities and... rating organizations (``NRSROs''). In addition, in accordance with the Dodd-Frank Act, the Commission is...

A cooperative reduction model for regional air pollution control in China that considers adverse health effects and pollutant reduction costs.

Science.gov (United States)

Xie, Yujing; Zhao, Laijun; Xue, Jian; Hu, Qingmi; Xu, Xiang; Wang, Hongbo

2016-12-15

How to effectively control severe regional air pollution has become a focus of global concern recently. The non-cooperative reduction model (NCRM) is still the main air pollution control pattern in China, but it is both ineffective and costly, because each province must independently fight air pollution. Thus, we proposed a cooperative reduction model (CRM), with the goal of maximizing the reduction in adverse health effects (AHEs) at the lowest cost by encouraging neighboring areas to jointly control air pollution. CRM has two parts: a model of optimal pollutant removal rates using two optimization objectives (maximizing the reduction in AHEs and minimizing pollutant reduction cost) while meeting the regional pollution control targets set by the central government, and a model that allocates the cooperation benefits (i.e., health improvement and cost reduction) among the participants according to their contributions using the Shapley value method. We applied CRM to the case of sulfur dioxide (SO 2 ) reduction in Yangtze River Delta region. Based on data from 2003 to 2013, and using mortality due to respiratory and cardiovascular diseases as the health endpoints, CRM saves 437 more lives than NCRM, amounting to 12.1% of the reduction under NCRM. CRM also reduced costs by US $65.8×10 6 compared with NCRM, which is 5.2% of the total cost of NCRM. Thus, CRM performs significantly better than NCRM. Each province obtains significant benefits from cooperation, which can motivate them to actively cooperate in the long term. A sensitivity analysis was performed to quantify the effects of parameter values on the cooperation benefits. Results shown that the CRM is not sensitive to the changes in each province's pollutant carrying capacity and the minimum pollutant removal capacity, but sensitive to the maximum pollutant reduction capacity. Moreover, higher cooperation benefits will be generated when a province's maximum pollutant reduction capacity increases. Copyright �

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

Science.gov (United States)

Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

Directory of Open Access Journals (Sweden)

Livia C L Lopes

2010-10-01

Full Text Available Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1 to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

Enrichment of anammox bacteria fro marine environment for the construction of a bioremediation reactor

Energy Technology Data Exchange (ETDEWEB)

Nakajima, Jun; Sakka, Makiko; Kimura, Tetsuya; Sakka, Kazuo [Mie Univ., Tsu (Japan). Graduate School of Bioresources; Furukawa, Kenji [Kumamoto Univ. (Japan). Dept. of Civil Engineering and Architecture

2008-01-15

In the global ocean nitrogen cycle, the anaerobic ammonium-oxidizing (anammox) process is recognized as important. In this study, we established an enrichment culture of marine anammox bacteria (MAB) in a column-type reactor. The reactor, which included a porous polyester non-woven fabric that had been placed at the sea floor in advance for enrichment, was continuously fed with NH{sub 4}Cl and NaNO{sub 2} for more than 1 year. Anammox activity in the MAB reactor was confirmed by {sup 15}N tracer analysis using {sup 15}NH{sub 4}Cl and Na{sup 14}NO{sub 2}. We identified two 16S rRNA genes in the amplified DNA fragments derived from MAB, which were highly homologous with those from Candidatus ''Scalindua wagneri'' and an uncultured planctomycete clone. Fluorescence in situ hybridization analysis using an anammox-specific probe also confirmed that MAB predominated in the reactor. To our knowledge, this is the first report on the establishment of an enrichment culture of anammox bacteria from the marine environment using a continuous culture system. (orig.)

Recognizing Body Dysmorphic Disorder (Dysmorphophobia)

Science.gov (United States)

Varma, Anukriti; Rastogi, Rajesh

2015-01-01

Dysmorphophobia is a psychiatric condition which frequently presents in the clinics of dermatologists and plastic surgeons. This disorder (also called body dysmorphic disorder) is troublesome to the patient whilst being confusing for the doctor. This commonly undiagnosed condition can be detected by a few simple steps. Timely referral to a psychiatrist benefits most patients suffering from it. This article describes with a case vignette, how to recognize body dysmorphic disorder presenting in the dermatological or aesthetic surgery set up. Diagnostic criteria, eitiology, approach to patient, management strategy and when to refer are important learning points. The importance of recognizing this disorder timely and referring the patient to the psychiatrist for appropriate treatment is crucial. This article covers all aspects of body dysmorphic disorder relevant to dermatologists and plastic surgeons and hopes to be useful in a better understanding of this disorder. PMID:26644741

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

Science.gov (United States)

Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

2017-08-01

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

46 CFR 188.10-59 - Recognized classification society.

Science.gov (United States)

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Recognized classification society. 188.10-59 Section 188... VESSELS GENERAL PROVISIONS Definition of Terms Used in This Subchapter § 188.10-59 Recognized classification society. This term means the American Bureau of Shipping or other classification society...

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables.

Science.gov (United States)

Moreno, María J; D'Arienzo, Pasquale; Manclús, Juan J; Montoya, Angel

2011-01-01

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I₅₀ value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

Science.gov (United States)

Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

2015-01-01

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus

DEFF Research Database (Denmark)

Fernandez-Alonso, M.; Lorenzo, G.; Perez, L.

1998-01-01

antibodies (MAbs), only 2 non-neutralizing MAbs, I10 (aa 139-153) and IP1H3 (aa 399-413), could be mapped to specific peptides in the pepscan of the gpG. Mapping of these MAbs was confirmed by immunoblotting with recombinant proteins and/or other synthetic peptides covering those sequences. None......Antibody Linear epitopes of the glycoprotein G (gpG) of the viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus of salmonids, were mapped by pepscan using overlapping 15-mer peptides covering the entire gpG sequence and ELISA with polyclonal and monoclonal murine and polyclonal trout...... antibodies. Among the regions recognized in the pepscan by the polyclonal antibodies (PAbs) were the previously identified phosphatidylserine binding heptad-repeats (Estepa & Coll 1996; Virology 216:60-70) and leucocyte stimulating peptides (Lorenzo et al. 1995; Virology 212:348-355). Among 17 monoclonal...

Production of human anti-HLA monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

1986-03-01

Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

High-Sensitivity Spectrophotometry.

Science.gov (United States)

Harris, T. D.

1982-01-01

Selected high-sensitivity spectrophotometric methods are examined, and comparisons are made of their relative strengths and weaknesses and the circumstances for which each can best be applied. Methods include long path cells, noise reduction, laser intracavity absorption, thermocouple calorimetry, photoacoustic methods, and thermo-optical methods.…

DISTRIBUTION OF GBM HEPARAN-SULFATE PROTEOGLYCAN CORE PROTEIN AND SIDE-CHAINS IN HUMAN GLOMERULAR-DISEASES

NARCIS (Netherlands)

VANDENBORN, J; VANDENHEUVEL, LPWJ; BAKKER, MAH; VEERKAMP, JH; ASSMANN, KJM; WEENING, JJ; BERDEN, JHM

Using monoclonal antibodies (mAbs) recognizing either the core protein or the heparan sulfate (HS) side chain of human GBM heparan sulfate proteoglycan (HSPG), we investigated their glomerular distribution on cryostat sections of human kidney tissues. The study involved 95 biopsies comprising twelve

Combined rapid (TUBEX test for typhoid-paratyphoid A fever based on strong anti-O12 response: design and critical assessment of sensitivity.

Directory of Open Access Journals (Sweden)

Meiying Yan

Full Text Available Rapid diagnostics can be accurate but, often, those based on antibody detection for infectious diseases are unwittingly underrated for various reasons. Herein, we described the development of a combined rapid test for two clinically-indistinguishable bacterial diseases, typhoid and paratyphoid A fever, the latter fast emerging as a global threat. By using monoclonal antibodies (mAbs to bacterial antigens of known chemical structures as probes, we were able to dissect the antibody response in patients at the level of monosaccharides. Thus, a mAb specific for a common lipopolysaccharide antigen (O12 found in both the causative organisms was employed to semi-quantify the amounts of anti-O12 antibodies present in both types of patients in an epitope-inhibition particle-based (TUBEX immunoassay. This colorimetric assay detected not only anti-O12 antibodies that were abundantly produced, but also, by steric hindrance, antibodies to an adjoining epitope (O9 or O2 in the typhoid or paratyphoid bacillus, respectively. Sensitivity and, particularly, reaction intensities, were significantly better than those obtained using an anti-O9 or anti-O2 mAb-probe in the examination of paired sera from 22 culture-confirmed typhoid patients (sensitivity, 81.8% vs 75.0% or single sera from 36 culture-confirmed paratyphoid patients (52.8% vs 28.6, respectively. Importantly, sensitivity was better (97.1% for typhoid, 75.0% for paratyphoid if allowance was made for the absence of relevant antibodies in certain specimens as determined by an independent, objective assay (ELISA--such specimens might have been storage-denatured (especially the older paratyphoid samples or procured from non-responders. Benchmarking against ELISA, which revealed high concordance between the two tests, was useful and more appropriate than comparing with culture methods as traditionally done, since antibody tests and culture target slightly different stages of these diseases. Paired sera

Reduction of graphene oxide at room temperature with vitamin C for RGO–TiO2 photoanodes in dye-sensitized solar cell

International Nuclear Information System (INIS)

Ding, Hui; Zhang, Sam; Chen, Ji-Tao; Hu, Xiao-Ping; Du, Zhao-Fu; Qiu, Yue-Xiu; Zhao, Dong-Liang

2015-01-01

Graphene has been used to enhance conversion efficiency in dye-sensitized solar cell (DSSC) through increased electronic transportation. Introduction of graphene into DSSC is realized through reduction of graphene oxide (GO) to reduced graphene or RGO in TiO 2 nanoparticles in solution. Chemical and hydrothermal reactions are two commonly used avenues. In the chemical route, toxic hydrazine and its derivatives are the typical reducing agent. Hydrothermal process is not toxic but requires prolonged heating. In this study, we mix flakes of GO with TiO 2 nanoparticles and use vitamin C to realize the reduction at room temperature. The RGO–TiO 2 composite films are then used as photoanode in DSSC. A conversion efficiency of 30% increase (to 7.89%) is obtained as compared to that of the pure TiO 2 photoanode. - Highlights: • We produce reduced graphene (RGO)–TiO 2 nanocomposite at room temperature. • The reduction is realized by vitamin C in a simple and environment-friendly way. • Reduced graphene provides a highway for electron transportation. • The RGO–P25 composite films are used as photoanode for DSSC. • Conversion efficiency of 30% increase (to 7.89%) is obtained by RGO–P25 photoanode

Genetic and antigenic characterization of Bungowannah virus, a novel pestivirus.

Science.gov (United States)

Kirkland, P D; Frost, M J; King, K R; Finlaison, D S; Hornitzky, C L; Gu, X; Richter, M; Reimann, I; Dauber, M; Schirrmeier, H; Beer, M; Ridpath, J F

2015-08-05

Bungowannah virus, a possible new species within the genus Pestivirus, has been associated with a disease syndrome in pigs characterized by myocarditis with a high incidence of stillbirths. The current analysis of the whole-genome and antigenic properties of this virus confirms its unique identity, and further suggests that this virus is both genetically and antigenically remote from previously recognized pestiviruses. There was no evidence of reactivity with monoclonal antibodies (mAbs) that are generally considered to be pan-reactive with other viruses in the genus, and there was little cross reactivity with polyclonal sera. Subsequently, a set of novel mAbs has been generated which allow detection of Bungowannah virus. The combined data provide convincing evidence that Bungowannah virus is a member of the genus Pestivirus and should be officially recognized as a novel virus species. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Sensitization to Gliadin Induces Moderate Enteropathy and Insulitis in Nonobese Diabetic-DQ8 Mice

Science.gov (United States)

Galipeau, Heather J.; Rulli, Nestor E.; Jury, Jennifer; Huang, Xianxi; Araya, Romina; Murray, Joseph A.; David, Chella S.; Chirdo, Fernando G.; McCoy, Kathy D.; Verdu, Elena F.

2012-01-01

Celiac disease (CD) is frequently diagnosed in patients with type 1 diabetes (T1D), and T1D patients can exhibit Abs against tissue transglutaminase, the auto-antigen in CD. Thus, gliadin, the trigger in CD, has been suggested to have a role in T1D pathogenesis. The objective of this study was to investigate whether gliadin contributes to enteropathy and insulitis in NOD-DQ8 mice, an animal model that does not spontaneously develop T1D. Gliadin-sensitized NOD-DQ8 mice developed moderate enteropathy, intraepithelial lymphocytosis, and barrier dysfunction, but not insulitis. Administration of anti-CD25 mAbs before gliadin-sensitization induced partial depletion of CD25+Foxp3+ T cells and led to severe insulitis, but did not exacerbate mucosal dysfunction. CD4+ T cells isolated from pancreatic lymph nodes of mice that developed insulitis showed increased proliferation and proinflammatory cytokines after incubation with gliadin but not with BSA. CD4+ T cells isolated from nonsensitized controls did not response to gliadin or BSA. In conclusion, gliadin sensitization induced moderate enteropathy in NOD-DQ8 mice. However, insulitis development required gliadin-sensitization and partial systemic depletion of CD25+Foxp3+ T cells. This humanized murine model provides a mechanistic link to explain how the mucosal intolerance to a dietary protein can lead to insulitis in the presence of partial regulatory T cell deficiency. PMID:21911598

Inhibition of Helicobacter pylori CagA-Induced Pathogenesis by Methylantcinate B from Antrodia camphorata

Directory of Open Access Journals (Sweden)

Chun-Jung Lin

2013-01-01

Full Text Available The bacterial pathogen Helicobacter pylori (Hp is the leading risk factor for the development of gastric cancer. Hp virulence factor, cytotoxin-associated gene A (CagA interacted with cholesterol-enriched microdomains and leads to induction of inflammation in gastric epithelial cells (AGS. In this study, we identified a triterpenoid methylantcinate B (MAB from the medicinal mushroom Antrodia camphoratawhich inhibited the translocation and phosphorylation of CagA and caused a reduction in hummingbird phenotype in HP-infected AGS cells. Additionally, MAB suppressed the Hp-induced inflammatory response by attenuation of NF-κB activation, translocation of p65 NF-κB, and phosphorylation of IκB-α, indicating that MAB modulates CagA-mediated signaling pathway. Additionally, MAB also suppressed the IL-8 luciferase activity and its secretion in HP-infected AGS cells. On the other hand, molecular structure simulations revealed that MAB interacts with CagA similarly to that of cholesterol. Moreover, binding of cholesterol to the immobilized CagA was inhibited by increased levels of MAB. Our results demonstrate that MAB is the first natural triterpenoid which competes with cholesterol bound to CagA leading to attenuation of Hp-induced pathogenesis of epithelial cells. Thus, this study indicates that MAB may have a scope to develop as a therapeutic candidate against Hp CagA-induced inflammation.

A sensitive impedance biosensor based on immunomagnetic separation and urease catalysis for rapid detection of Listeria monocytogenes using an immobilization-free interdigitated array microelectrode.

Science.gov (United States)

Chen, Qi; Lin, Jianhan; Gan, Chengqi; Wang, Yuhe; Wang, Dan; Xiong, Yonghua; Lai, Weihua; Li, Yuntao; Wang, Maohua

2015-12-15

In this study, we described a novel impedance biosensor combining immunomagnetic separation with urease catalysis for sensitive detection of foodborne bacteria using Listeria monocytogenes as model and an immobilization-free microelectrode as detector. The monoclonal antibodies (MAbs) were immobilized on the surface of the magnetic nanoparticles (MNPs) with the diameter of 180 nm by biotin-streptavidin system for specifically and efficiently separating Listeria cells from sample background. The polyclonal antibodies (PAbs) and the urease were modified onto the surface of the gold nanoparticles (AuNPs) with the diameter of 20 nm and the modified AuNPs were used to react with Listera to form the MNP-MAb-Listeria-PAb-AuNP-urease sandwich complexes. The urease in the complexes could catalyze the hydrolysis of the urea into ammonium carbonate and this led to an increase in the ionic strength of the media, which could be detected by the microelectrode. The magnetic separation efficiencies for L. monocytogenes at the concentrations ranging from 3.0×10(1) to 3.0×10(4) CFU/mL were over 95% for the pure cultures and over 85% for the spiked lettuce samples. The lower detection limit of this biosensor for L. monocytogenes was found to be 300 CFU/mL in both the pure cultures and the spiked lettuce samples. The microelectrode was demonstrated to be reusable for over 50 times with thorough cleaning by deionized water. This biosensor showed its potential to provide a simple, low-cost and sensitive method for rapid screening of foodborne pathogens and could be extended for detection of other biological or chemical targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

Directory of Open Access Journals (Sweden)

Cameron Martin

Full Text Available Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb, designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs from these species relative to cells incubated with an isotype control (p<0.001. In addition, the mAb induced significant nitric oxide (p<0.0001 release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001 IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

Monoclonal antibodies to the major Lolium perenne (rye grass) pollen allergen Lol p I (Rye I).

Science.gov (United States)

Kahn, C R; Marsh, D G

1986-12-01

Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I (Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I-Lol I. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, Kd, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 X 10(-6) L/M. This MAb recognized the related 27,000-30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I (Cyn d I), which by conventional analysis using polyclonal anti-Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

Directory of Open Access Journals (Sweden)

Davide Corti

2010-01-01

Full Text Available The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Molecular characterization of two sub-family specific monoclonal antibodies to meningococcal Factor H binding protein

Directory of Open Access Journals (Sweden)

C. Lo Passo

2018-04-01

Full Text Available Factor H binding protein (FHbp is a component of two licensed vaccines for prevention of sepsis and meningitis caused by serogroup B meningococci. FHbp binds human Factor H (FH, which contributes to evasion of host immunity and FHbp sequence variants can be classified into two sub-families. Antibodies against FHbp elicit complement-mediated killing and can inhibit recruitment of FH to the bacterial surface. We report epitope mapping studies of two murine IgG mAbs, designated JAR 31 and JAR 36, isolated from a mouse immunized with FHbp in sub-family A, which is present in ∼30–40% of invasive isolates. In the present study, we tested the reactivity of mAbs JAR 31 and JAR 36 with seven natural FHbp sequence variants from different phylogenic groups. We screened bacteriophage-displayed peptide libraries to identify amino acid residues contributing to the JAR 36 epitope. Based on the reactivities of mAbs JAR 31 and JAR 36 with the seven FHbp variants, and the frequent occurrences of aspartate (D and lysine (K residues in the JAR 36-bound phage peptides, we selected six residues in the carboxyl-terminal region of FHbp for replacement with alanine (A. The D201A and K203A substitutions respectively eliminated and decreased binding of mAbs JAR 31 and JAR 36 to FHbp. These substitutions did not affect binding of the control mAb JAR 33 or of human FH. JAR 31 or JAR 36 mediated cooperative complement-mediated bactericidal activity with other anti-FHbp mAbs. The identification of two amino acid residues involved in the epitopes recognized by these anti-FHbp mAbs may contribute to a more complete understanding of the spatial requirements for cooperative anti-FHbp mAb bactericidal activity. Keywords: Biochemistry, Immunology, Microbiology, Molecular biology

Green-house gasses reduction in the energy sector

International Nuclear Information System (INIS)

Todorovski, Mirko; Markovska, Natasha; Boshevski, Tome; Pop-Jordanov, Jordan

2004-01-01

As a follow-up activity of the Macedonian First National Communication under the UNFC-CC, Technology Needs Assessment is conducted, evaluating by GACMO model the measures related mainly to energy efficient and renewable energy technologies. An abatement cost curve is constructed and used as an illustrative tool for recognizing priorities in GHG abatement policy. About half of the measures are shown to be of 'win-win' type, reducing 3% of the baseline GHG emissions, while the total reduction which can be achieved by all measures amounts to 20%. For each measure difficulties for implementation are identified, being the highest for the measures with largest GHG abatement potential. Generally, these difficulties include lack of financing and low prospects for attracting foreign investments as well as legislative and administrative barriers. It must be recognized that climate change issues could not be of high priority in a country with economy in transition. Consequently, in our case the main issues which should be integrated within the framework of ongoing transition reforms, in an effort to implement a GHG emission reduction policy, would be: institutional capacity building, attracting foreign investment, emphasis on energy efficiency and considering the switch toward less carbon intensive fuels. (Author)

Comparison of Simulated PEC Probe Performance for Detecting Wall Thickness Reduction

International Nuclear Information System (INIS)

Shin, Young Kil; Choi, Dong Myung; Jung, Hee Sung

2009-01-01

In this paper, four different types of pulsed eddy current(PEC) probe are designed and their performance of detecting wall thickness reduction is compared. By using the backward difference method in time and the finite element method in space, PEC signals from various thickness and materials are numerically calculated and three features of the signal are selected. Since PEC signals and features are obtained by various types and sizes of probe, the comparison is made through the normalized features which reflect the sensitivity of the feature to thickness reduction. The normalized features indicate that the shielded reflection probe provides the best sensitivity to wall thickness reduction for all three signal features. Results show that the best sensitivity to thickness reduction can be achieved by the peak value, but also suggest that the time to peak can be a good candidate because of its linear relationship with the thickness variation.

Recognizing Multi-user Activities using Body Sensor Networks

DEFF Research Database (Denmark)

Gu, Tao; Wang, Liang; Chen, Hanhua

2011-01-01

The advances of wireless networking and sensor technology open up an interesting opportunity to infer human activities in a smart home environment. Existing work in this paradigm focuses mainly on recognizing activities of a single user. In this work, we address the fundamental problem...... activity classes of data—for building activity models and design a scalable, noise-resistant, Emerging Pattern based Multi-user Activity Recognizer (epMAR) to recognize both single- and multi-user activities. We develop a multi-modal, wireless body sensor network for collecting real-world traces in a smart...... home environment, and conduct comprehensive empirical studies to evaluate our system. Results show that epMAR outperforms existing schemes in terms of accuracy, scalability and robustness....

Improved foot sensitivity and pain reduction in patients with peripheral neuropathy after treatment with monochromatic infrared photo energy--MIRE.

Science.gov (United States)

Harkless, Lawrence B; DeLellis, Salvatore; Carnegie, Dale H; Burke, Thomas J

2006-01-01

The medical records of 2239 patients (mean age=73 years) with established peripheral neuropathy (PN) were examined to determine whether treatment with MIRE was, in fact, associated with increased foot sensitivity to the Semmes Weinstein monofilament (SWM) 5.07 and a reduction in neuropathic pain. The PN in 1395 of these patients (62%) was due to diabetes. Prior to treatment with MIRE, of the 10 tested sites (5 on each foot), 7.1+/-2.9 were insensitive to the SWM 5.07, and 2078 patients (93%) exhibited loss of protective sensation defined by Medicare as a loss of sensation at two or more sites on either foot. After treatment, the number of insensate sites on both feet decreased to 2.4+/-2.6, an improvement of 66%. Of the 2078 (93%) patients initially presenting with loss of protective sensation, 1106 (53%) no longer had loss of protective sensation after treatment (P<.0001); 1563 patients (70%) also exhibited neuropathic pain in addition to sensory impairment. Prior to treatment with MIRE, pain measured on the 11-point visual analogue scale (VAS) was 7.2+/-2.2 points, despite the use of a variety of pain-relieving therapeutic agents. After treatment with MIRE, pain was reduced by 4.8+/-2.4 points, a 67% reduction. Therefore, MIRE appears to be associated with significant clinical improvement in foot sensation and, simultaneously, a reduction in neuropathic pain in a large cohort of primarily Medicare aged, community-dwelling patients, initially diagnosed with PN. The quality of life associated with these two outcomes cannot be underappreciated.

Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

Energy Technology Data Exchange (ETDEWEB)

Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

1998-01-01

Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with {sup 99m}Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with {sup 99m}Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 {+-} 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.

Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

1998-01-01

Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99m Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99m Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 ± 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported

Sensitizing Young English Language Learners Towards Environmental Care

Science.gov (United States)

Castillo, Rigoberto; Rojas, María del Pilar

2014-01-01

This paper reports an action research study aimed at understanding how to sensitize young English language learners towards caring for the environment. The pedagogical intervention in a 5th grade class consisted in the use of creative writing strategies to express learners' ideas. Three stages were followed: "recognizing facts,"…

Synthesis and characterization of (Ni{sub 1−x}Co{sub x})Se{sub 2} based ternary selenides as electrocatalyst for triiodide reduction in dye-sensitized solar cells

Energy Technology Data Exchange (ETDEWEB)

Theerthagiri, J.; Senthil, R.A. [Solar Energy Lab, Department of Chemistry, Thiruvalluvar University, Vellore 632115 (India); Buraidah, M.H. [Centre for Ionics University of Malaya, Department of Physics, University of Malaya, Kuala Lumpur 50603 (Malaysia); Raghavender, M. [Department of Physics, Yogi Vemana University, Kadapa 516003, Andhra Pradesh (India); Madhavan, J., E-mail: jagan.madhavan@gmail.com [Solar Energy Lab, Department of Chemistry, Thiruvalluvar University, Vellore 632115 (India); Arof, A.K. [Centre for Ionics University of Malaya, Department of Physics, University of Malaya, Kuala Lumpur 50603 (Malaysia)

2016-06-15

Ternary metal selenides of (Ni{sub 1−x}Co{sub x})Se{sub 2} with 0≤x≤1 were synthesized by using one-step hydrothermal reduction route. The synthesized metal selenides were utilized as an efficient, low-cost platinum free counter electrode for dye-sensitized solar cells. The cyclic voltammetry and electrochemical impedance spectroscopy studies revealed that the Ni{sub 0.5}Co{sub 0.5}Se{sub 2} counter electrode exhibited higher electrocatalytic activity and lower charge transfer resistance at the counter electrode/electrolyte interface than the other compositions for reduction of triiodide to iodide. Ternary selenides of Ni{sub 0.5}Co{sub 0.5}Se{sub 2} offer a synergistic effect to the electrocatalytic activity for the reduction of triiodide that might be due to an increase in active catalytic sites and small charge transfer resistance. The DSSC with Ni{sub 0.5}Co{sub 0.5}Se{sub 2} counter electrode achieved a high power conversion efficiency of 6.02%, which is comparable with that of conventional platinum counter electrode (6.11%). This present investigation demonstrates the potential application of Ni{sub 0.5}Co{sub 0.5}Se{sub 2} as counter electrode in dye-sensitized solar cells.

Equipping African American Clergy to Recognize Depression.

Science.gov (United States)

Anthony, Jean Spann; Morris, Edith; Collins, Charles W; Watson, Albert; Williams, Jennifer E; Ferguson, Bʼnai; Ruhlman, Deborah L

2016-01-01

Many African Americans (AAs) use clergy as their primary source of help for depression, with few being referred to mental health providers. This study used face-to-face workshops to train AA clergy to recognize the symptoms and levels of severity of depression. A pretest/posttest format was used to test knowledge (N = 42) about depression symptoms. Results showed that the participation improved the clergy's ability to recognize depression symptoms. Faith community nurses can develop workshops for clergy to improve recognition and treatment of depression.

Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

Directory of Open Access Journals (Sweden)

Dickson Dennis W

2009-10-01

Full Text Available Abstract Background Insulin-degrading enzyme (IDE is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs targeting natively folded human and rodent IDE. Results Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. Conclusion We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

Development of NATO's recognized environmental picture

Science.gov (United States)

Teufert, John F.; Trabelsi, Mourad

2006-05-01

An important element for the fielding of a viable, effective NATO Response Force (NRF) is access to meteorological, oceanographic, geospatial data (GEOMETOC) and imagery. Currently, the available GEOMETOC information suffers from being very fragmented. NATO defines the Recognised Environmental Picture as controlled information base for GEOMETOC data. The NATO REP proposes an architecture that is both flexible and open. The focus lies on enabling a network-centric approach. The key into achieving this is relying on using open, well recognized standards that apply to both the data exchange protocols and the data formats. Communication and information exchange based on open standards enables system interoperability. Diverse systems, each with unique, specialized contributions to an increased understanding of the battlespace, can now cooperate to a manageable information sphere. By clearly defining responsibilities in the generation of information, a reduction in data transfer overhead is achieved . REP identifies three main stages in the dissemination of GEOMETOC data. These are Collection, Fusion (and Analysis) and Publication. A REP architecture has been successfully deployed during the NATO Coalition Warrior Interoperability Demonstration (CWID) in Lillehammer, Norway during June 2005. CWID is an annual event to validate and improve the interoperability of NATO and national Consultation and command, control, communications, computers, intelligence, surveillance, and reconnaissance (C4ISR) systems. With a test case success rate of 84%, it was able to provide relevant GEOMETOC support to the main NRF component headquarters. In 2006, the REP architecture will be deployed and validated during the NATO NRF Steadfast live exercises.

Energy dependence corrections to MOSFET dosimetric sensitivity

International Nuclear Information System (INIS)

Cheung, T.; Yu, P.K.N.; Butson, M.J.; Illawarra Cancer Care Centre, Crown St, Wollongong

2009-01-01

Metal Oxide Semiconductor Field Effect Transistors (MOSFET's) are dosimeters which are now frequently utilized in radiotherapy treatment applications. An improved MOSFET, clinical semiconductor dosimetry system (CSDS) which utilizes improved packaging for the MOSFET device has been studied for energy dependence of sensitivity to x-ray radiation measurement. Energy dependence from 50 kVp to 10 MV x-rays has been studied and found to vary by up to a factor of 3.2 with 75 kVp producing the highest sensitivity response. The detectors average life span in high sensitivity mode is energy related and ranges from approximately 100 Gy for 75 kVp x-rays to approximately 300 Gy at 6MV x-ray energy. The MOSFET detector has also been studied for sensitivity variations with integrated dose history. It was found to become less sensitive to radiation with age and the magnitude of this effect is dependant on radiation energy with lower energies producing a larger sensitivity reduction with integrated dose. The reduction in sensitivity is however approximated reproducibly by a slightly non linear, second order polynomial function allowing corrections to be made to reading to account for this effect to provide more accurate dose assessments both in phantom and in-vivo.

Energy dependence corrections to MOSFET dosimetric sensitivity.

Science.gov (United States)

Cheung, T; Butson, M J; Yu, P K N

2009-03-01

Metal Oxide Semiconductor Field Effect Transistors (MOSFET's) are dosimeters which are now frequently utilized in radiotherapy treatment applications. An improved MOSFET, clinical semiconductor dosimetry system (CSDS) which utilizes improved packaging for the MOSFET device has been studied for energy dependence of sensitivity to x-ray radiation measurement. Energy dependence from 50 kVp to 10 MV x-rays has been studied and found to vary by up to a factor of 3.2 with 75 kVp producing the highest sensitivity response. The detectors average life span in high sensitivity mode is energy related and ranges from approximately 100 Gy for 75 kVp x-rays to approximately 300 Gy at 6 MV x-ray energy. The MOSFET detector has also been studied for sensitivity variations with integrated dose history. It was found to become less sensitive to radiation with age and the magnitude of this effect is dependant on radiation energy with lower energies producing a larger sensitivity reduction with integrated dose. The reduction in sensitivity is however approximated reproducibly by a slightly non linear, second order polynomial function allowing corrections to be made to readings to account for this effect to provide more accurate dose assessments both in phantom and in-vivo.

Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup.

Science.gov (United States)

Chen, Tsung-Chi; Huang, Ching-Wen; Kuo, Yan-Wen; Liu, Fang-Lin; Yuan, Chao-Hsiu Hsuan; Hsu, Hei-Ti; Yeh, Shyi-Dong

2006-12-01

ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.

Recognizing and Managing Interpersonal Conflict.

Science.gov (United States)

Deane, Nancy; Hovland, Michael

1993-01-01

Practical advice is offered, to managers and supervisors at any level, on recognizing and analyzing interpersonal conflicts, managing such conflicts and making them productive, and ensuring that performance reviews result in progress for both supervisor and employee. Conflict is seen as inevitable, an opportunity to take action, and manageable.…

Recognizing Prefixes in Scientific Quantities

Science.gov (United States)

Sokolowski, Andrzej

2015-01-01

Although recognizing prefixes in physical quantities is inherent for practitioners, it might not be inherent for students, who do not use prefixes in their everyday life experiences. This deficiency surfaces in AP Physics exams. For example, readers of an AP Physics exam reported "a common mistake of incorrectly converting nanometers to…

Reduction of graphene oxide at room temperature with vitamin C for RGO–TiO{sub 2} photoanodes in dye-sensitized solar cell

Energy Technology Data Exchange (ETDEWEB)

Ding, Hui [Research Institute of Functional Materials, Central Iron and Steel Research Institute, No. 76 Xueyuan Nanlu, Haidian, Beijing 100081 (China); Zhang, Sam, E-mail: MSYZhang@ntu.edu.sg [School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Chen, Ji-Tao [College of Chemistry and Molecular Engineering, Peking University, No. 5 Yiheyuan Road, Haidian District, Beijing 100871 (China); Hu, Xiao-Ping [Research & Development Center, Advanced Technology & Materials Co., Ltd., No. 76 Xueyuan Nanlu, Haidian, Beijing 100081 (China); Du, Zhao-Fu; Qiu, Yue-Xiu; Zhao, Dong-Liang [Research Institute of Functional Materials, Central Iron and Steel Research Institute, No. 76 Xueyuan Nanlu, Haidian, Beijing 100081 (China)

2015-06-01

Graphene has been used to enhance conversion efficiency in dye-sensitized solar cell (DSSC) through increased electronic transportation. Introduction of graphene into DSSC is realized through reduction of graphene oxide (GO) to reduced graphene or RGO in TiO{sub 2} nanoparticles in solution. Chemical and hydrothermal reactions are two commonly used avenues. In the chemical route, toxic hydrazine and its derivatives are the typical reducing agent. Hydrothermal process is not toxic but requires prolonged heating. In this study, we mix flakes of GO with TiO{sub 2} nanoparticles and use vitamin C to realize the reduction at room temperature. The RGO–TiO{sub 2} composite films are then used as photoanode in DSSC. A conversion efficiency of 30% increase (to 7.89%) is obtained as compared to that of the pure TiO{sub 2} photoanode. - Highlights: • We produce reduced graphene (RGO)–TiO{sub 2} nanocomposite at room temperature. • The reduction is realized by vitamin C in a simple and environment-friendly way. • Reduced graphene provides a highway for electron transportation. • The RGO–P25 composite films are used as photoanode for DSSC. • Conversion efficiency of 30% increase (to 7.89%) is obtained by RGO–P25 photoanode.

An own-age bias in recognizing faces with horizontal information

Directory of Open Access Journals (Sweden)

Andreas Schaich

2016-11-01

Full Text Available Horizontal information, as a result of a selective filtering process, are essential in younger adults’ (YA ability to recognize human faces. Obermeyer, Kolling, Schaich, and Knopf (2012 recently reported impaired recognition of faces with horizontal information in older adults (OA suggesting age-variant processing. Two yet unconsidered factors (stimulus age and exposure duration that may have influenced previous results, were investigated in this study. Forty-seven YA (18-35yrs and 49 OA (62-83yrs were tested in a 2x2x2x2 mixed design with the between-subjects factors age group (YA vs OA and stimulus age (young faces vs older faces and the within-subjects factors filter (filtered (HF faces vs unfiltered faces (UF and exposure duration (0.8s vs 8s. Subjects were presented morph videos between pairs of faces: A starting face gradually merged into either the previously encoded target face or a control face. As expected, results showed an increase in recognition sensitivity (d’ with longer exposure duration in YA with both younger and older HF faces. OA however were unable to recognize filtered young faces not even with increased exposure duration. Furthermore, only elderly participants showed more accurate recognition with faces of their own age relative to other-age faces (own-age bias, OAB. For YA no OAB was observed. Filtered face recognition was significantly correlated with unfiltered recognition in YA but not in OA. It is concluded, that processing of horizontal information changes at a higher age. Presenting filtered or unfiltered faces both targets convergent face-specific processing only in YA but not in OA.

Development of a Monoclonal Antibody-Based Sandwich ELISA for Peanut Allergen Ara h 1 in Food

Directory of Open Access Journals (Sweden)

Chuanlai Xu

2013-07-01

Full Text Available We have established a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA based on two monoclonal antibodies (mAb to measure the content of the major peanut allergen Ara h 1 in foods. Two mAbs were selected out of 12 murine hybridoma cells secreting Ara h 1-specific antibody. Using mAb 6 as the capture antibody and HRP-labelled mAb 4 as the detection antibody, the limit of detection (LOD the assay was 0.34 ng/mL. Cross-reaction analysis showed that this method was strongly specific and had no cross-reactions with Ara h 2, pea protein or soy protein. Sample analysis showed that this ELISA was a useful tool to monitor peanut allergens in food products by measuring Ara h 1 content.

Use of a charge reducing agent to enable intact mass analysis of cysteine-linked antibody-drug-conjugates by native mass spectrometry

Directory of Open Access Journals (Sweden)

Kamila J. Pacholarz

2016-06-01

Full Text Available Antibody-drug-conjugates (ADC are a growing class of anticancer biopharmaceuticals. Conjugation of cysteine linked ADCs, requires initial reduction of mAb inter-chain disulfide bonds, as the drugs are attached via thiol chemistry. This results in the active mAb moiety being transformed from a covalently linked tetramer to non-covalently linked complexes, which hinders precise determination of drug load with LC–MS. Here, we show how the addition of the charge reducing agent triethylammonium acetate (TEAA preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination by direct infusion native MS.

Phase Noise Tolerant QPSK Receiver Using Phase Sensitive Wavelength Conversion

DEFF Research Database (Denmark)

Da Ros, Francesco; Xu, Jing; Lei, Lei

2013-01-01

A novel QPSK receiver based on a phase noise reduction pre-stage exploiting PSA in a HNLF and balanced detection is presented. Receiver sensitivity improvement over a conventional balanced receiver is demonstrated.......A novel QPSK receiver based on a phase noise reduction pre-stage exploiting PSA in a HNLF and balanced detection is presented. Receiver sensitivity improvement over a conventional balanced receiver is demonstrated....

Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

DEFF Research Database (Denmark)

Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

2009-01-01

The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

Susceptibility of different subsets of immature thymocytes to apoptosis induced by anti-TCRmAb

International Nuclear Information System (INIS)

Li Hongmei; Zhong Renqian; Yu Jiaping; Kong Xiantao; Chen Weifeng

2003-01-01

To analysis the susceptibility of different subsets of immature mice thymocytes to apoptosis induced by anti-TCRmAbs in vitro apoptosis was induced in unfractionated mice thymocytes by anti-TCRmAb. In Vivo apoptosis was induced in BALB/c mice by anti-TCR mAb, and thymocytes were examined by FACS. Results showed that CD4 + CD8 + DP thymocytes and CD4 - CD8 + CD3 - thymocytes were equally sensitive to apoptosis after treatment with the anti-TCR mAb. In sharp contrast, the early migrants or precursor containing thymocytes which are CD4 - CD8 - CD3 - TN have a lower spontaneous apoptosis rate and were relatively resistant to the anti-TCR mAb. The findings showed a breakpoint in thymocyte sensitivity to apoptosis which occurs after the onset of CD4 - CD8 + CD3 expression, suggesting that susceptibility of thymocytes to apoptosis is developmentally regulated

FCγ Chimeric Receptor-Engineered T Cells: Methodology, Advantages, Limitations, and Clinical Relevance

Directory of Open Access Journals (Sweden)

Giuseppe Sconocchia

2017-04-01

Full Text Available For many years, disappointing results have been generated by many investigations, which have utilized a variety of immunologic strategies to enhance the ability of a patient’s immune system to recognize and eliminate malignant cells. However, in recent years, immunotherapy has been used successfully for the treatment of hematologic and solid malignancies. The impressive clinical responses observed in many types of cancer have convinced even the most skeptical clinical oncologists that a patient’s immune system can recognize and reject his tumor if appropriate strategies are implemented. The success immunotherapy is due to the development of at least three therapeutic strategies. They include tumor-associated antigen (TAA-specific monoclonal antibodies (mAbs, T cell checkpoint blockade, and TAA-specific chimeric antigen receptors (CARs T cell-based immunotherapy. However, the full realization of the therapeutic potential of these approaches requires the development of strategies to counteract and overcome some limitations. They include off-target toxicity and mechanisms of cancer immune evasion, which obstacle the successful clinical application of mAbs and CAR T cell-based immunotherapies. Thus, we and others have developed the Fc gamma chimeric receptors (Fcγ-CRs-based strategy. Like CARs, Fcγ-CRs are composed of an intracellular tail resulting from the fusion of a co-stimulatory molecule with the T cell receptor ζ chain. In contrast, the extracellular CAR single-chain variable fragment (scFv, which recognizes the targeted TAA, has been replaced with the extracellular portion of the FcγRIIIA (CD16. Fcγ-CR T cells have a few intriguing features. First, given in combination with mAbs, Fcγ-CR T cells mediate anticancer activity in vitro and in vivo by an antibody-mediated cellular cytotoxicity mechanism. Second, CD16-CR T cells can target multiple cancer types provided that TAA-specific mAbs with the appropriate specificity are available

Orientation of llama antibodies strongly increases sensitivity of biosensors

NARCIS (Netherlands)

Trilling, A.K.; Hesselink, T.; Houwelingen, van A.; Cordewener, J.H.G.; Jongsma, M.A.; Schoffelen, S.; Hest, van J.C.M.; Zuilhof, J.T.; Beekwilder, J.

2014-01-01

Sensitivity of biosensors depends on theorientation of bio-receptors on the sensor surface.The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV)

Dimethylarginine Dimethylaminohydrolase Overexpression enhances Insulin Sensitivity

Science.gov (United States)

Sydow, Karsten; Mondon, Carl E.; Schrader, Joerg; Konishi, Hakuoh; Cooke, John P.

2011-01-01

Objective Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity. Methods and Results To test this hypothesis we employed a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22–70 weeks) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistence index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2μM) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle. Conclusions These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity. PMID:18239148

Immune Cells in Blood Recognize Tumors

Science.gov (United States)

NCI scientists have developed a novel strategy for identifying immune cells circulating in the blood that recognize specific proteins on tumor cells, a finding they believe may have potential implications for immune-based therapies.

Production, Characterization and Antioxidant Potential of Protease from Streptomyces sp. MAB18 Using Poultry Wastes

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Panchanathan Manivasagan

2013-01-01

Full Text Available Poultry waste is an abundant renewable source for the recovery of several value-added metabolites with potential industrial applications. This study describes the production of protease on poultry waste, with the subsequent use of the same poultry waste for the extraction of antioxidants. An extracellular protease-producing strain was isolated from Cuddalore coast, India, and identified as Streptomyces sp. MAB18. Its protease was purified 17.13-fold with 21.62% yield with a specific activity of 2398.36 U/mg and the molecular weight was estimated as 43 kDa. The enzyme was optimally active at pH 8–10 and temperature 50–60°C and it was most stable up to pH 12 and 6–12% of NaCl concentration. The enzyme activity was reduced when treated with Hg2+, Pb2+, and SDS and stimulated by Fe2+, Mg2+, Triton X-100, DMSO (dimethyl sulfoxide, sodium sulphite, and β-mercaptoethanol. Furthermore, the antioxidant activities of protease were evaluated using in vitro antioxidant assays, such as DPPH radical-scavenging activity, O2 scavenging activity, NO scavenging activity, Fe2+ chelating activity, and reducing power. The enzyme showed important antioxidant potential with an IC50 value of 78±0.28 mg/mL. Results of the present study indicate that the poultry waste-derived protease may be useful as supplementary protein and antioxidant in the animal feed formulations.

Application of decision tree technique to sensitivity analysis for results of radionuclide migration calculations. Research documents

International Nuclear Information System (INIS)

Nakajima, Kunihiko; Makino, Hitoshi

2005-03-01

Uncertainties are always present in the parameters used for the nuclide migration analysis in the geological disposal system. These uncertainties affect the result of such analyses, e.g., and the identification of dominant nuclides. It is very important to identify the parameters causing the significant impact on the results, and to investigate the influence of identified parameters in order to recognize R and D items with respect to the development of geological disposal system and understanding of the system performance. In our study, the decision tree analysis technique was examined in the sensitivity analysis as a method for investigation of the influences of the parameters and for complement existing sensitivity analysis. As a result, results obtained from Monte Carlo simulation with parameter uncertainties could be distinguished with not only important parameters but also with their quantitative conditions (e.g., ranges of parameter values). Furthermore, information obtained from the decision tree analysis could be used 1) to categorize the results obtained from the nuclide migration analysis for a given parameter set, 2) to show prospective effect of reduction to parameter uncertainties on the results. (author)

Why sensitive bacteria are resistant to hospital infection control [version 2; referees: 2 approved

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Esther van Kleef

2017-11-01

Full Text Available Background: Large reductions in the incidence of antibiotic-resistant strains of Staphylococcus aureus and Clostridium difficile have been observed in response to multifaceted hospital-based interventions. Reductions in antibiotic-sensitive strains have been smaller or non-existent. It has been argued that since infection control measures, such as hand hygiene, should affect resistant and sensitive strains equally, observed changes must have largely resulted from other factors, including changes in antibiotic use. We used a mathematical model to test the validity of this reasoning. Methods: We developed a mechanistic model of resistant and sensitive strains in a hospital and its catchment area. We assumed the resistant strain had a competitive advantage in the hospital and the sensitive strain an advantage in the community. We simulated a hospital hand hygiene intervention that directly affected resistant and sensitive strains equally. The annual incidence rate ratio (IRR associated with the intervention was calculated for hospital- and community-acquired infections of both strains. Results: For the resistant strain, there were large reductions in hospital-acquired infections (0.1 ≤ IRR ≤ 0.6 and smaller reductions in community-acquired infections (0.2 ≤ IRR ≤  0.9. These reductions increased in line with increasing importance of nosocomial transmission of the strain. For the sensitive strain, reductions in hospital acquisitions were much smaller (0.6 ≤ IRR ≤ 0.9, while communityacquisitions could increase or decrease (0.9 ≤ IRR ≤ 1.2. The greater the importance of the community environment for the transmission of the sensitive strain, the smaller the reductions. Conclusions: Counter-intuitively, infection control interventions, including hand hygiene, can have strikingly discordant effects on resistant and sensitive strains even though they target them equally, following differences in their adaptation to hospital and

Memory device sensitivity trends in aircraft's environment

International Nuclear Information System (INIS)

Bouchet, T.; Fourtine, S.; Calvet, M.C.

1999-01-01

The authors present the SEU (single event upset) sensitivity of 31 SRAM (static random access memory) and 8 DRAM (dynamic random access memory) according to their technologies. 2 methods have been used to compute the SEU rate: the NCS (neutron cross section) method and the BGR (burst generation rate) method, the physics data required by both methods have been either found in scientific literature or directly measured. The use of new technologies implies a quicker time response through a dramatic reduction of chip size and of the amount of energy representing 1 bit. The reduction of size makes less particles are likely to interact with the chip but the reduction of the critical charge implies that these interactions are more likely to damage the chip. The SEU sensitivity is then parted between these 2 opposed trends. Results show that for technologies beyond 0,18 μm these 2 trends balance roughly. Nevertheless the feedback experience shows that the number of errors is increasing. This is due to the fact that avionics requires more and more memory to perform numerical functions, the number of bits is increasing so is the risk of errors. As far as SEU is concerned, RAM devices are less and less sensitive comparatively for 1 bit, and DRAM seem to be less sensitive than SRAM. (A.C.)

Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.

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Yali Qin

Full Text Available We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs against Human Immunodeficiency Virus type-1 (HIV-1 in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR, followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37 exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.

Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

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Jinfeng Ti

Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

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Yukihiro Shoyama

2013-01-01

Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.

Science.gov (United States)

Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

2008-08-31

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

In vivo sensitivity of Phakopsora pachyrhizi to DMI and QoI fungicides

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Erlei Melo Reis

2015-03-01

Full Text Available In in vivo experiments the sensitivity of 18 isolates of Phakopsora pachyrhizi from several regions of Brazil to IDM fungicides (cyproconazole, epoxiconazole and tebuconazole and an IQE (pyraclostrobin were evaluated. The assessments were based on leaflet uredia density. Inhibitory concentration (IC50 and sensitivity reduction factor were determined for all fungicide x strain interactions. Tebuconazole sensitivity reduction was detected for most fungus isolates. In contrast, there was no fungicide shift in sensitivity of the fungus to pyraclostrobin. We conclude that the control failure of soybean rust found in some farms is due to the reduced sensitivity of the fungus to the IDM fungicide and that it remains sensitive to pyraclostrobin.

HIV p24 as scaffold for presenting conformational HIV Env antigens.

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Maria Tagliamonte

Full Text Available Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4(+ and CD8(+ T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes.

Pediatric rheumatology: An under-recognized subspecialty in India

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Akhila Kavirayani

2017-01-01

Full Text Available Pediatrics in India at the levels of both undergraduate and postgraduate training is often viewed upon as an acute disease specialty with little emphasis on chronic medical musculoskeletal diseases. Pediatric rheumatology is an under-recognized subspecialty of pediatrics which deals specifically with childhood arthritis, noninflammatory joint pains, connective tissue diseases, autoimmune diseases, vasculitis, and other rare inflammatory disorders. This article aims to give a bird's eye view of the repertoire of commonly encountered problems seen by a pediatric rheumatologist, via a classical case vignette for each topic followed by discussion. There is also mention of some rare diseases managed within pediatric rheumatology to give a flavor of the spectrum of diseases encountered. This is to raise awareness of the importance of pediatric rheumatology as a subspecialty within India and to prompt readers to seek specialist advice when encountering challenging cases. Pediatric rheumatologists network and work collaboratively with many other specialties such as ophthalmology, dermatology, neurology, orthopedics, nephrology, infectious diseases, immunology, and gastroenterology for combined care of diverse conditions. There is an unmet need in India to develop a training program for pediatric rheumatology so that shared care pathways with sensitized pediatricians and other specialists can be developed nationwide, to serve these children better to achieve optimal outcomes.

IgA attenuates anaphylaxis and subsequent immune responses in mice: possible application of IgA to vaccines.

Science.gov (United States)

Yamaki, Kouya; Nakashima, Takayuki; Miyatake, Kenji; Ishibashi, Yuki; Ito, Ayaka; Kuranishi, Ayu; Taguchi, Akihito; Morioka, Ayumi; Yamamoto, Midori; Yoshino, Shin

2014-01-01

Administration of the influenza vaccination to patients with an egg allergy is major health concern. Contaminating egg antigens occasionally induce severe anaphylactic shock in these patients following administration of the vaccination; therefore, the development of a safer vaccination is needed. In the present study, we investigated whether a mixture of four newly and previously generated anti-ovalbumin (OVA) IgA monoclonal antibodies (mAbs) could inhibit both anaphylactic shock upon a subcutaneous OVA challenge and subsequent further sensitization against OVA in passively anti-OVA IgE-sensitized mice and actively sensitized mice with an injection of OVA. The prevention of anaphylaxis by anti-OVA IgA mAbs was suggested to be mediated through the inhibition of OVA binding to allergenic antibodies such as anti-OVA IgE on mast cells and deceleration of the rate of OVA penetration from the injected site into the systemic circulation. Anti-OVA IgA mAbs inhibited further sensitization against OVA in mice actively sensitized with OVA, but did not affect sensitization against the unrelated antigen, phosphorylcholine-keyhole limpet hemocyanin co-injected with OVA. Our findings indicate that adding the anti-egg antigen IgA to the influenza vaccine should reduce not only the risk of inducing anaphylactic shock, but also undesired further sensitization against egg antigens following the vaccination without affecting the intended beneficial effect of the vaccine, namely the upregulation of immune responses to influenza viruses.

Interactions between ibrutinib and anti-CD20 antibodies; competing effects on the outcome of combination therapy

Science.gov (United States)

Skarzynski, Martin; Niemann, Carsten U; Lee, Yuh Shan; Martyr, Sabrina; Maric, Irina; Salem, Dalia; Stetler-Stevenson, Maryalice; Marti, Gerald E; Calvo, Katherine R; Yuan, Constance; Valdez, Janet; Soto, Susan; Farooqui, Mohammed Z.H.; Herman, Sarah E.M.; Wiestner, Adrian

2015-01-01

Purpose Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAbs) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in pre-clinical studies in vitro ibrutinib was reported to decrease CD20 expression and inhibits cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. Experimental Design Patients received single agent ibrutinib (420mg daily) on an investigator-initiated phase 2 trial. Serial blood samples were collected pre-treatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. Results We demonstrate that CD20 expression on ibrutinib was rapidly and persistently down-regulated (median reduction 74%, day 28, Pibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pre-treatment cells (median reduction 75%, Pibrutinib, providing a likely mechanism for the preserved C3d opsonization. Additionally, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. Conclusions Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal anti-tumor effects of such combinations requires further investigation. PMID:26283682

Interactions between Ibrutinib and Anti-CD20 Antibodies: Competing Effects on the Outcome of Combination Therapy.

Science.gov (United States)

Skarzynski, Martin; Niemann, Carsten U; Lee, Yuh Shan; Martyr, Sabrina; Maric, Irina; Salem, Dalia; Stetler-Stevenson, Maryalice; Marti, Gerald E; Calvo, Katherine R; Yuan, Constance; Valdez, Janet; Soto, Susan; Farooqui, Mohammed Z H; Herman, Sarah E M; Wiestner, Adrian

2016-01-01

Clinical trials of ibrutinib combined with anti-CD20 monoclonal antibodies (mAb) for chronic lymphocytic leukemia (CLL) report encouraging results. Paradoxically, in preclinical studies, in vitro ibrutinib was reported to decrease CD20 expression and inhibit cellular effector mechanisms. We therefore set out to investigate effects of in vivo ibrutinib treatment that could explain this paradox. Patients received single-agent ibrutinib (420 mg daily) on an investigator-initiated phase II trial. Serial blood samples were collected pretreatment and during treatment for ex vivo functional assays to examine the effects on CLL cell susceptibility to anti-CD20 mAbs. We demonstrate that CD20 expression on ibrutinib was rapidly and persistently downregulated (median reduction 74%, day 28, P ibrutinib were less susceptible to anti-CD20 mAb-mediated complement-dependent cytotoxicity than pretreatment cells (median reduction 75%, P ibrutinib, providing a likely mechanism for the preserved C3d opsonization. In addition, ibrutinib significantly inhibited trogocytosis, a major contributor to antigen loss and tumor escape during mAb therapy. Our data indicate that ibrutinib promotes both positive and negative interactions with anti-CD20 mAbs, suggesting that successfully harnessing maximal antitumor effects of such combinations requires further investigation. ©2015 American Association for Cancer Research.

[Violence against elderly people. Recognize--Sensitize--Act!].

Science.gov (United States)

Hirsch, Rolf D

2016-01-01

Elder abuse is--especially in view of the demographic development--a topic that is still neglected socially and in health policy, but also in terms of scientific research. There are different definitions of violence and these can be difficult to formulate, depending on the field. In gerontology, a rather broad frame is usually used to describe the phenomenon of violence. Its shapes are multilayered and diverse (e.g., physical, psychological, restriction of freedom, neglect, financial exploitation, and structural and cultural). In principle, any act of violence is also a breach of the law. Violence can occur in public places and in family and institutional settings (e.g., hospital and outpatient and inpatient care for the elderly). The statistical occurence in family settings is around 25% and in institutional settings between 11 and 24%. Acts of violence are usually an expression of helplessness, shame, overwork, poor support and lack of knowledge of alternatives. Often there is a pathological relationship, in which the roles of "perpetrators" and "victims" can change. Acts of violence have massive consequences for those affected. Preventative measures to reduce violence have various points of departure (e.g., company, region, institution, professional). So far, there are hardly any points of contact and professional assistance for elderly victims.

Characterization of a maternal type VI collagen in Xenopus embryos suggests a role for collagen in gastrulation

NARCIS (Netherlands)

Otte, A. P.; Roy, D.; Siemerink, M.; KOSTER, C. H.; Hochstenbach, F.; Timmermans, A.; Durston, A. J.

1990-01-01

We characterized a novel extracellular matrix element that is present in the earliest developmental stages of Xenopus laevis, and is recognized by an mAb 3D7. Based on amino acid composition, breakdown patterns by bacterial collagenases, and the molecular weights of the components of the antigen

Mapping the binding site of a cross-reactive Plasmodium falciparum PfEMP1 monoclonal antibody inhibitory of ICAM-1 binding

DEFF Research Database (Denmark)

Lennartz, Frank; Bengtsson, Anja; Olsen, Rebecca W

2015-01-01

The virulence of Plasmodium falciparum is linked to the ability of infected erythrocytes (IE) to adhere to the vascular endothelium, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). In this article, we report the functional characterization of an mAb that recognizes a panel of P...

Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

Science.gov (United States)

Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

2014-02-01

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

Radiation exposure reduction by use of Kevlar cassettes in the neonatal nursery

International Nuclear Information System (INIS)

Herman, M.W.; Mak, H.K.; Lachman, R.S.

1987-01-01

A study was performed to determine whether the use of Kevlar cassettes in the neonatal intensive care nursery would reduce radiation exposure to patients. The radiation dose to the neonates was measured by using thermoluminescent dosimeters. In addition, the attenuation of the Kevlar cassettes and the sensitivity of the film-screen combination were compared with the previously used system. The greatest radiation reduction using a mobile X-ray unit was 27%; based on sensitivity measurements, the theoretical reduction averaged 38%. The reduction in radiation exposure resulted from reduced attenuation by the Kevlar cassette

A four-day Western-style dietary intervention causes reductions in hippocampal-dependent learning and memory and interoceptive sensitivity.

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Tuki Attuquayefio

Full Text Available In animals, a Western style diet-high in saturated fat and added sugar-causes impairments in hippocampal-dependent learning and memory (HDLM and perception of internal bodily state (interoception. In humans, while there is correlational support for a link between Western-style diet, HDLM, and interoception, there is as yet no causal data. Here, healthy individuals were randomly assigned to consume either a breakfast high in saturated fat and added sugar (Experimental condition or a healthier breakfast (Control condition, over four consecutive days. Tests of HDLM, interoception and biological measures were administered before and after breakfast on the days one and four, and participants completed food diaries before and during the study. At the end of the study, the Experimental condition showed significant reductions in HDLM and reduced interoceptive sensitivity to hunger and fullness, relative to the Control condition. The Experimental condition also showed a markedly different blood glucose and triglyceride responses to their breakfast, relative to Controls, with larger changes in blood glucose across breakfast being associated with greater reductions in HDLM. The Experimental condition compensated for their energy-dense breakfast by reducing carbohydrate intake, while saturated fat intake remained consistently higher than Controls. This is the first experimental study in humans to demonstrate that a Western-style diet impacts HDLM following a relatively short exposure-just as in animals. The link between diet-induced HDLM changes and blood glucose suggests one pathway by which diet impacts HDLM in humans.

Sensory sensitivity and identification and hedonic assessment ofolfactory stimuli

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Borys Ruszpel

2012-06-01

Full Text Available Conducted research had an exploratory character. It was focused on connections between temperament and olfactory functioning – in particular, identification and affective assessment of olfactory stimuli. Main research question dealt with potential correlations between sensory sensitivity (dimension of temperamental questionnaire FCZ‑KT with declarative and objective ability to identify presented odours and their assessment. Fifty four schoolgirls from one of the Warsaw sec‑ ondary schools participated in the research and they were asked for filling in the FCZ‑KT questionnaire and evaluating each of 16 smell samples. Analyses revealed a significant positive correlation between declared familiarity and accurate odours’ identification (odours that were subjectively known were recognized more accurately than unknown and a posi‑ tive correlation between declared familiarity and affective assessment (odours that were known were assessed as more pleasant than unknown. Sensory sensitivity was not correlated neither with declarative nor real ability to identify smells, however sensory sensitivity was positively correlated with affective assessment (the higher scores on sensory sensitivity dimension, the more pleasantly assessed odours in general. Analyses revealed a number of connections between other dimensions of FCZ‑KT questionnaire (perseverance, liveliness, stamina and the ability (both objective and subjective to correctly identify odours which were most difficult to recognize. Completed project might be perceived as a starting point for further research concerning relationships between temperament, olfactory functioning, and food preferences among patients diagnosed with eating disorders such as anorexia nervosa, bulimia nervosa, and obesity.

E-cigarette Regulation and Harm Reduction: The Case of Hong Kong.

Science.gov (United States)

Churk, Shue Sing

Harm reduction is an internationally recognized tobacco control strategy. E-cigarettes, being a less harmful alternative to smoking, have the potential to achieve harm reduction. Within this context, this article critiques Hong Kong’s legal regime governing e-cigarettes and the proposed prohibition of the product. It is argued that the current law is uncertain and inadequate. Although a reform of laws relating to e-cigarettes is needed, it is argued that banning the product altogether as a means to reducing the harm of tobacco use is unsupported by evidence.

UMTRA Project-Level Cost Reduction/Productivity Improvement Program manual

International Nuclear Information System (INIS)

1995-06-01

Mission of the Uranium Mill Tailings Remedial Action (UMTRA) Project Cost Reduction/Productivity Improvement Program (CR/PIP) is to contribute to the UMTRA Project's environmental restoration mission by providing the means to achieve and recognize continuous improvements and cost savings. This manual includes program definition, description of UMTRA project organizational responsibilities and interfaces with existing project functions, guidance to contractors, and definition of project-level functions

Recognizing Textual Entailment: Challenges in the Portuguese Language

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Gil Rocha

2018-03-01

Full Text Available Recognizing textual entailment comprises the task of determining semantic entailment relations between text fragments. A text fragment entails another text fragment if, from the meaning of the former, one can infer the meaning of the latter. If such relation is bidirectional, then we are in the presence of a paraphrase. Automatically recognizing textual entailment relations captures major semantic inference needs in several natural language processing (NLP applications. As in many NLP tasks, textual entailment corpora for English abound, while the same is not true for more resource-scarce languages such as Portuguese. Exploiting what seems to be the only Portuguese corpus for textual entailment and paraphrases (the ASSIN corpus, in this paper, we address the task of automatically recognizing textual entailment (RTE and paraphrases from text written in the Portuguese language, by employing supervised machine learning techniques. We employ lexical, syntactic and semantic features, and analyze the impact of using semantic-based approaches in the performance of the system. We then try to take advantage of the bi-dialect nature of ASSIN to compensate its limited size. With the same aim, we explore modeling the task of recognizing textual entailment and paraphrases as a binary classification problem by considering the bidirectional nature of paraphrases as entailment relationships. Addressing the task as a multi-class classification problem, we achieve results in line with the winner of the ASSIN Challenge. In addition, we conclude that semantic-based approaches are promising in this task, and that combining data from European and Brazilian Portuguese is less straightforward than it may initially seem. The binary classification modeling of the problem does not seem to bring advantages to the original multi-class model, despite the outstanding results obtained by the binary classifier for recognizing textual entailments.

Burnout: Recognize and Reverse.

Science.gov (United States)

Anne, Samantha

2014-07-01

Physician burnout may be underrecognized and can cause significant detrimental effects on personal health and job satisfaction. Burnout has been associated with medical errors, alcohol and drug abuse, and neglect and abandonment of career goals. With self-awareness, development of coping mechanisms, and the adoption of a strong social and professional support network, burnout can be combated. This article focuses on recognizing characteristics of burnout and providing strategies to cope to avoid reaching a high degree of burnout. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2014.

Therapeutic efficacy of antibodies lacking Fcγ receptor binding against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies [corrected].

Directory of Open Access Journals (Sweden)

Katherine L Williams

2013-02-01

Full Text Available Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS are life-threatening complications following infection with one of the four serotypes of dengue virus (DENV. At present, no vaccine or antiviral therapies are available against dengue. Here, we characterized a panel of eight human or mouse-human chimeric monoclonal antibodies (MAbs and their modified variants lacking effector function and dissected the mechanism by which some protect against antibody-enhanced lethal DENV infection. We found that neutralizing modified MAbs that recognize the fusion loop or the A strand epitopes on domains II and III of the envelope protein, respectively, act therapeutically by competing with and/or displacing enhancing antibodies. By analyzing these relationships, we developed a novel in vitro suppression-of-enhancement assay that predicts the ability of modified MAbs to act therapeutically against antibody-enhanced disease in vivo. These studies provide new insight into the biology of DENV pathogenesis and the requirements for antibodies to treat lethal DENV disease.

Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies.

Science.gov (United States)

Lee, Warren; Syed Atif, Ali; Tan, Soo Choon; Leow, Chiuan Herng

2017-08-01

The advantages of chicken (Gallus gallus domesticus) antibodies as immunodiagnostic and immunotherapeutic biomolecules has only been recently recognized. Even so, chicken antibodies remain less-well characterized than their mammalian counterparts. This review aims at providing a current overview of the structure, function, development and generation of chicken antibodies. Additionally, brief but comprehensive insights into current knowledge pertaining to the immunogenetic framework and diversity-generation of the chicken immunoglobulin repertoire which have contributed to the establishment of recombinant chicken mAb-generating methods are discussed. Focus is provided on the current methods used to generate antibodies from chickens with added emphasis on the generation of recombinant chicken mAbs and its derivative formats. The advantages and limitations of established protocols for the generation of chicken mAbs are highlighted. The various applications of recombinant chicken mAbs and its derivative formats in immunodiagnostics and immunotherapy are further detailed. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of genetic engineering on the pharmacokinetics of antibodies

International Nuclear Information System (INIS)

Colcher, D.; Goel, A.; Pavlinkova, G.; Beresford, G.; Booth, B.; Batra, S.K.

1999-01-01

Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of the antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effectors functions. Advanced in genetic engineering have provided rapid progress the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetics studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment

Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.

Science.gov (United States)

Davis, W C; Naessens, J; Brown, W C; Ellis, J A; Hamilton, M J; Cantor, G H; Barbosa, J I; Ferens, W; Bohach, G A

1996-08-01

Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.

Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

Science.gov (United States)

The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells

DEFF Research Database (Denmark)

Drasbek, Mette; Christiansen, Gunna; Drasbek, Kim Ryun

2007-01-01

The human pathogen Mycoplasma pneumoniae can cause atypical pneumonia through adherence to epithelial cells in the respiratory tract. The major immunogenic protein, P1, participates in the attachment of the bacteria to the host cells. To investigate the adhesion properties of P1, a recombinant...... protein (rP1-II) covering amino acids 1107-1518 of the P1 protein was produced. This protein inhibited the adhesion of M. pneumoniae to human HEp-2 cells, as visualized in a competitive-binding assay using immunofluorescence microscopy. Previous studies have shown that mAbs that recognize two epitopes...... overlapping synthetic peptides covering the whole of rP1-II were evaluated in the competitive-binding assay using immunofluorescence microscopy. A reduction in the number of M. pneumoniae microcolonies was seen, which was confirmed for five peptides using a POLARstar OPTIMA reader to measure fluorescence...

Evolving GIS technologies in nature conservation and the spatial planning strategy of Tara NP (Serbia as a potential UNECSO MAB reserve

Directory of Open Access Journals (Sweden)

Radović Dejan

2008-01-01

Full Text Available Mt. Tara NP was nominated in 2004 within the UNESCO - ROSTE programme, for Man and the Biosphere (MAB Reserve status in Serbia as transboundary 'Peace Park' status between Serbia and Bosnia & Herzegovina. Mt. Tara is one of the most important centres of Balkans and European ecosystems and species diversity. They represent a unique example of well preserved forests in south eastern Europe with numerous endemic and relict species of flora and fauna. In this floristic diversity of Mt Tara of the greatest interest is the Serbian (Pančić's spruce Picea omorika. Mt. Tara NP is characterized by specific geomorphologic, hydrologic, geologic, soil and climatic features. The geographical information system (GIS that we have created has proved an excellent tool for the spatial planning strategy in assessment and conservation of all natural characteristics of Mt. Tara NP, and is helpful to Park management for sustainable use of landscape resources. GIS of Mt. Tara NP includes data on natural, artificial and management themes.

Drawing and Recognizing Chinese Characters with Recurrent Neural Network.

Science.gov (United States)

Zhang, Xu-Yao; Yin, Fei; Zhang, Yan-Ming; Liu, Cheng-Lin; Bengio, Yoshua

2018-04-01

Recent deep learning based approaches have achieved great success on handwriting recognition. Chinese characters are among the most widely adopted writing systems in the world. Previous research has mainly focused on recognizing handwritten Chinese characters. However, recognition is only one aspect for understanding a language, another challenging and interesting task is to teach a machine to automatically write (pictographic) Chinese characters. In this paper, we propose a framework by using the recurrent neural network (RNN) as both a discriminative model for recognizing Chinese characters and a generative model for drawing (generating) Chinese characters. To recognize Chinese characters, previous methods usually adopt the convolutional neural network (CNN) models which require transforming the online handwriting trajectory into image-like representations. Instead, our RNN based approach is an end-to-end system which directly deals with the sequential structure and does not require any domain-specific knowledge. With the RNN system (combining an LSTM and GRU), state-of-the-art performance can be achieved on the ICDAR-2013 competition database. Furthermore, under the RNN framework, a conditional generative model with character embedding is proposed for automatically drawing recognizable Chinese characters. The generated characters (in vector format) are human-readable and also can be recognized by the discriminative RNN model with high accuracy. Experimental results verify the effectiveness of using RNNs as both generative and discriminative models for the tasks of drawing and recognizing Chinese characters.

Recognizing Variable Environments The Theory of Cognitive Prism

CERN Document Server

Dong, Tiansi

2012-01-01

Normal adults do not have any difficulty in recognizing their homes. But can artificial systems do in the same way as humans? This book collects interdisciplinary evidences and presents an answer from the perspective of computing, namely, the theory of cognitive prism. To recognize an environment, an intelligent system only needs to classify objects, structures them based on the connection relation (not through measuring!), subjectively orders the objects, and compares with the target environment, whose knowledge is similarly structured. The intelligent system works, therefore, like a prism: when a beam of light (a scene) reaches (is perceived) to an optical prism (by an intelligent system), some light (objects) is reflected (are neglected), those passed through (the recognized objects) are distorted (are ordered differently). So comes the term 'cognitive prism'! Two fundamental propositions used in the theory can be informally stated as follow: an orientation relation is a kind of distance comparison relatio...

Preliminary Studies on the Development of Monoclonal Antibodies Against Mycelia of Ganoderma boninense, the Causal Pathogen of Basal Stem Rot of Oil Palm

Directory of Open Access Journals (Sweden)

Shamala, S.

2006-01-01

Full Text Available This study aimed to raise specific MAbs against G. boninense, the causal pathogen of basal stem rot (BSR of oil palm. Crude mycelium extract of G. boninense was used as immunogen to generate MAbs. Mycelium was harvested from liquid culture and freeze-dried followed by re-suspension in phosphate buffer saline (PBS. Two 10-week old BALB-C mice were immunized with the mycelial extract. The mice were boosted once before harvesting their spleens for fusion. The MAbs were fused with myeloma cells from BALB-C mice. Initial screening was carried out using plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA with mycelial immunogen of G. boninense. The MAbs with positive signals were verified via secondary screening and cloned for cross-reactivity test. Cross-reactivity testing was carried out with 2 other fungi namely; Trichoderma and Botrytis along with 2 different species of Ganoderma commonly found in oil palm plantations namely; G. zonatum, and G. miniatocinctum. This study found that the MAbs raised against G. boninense were not specific as the MAbs gave positive signals through the cross-reactivity test with all fungi tested in the cross-reactivity. Future work would be using these MAbs in a co-immunization program whereby the generated Ganoderma sp generic monoclonal antibody will be pre-mixed with the G. boninense mycelium immunogen to allow reduction in the potential cross-reactivity of newly generated antibodies with Ganoderma sp. Our efforts are also currently directed at optimizing the immunogen preparation for the production of MAbs specific to G. boninense.

Super-Memorizers Are Not Super-Recognizers

NARCIS (Netherlands)

Ramon, M.; Miellet, S.; Dzieciol, A.M.; Konrad, B.N; Dresler, M.; Caldara, R.

2016-01-01

Humans have a natural expertise in recognizing faces. However, the nature of the interaction between this critical visual biological skill and memory is yet unclear. Here, we had the unique opportunity to test two individuals who have had exceptional success in the World Memory Championships,

Stress Sensitization of Ethanol Withdrawal-Induced Reduction in Social Interaction: Inhibition by CRF-1 and Benzodiazepine Receptor Antagonists and a 5-HT1A-Receptor Agonist

OpenAIRE

Breese, George R; Knapp, Darin J; Overstreet, David H

2004-01-01

Repeated withdrawals from chronic ethanol sensitize the withdrawal-induced reduction in social interaction behaviors. This study determined whether stress might substitute for repeated withdrawals to facilitate withdrawal-induced anxiety-like behavior. When two 1-h periods of restraint stress were applied at 1-week intervals to rats fed control diet, social interaction was reduced upon withdrawal from a subsequent 5-day exposure to ethanol diet. Neither this ethanol exposure alone nor exposur...

Energy Savings from Industrial Water Reductions

Energy Technology Data Exchange (ETDEWEB)

Rao, Prakash; McKane, Aimee; de Fontaine, Andre

2015-08-03

Although it is widely recognized that reducing freshwater consumption is of critical importance, generating interest in industrial water reduction programs can be hindered for a variety of reasons. These include the low cost of water, greater focus on water use in other sectors such as the agriculture and residential sectors, high levels of unbilled and/or unregulated self-supplied water use in industry, and lack of water metering and tracking capabilities at industrial facilities. However, there are many additional components to the resource savings associated with reducing site water use beyond the water savings alone, such as reductions in energy consumption, greenhouse gas emissions, treatment chemicals, and impact on the local watershed. Understanding and quantifying these additional resource savings can expand the community of businesses, NGOs, government agencies, and researchers with a vested interest in water reduction. This paper will develop a methodology for evaluating the embedded energy consumption associated with water use at an industrial facility. The methodology developed will use available data and references to evaluate the energy consumption associated with water supply and wastewater treatment outside of a facility’s fence line for various water sources. It will also include a framework for evaluating the energy consumption associated with water use within a facility’s fence line. The methodology will develop a more complete picture of the total resource savings associated with water reduction efforts and allow industrial water reduction programs to assess the energy and CO2 savings associated with their efforts.

Highly sensitive and rapid bacteria detection using molecular beacon-Au nanoparticles hybrid nanoprobes.

Science.gov (United States)

Cao, Jing; Feng, Chao; Liu, Yan; Wang, Shouyu; Liu, Fei

2014-07-15

Since many diseases are caused by pathogenic bacterial infections, accurate and rapid detection of pathogenic bacteria is in urgent need to timely apply appropriate treatments and to reduce economic costs. To end this, we designed molecular beacon-Au nanoparticle hybrid nanoprobes to improve the bacterial detection efficiency and sensitivity. Here, we show that the designed molecular beacon modified Au nanoparticles could specifically recognize synthetic DNAs targets and can readily detect targets in clinical samples. Moreover, the hybrid nanoprobes can recognize Escherichia coli within an hour at a concentration of 10(2) cfu/ml, which is 1000-folds sensitive than using molecular beacon directly. Our results show that the molecular beacon-Au nanoparticle hybrid nanoprobes have great potential in medical and biological applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody.

Science.gov (United States)

Zhou, E M; Kisil, F T

1995-03-01

A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen.

Air Layer Drag Reduction

Science.gov (United States)

Ceccio, Steven; Elbing, Brian; Winkel, Eric; Dowling, David; Perlin, Marc

2008-11-01

A set of experiments have been conducted at the US Navy's Large Cavitation Channel to investigate skin-friction drag reduction with the injection of air into a high Reynolds number turbulent boundary layer. Testing was performed on a 12.9 m long flat-plate test model with the surface hydraulically smooth and fully rough at downstream-distance-based Reynolds numbers to 220 million and at speeds to 20 m/s. Local skin-friction, near-wall bulk void fraction, and near-wall bubble imaging were monitored along the length of the model. The instrument suite was used to access the requirements necessary to achieve air layer drag reduction (ALDR). Injection of air over a wide range of air fluxes showed that three drag reduction regimes exist when injecting air; (1) bubble drag reduction that has poor downstream persistence, (2) a transitional regime with a steep rise in drag reduction, and (3) ALDR regime where the drag reduction plateaus at 90% ± 10% over the entire model length with large void fractions in the near-wall region. These investigations revealed several requirements for ALDR including; sufficient volumetric air fluxes that increase approximately with the square of the free-stream speed, slightly higher air fluxes are needed when the surface tension is reduced, higher air fluxes are required for rough surfaces, and the formation of ALDR is sensitive to the inlet condition.

Identification of two novel rabbit hemorrhagic disease virus (RHDV) B cell epitopes and evaluation of its immunoprotection against RHDV.

Science.gov (United States)

DeSheng, Kong; HuaiRan, Liu; JiaSen, Liu; Zuo, Yu; Qian, Jiang; DongChun, Guo; XiaoLiang, Hu; FengJie, Wang; QianQian, Huang; LianDong, Qu

2015-07-01

The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD4, AG10, BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG10 and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG10 and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG10 and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG10 and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG10 and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.

Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro.

Science.gov (United States)

Wilke, Georgia; Ravindran, Soumya; Funkhouser-Jones, Lisa; Barks, Jennifer; Wang, Qiuling; VanDussen, Kelli L; Stappenbeck, Thaddeus S; Kuhlenschmidt, Theresa B; Kuhlenschmidt, Mark S; Sibley, L David

2018-06-27

Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti- Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology. IMPORTANCE Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and

Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65.

Science.gov (United States)

Liu, Maojun; DU, Gaimei; Zhang, Yue; Wu, Yuzi; Wang, Haiyan; Li, Bin; Bai, Yun; Feng, Zhixin; Xiong, Qiyan; Bai, Fangfang; Browning, Glenn F; Shao, Guoqing

2016-09-01

Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection.

Predictive brain networks for major depression in a semi-multimodal fusion hierarchical feature reduction framework.

Science.gov (United States)

Yang, Jie; Yin, Yingying; Zhang, Zuping; Long, Jun; Dong, Jian; Zhang, Yuqun; Xu, Zhi; Li, Lei; Liu, Jie; Yuan, Yonggui

2018-02-05

Major depressive disorder (MDD) is characterized by dysregulation of distributed structural and functional networks. It is now recognized that structural and functional networks are related at multiple temporal scales. The recent emergence of multimodal fusion methods has made it possible to comprehensively and systematically investigate brain networks and thereby provide essential information for influencing disease diagnosis and prognosis. However, such investigations are hampered by the inconsistent dimensionality features between structural and functional networks. Thus, a semi-multimodal fusion hierarchical feature reduction framework is proposed. Feature reduction is a vital procedure in classification that can be used to eliminate irrelevant and redundant information and thereby improve the accuracy of disease diagnosis. Our proposed framework primarily consists of two steps. The first step considers the connection distances in both structural and functional networks between MDD and healthy control (HC) groups. By adding a constraint based on sparsity regularization, the second step fully utilizes the inter-relationship between the two modalities. However, in contrast to conventional multi-modality multi-task methods, the structural networks were considered to play only a subsidiary role in feature reduction and were not included in the following classification. The proposed method achieved a classification accuracy, specificity, sensitivity, and area under the curve of 84.91%, 88.6%, 81.29%, and 0.91, respectively. Moreover, the frontal-limbic system contributed the most to disease diagnosis. Importantly, by taking full advantage of the complementary information from multimodal neuroimaging data, the selected consensus connections may be highly reliable biomarkers of MDD. Copyright © 2017 Elsevier B.V. All rights reserved.

Sensitization and allergy to Cannabis sativa leaves in a population of tomato (Lycopersicon esculentum)-sensitized patients.

Science.gov (United States)

de Larramendi, Carlos Hernando; Carnés, Jerónimo; García-Abujeta, José Luís; García-Endrino, Ana; Muñoz-Palomino, Elena; Huertas, Angel Julio; Fernández-Caldas, Enrique; Ferrer, Angel

2008-01-01

Cases of allergy to Cannabis sativa have occasionally been reported, but both the allergenic profile and eventual cross-reactivity pattern remain unknown. To analyze the allergenic profile of a population of patients from Spain sensitized to C. sativa and to characterize the C. sativa leaf extract. A total of 32 subjects were enrolled in the study: group A, 10 individuals sensitized to tomato, reporting reactions by contact or inhalation to Cannabis; group B, 14 individuals sensitized to tomato, without reactions to Cannabis; group C, 8 individuals not sensitized to tomato and without reactions to Cannabis. Sensitivity to Cannabis, tomato and peach peel, Platanus hybrida and Artemisia vulgaris pollen extracts was measured by skin tests and specific IgE. Individual immunoblots and inhibition experiments with a pool of sera were conducted. All tomato-sensitized subjects (and 1 negative) had positive skin tests to C. sativa leaves and hashish. Specific IgE to C. sativa and peach peel was more common than to tomato. Immunoblot experiments showed 2 prominent bands of 10 and 14 kDa and 2 weakly recognized bands of 30 and 45 kDa. Tomato, peach and A. vulgaris extracts inhibited most of the bands present in C. sativa. P. hybrida inhibited only the high-molecular-weight bands. Sensitization to C. sativa with or without symptoms is frequent among patients in Spain sensitized to tomato. C. sativa leaves are a potential allergenic source and their allergens may cross-react with other allergenic sources from plants (fruit peels and pollen). (c) 2008 S. Karger AG, Basel

The roles of categorical and coordinate spatial relations in recognizing buildings.

Science.gov (United States)

Palermo, Liana; Piccardi, Laura; Nori, Raffaella; Giusberti, Fiorella; Guariglia, Cecilia

2012-11-01

Categorical spatial information is considered more useful for recognizing objects, and coordinate spatial information for guiding actions--for example, during navigation or grasping. In contrast with this assumption, we hypothesized that buildings, unlike other categories of objects, require both categorical and coordinate spatial information in order to be recognized. This hypothesis arose from evidence that right-brain-damaged patients have deficits in both coordinate judgments and recognition of buildings and from the fact that buildings are very useful for guiding navigation in urban environments. To test this hypothesis, we assessed 210 healthy college students while they performed four different tasks that required categorical and coordinate judgments and the recognition of common objects and buildings. Our results showed that both categorical and coordinate spatial representations are necessary to recognize a building, whereas only categorical representations are necessary to recognize an object. We discuss our data in view of a recent neural framework for visuospatial processing, suggesting that recognizing buildings may specifically activate the parieto-medial-temporal pathway.

Antarctic skuas recognize individual humans.

Science.gov (United States)

Lee, Won Young; Han, Yeong-Deok; Lee, Sang-Im; Jablonski, Piotr G; Jung, Jin-Woo; Kim, Jeong-Hoon

2016-07-01

Recent findings report that wild animals can recognize individual humans. To explain how the animals distinguish humans, two hypotheses are proposed. The high cognitive abilities hypothesis implies that pre-existing high intelligence enabled animals to acquire such abilities. The pre-exposure to stimuli hypothesis suggests that frequent encounters with humans promote the acquisition of discriminatory abilities in these species. Here, we examine individual human recognition abilities in a wild Antarctic species, the brown skua (Stercorarius antarcticus), which lives away from typical human settlements and was only recently exposed to humans due to activities at Antarctic stations. We found that, as nest visits were repeated, the skua parents responded at further distances and were more likely to attack the nest intruder. Also, we demonstrated that seven out of seven breeding pairs of skuas selectively responded to a human nest intruder with aggression and ignored a neutral human who had not previously approached the nest. The results indicate that Antarctic skuas, a species that typically inhabited in human-free areas, are able to recognize individual humans who disturbed their nests. Our findings generally support the high cognitive abilities hypothesis, but this ability can be acquired during a relatively short period in the life of an individual as a result of interactions between individual birds and humans.

Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

Directory of Open Access Journals (Sweden)

Hardy Michele E

2008-01-01

Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

Production, characterization and application of monoclonal antibodies to the coelomocytes of sea urchin Strongylocentrotus intermedius.

Science.gov (United States)

Wang, Yinan; Meng, Shaodong; Zhang, Jialin; Ding, Jun; Li, Qiang

2018-04-01

Sea urchin is one of marine animals with high economic and great scientific research values. Axial organ is a glandular organ that has been presumed as coelomocytes origin site. In this paper, two monoclonal antibodies (3G10 and 6B3) against coelomocytes of sea urchin Strongylocentrotus intermedius were developed by hybridoma technique. The mAbs were characterized by indirect immunofluorescence assay test (IIFAT), flow cytometry (FCM) and western blot assay. Results showed that mAb 3G10 recognized a protein of a molecular weight of 17 kDa in the spherule cells, while mAb 6B3 reacted with a protein of a molecular weight of 35 kDa in the phagocytes. Furthermore, specificity analysis revealed that the two mAbs could react with the coelomocytes of sea urchin S. nudus and Hemicentrotus pulcherrimus, but not with those of other common echinoderms including sea cucumber Apostichopus japonicus and starfish Asterias rollestoni. To determine whether the coelomocytes exist in the axial organ of sea urchin, the IIFAT assays were carried out based on the two mAbs. Result showed that positive fluorescence signals were distributed in the organ. It was revealed that the axial organ was rich in coelomocytes, which suggests that the organ may play as a producing source or reservoir in the ontogenesis of coelomocytes of sea urchin. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of Endotrypanum Parasites Using Specific Monoclonal Antibodies

Directory of Open Access Journals (Sweden)

Ramos Franco Antonia Maria

1997-01-01

Full Text Available A large number of Endotrypanum stocks (representing an heterogeneous population of strains have been screened against a panel of monoclonal antibodies (MAbs derived for selected species of Endotrypanum or Leishmania, to see whether this approach could be used to group/differentiate further among these parasites. Using different immunological assay systems, MAbs considered specific for the genus Endotrypanum (E-24, CXXX-3G5-F12 or strain M6159 of E. schaudinni (E-2, CXIV-3C7-F5 reacted variably according to the test used but in the ELISA or immunofluorescence assay both reacted with all the strains tested. Analyses using these MAbs showed antigenic diversity occurring among the Endotrypanum strains, but no qualitative or quantitative reactivity pattern could be consistently related to parasite origin (i.e., host species involved or geographic area of isolation. Western blot analyses of the parasites showed that these MAbs recognized multiple components. Differences existed either in the epitope density or molecular forms associated with the antigenic determinants and therefore allowed the assignment of the strains to specific antigenic groups. Using immunofluorescence or ELISA assay, clone E-24 produced reaction with L. equatorensis (which is a parasite of sloth and rodent, but not with other trypanosomatids examined. Interestingly, the latter parasite and the Endotrypanum strains cross-reacted with a number of MAbs that were produced against members of the L. major-L. tropica complex

An Investigation of the Critical Events and Influential Factors to the Evolution of the U.S. Man and the Biosphere Program

Science.gov (United States)

Thomsen, Jennifer M.

2018-04-01

The UNESCO Man and the Biosphere program has operated for 45 years as an international program that started in the 1970s to conserve biotic communities and provide areas for research, education, and training. The program later evolved in the 1990s to address social and environmental issues in a sustainable manner across a landscape. This program was one of the first efforts that recognized the importance of working beyond park and protected area boundaries and the need to sustain livelihoods as much as the resources. In the MAB program's infancy, the United States (U.S.) was a major advocate and leader with more than 45 biosphere reserves, most of them established in or around 1976. Yet, many political, economic, and other external factors influenced the U.S. MAB involvement in subsequent years. Consequently, the U.S. has remained largely inactive in the international MAB network for the past fifteen years until a recent push to revive the program under the leadership of the State Department and the National Park Service. Through in-depth research on two longterm U.S. biosphere reserves, this paper provides a description of the key events impacting the U.S. MAB program over the past several decades and discusses the influential role of politics, a public image, and the perceptions of international designations. Through the lessons presented in this paper, recommendations are provided to support the revival of the MAB program in the U.S.

Identification of a novel linear B-cell epitope in the UL26 and UL26.5 proteins of Duck Enteritis Virus

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Kong Xiangang

2010-09-01

Full Text Available Abstract Background The Unique Long 26 (UL26 and UL26.5 proteins of herpes simplex virus are known to function during the assembly of the viruses. However, for duck enteritis virus (DEV, which is an unassigned member of the family Herpesviridae, little information is available about the function of the two proteins. In this study, the C-terminus of DEV UL26 protein (designated UL26c, which contains the whole of UL26.5, was expressed, and the recombinant UL26c protein was used to immunize BALB/c mice to generate monoclonal antibodies (mAb. The mAb 1C8 was generated against DEV UL26 and UL26.5 proteins and used subsequently to map the epitope in this region. Both the mAb and its defined epitope will provide potential tools for further study of DEV. Results A mAb (designated 1C8 was generated against the DEV UL26c protein, and a series of 17 partially overlapping fragments that spanned the DEV UL26c were expressed with GST tags. These peptides were subjected to enzyme-linked immunosorbent assay (ELISA and western blotting analysis using mAb 1C8 to identify the epitope. A linear motif, 520IYYPGE525, which was located at the C-terminus of the DEV UL26 and UL26.5 proteins, was identified by mAb 1C8. The result of the ELISA showed that this epitope could be recognized by DEV-positive serum from mice. The 520IYYPGE525 motif was the minimal requirement for reactivity, as demonstrated by analysis of the reactivity of 1C8 with several truncated peptides derived from the motif. Alignment and comparison of the 1C8-defined epitope sequence with those of other alphaherpesviruses indicated that the motif 521YYPGE525 in the epitope sequence was conserved among the alphaherpesviruses. Conclusion A mAb, 1C8, was generated against DEV UL26c and the epitope-defined minimal sequence obtained using mAb 1C8 was 520IYYPGE525. The mAb and the identified epitope may be useful for further study of the design of diagnostic reagents for DEV.

Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies

International Nuclear Information System (INIS)

Escobar, Normando Iznaga; Morales, Alejo; Nunez, Gilda

1996-01-01

A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (μg/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 μg, the lowest detection limit for cysteine quantification was 0.03 μg. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples

Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Escobar, Normando Iznaga; Morales, Alejo; Nunez, Gilda

1996-07-01

A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration ({mu}g/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 {mu}g, the lowest detection limit for cysteine quantification was 0.03 {mu}g. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.

Tuning of CO2 Reduction Selectivity on Metal Electrocatalysts.

Science.gov (United States)

Wang, Yuhang; Liu, Junlang; Wang, Yifei; Al-Enizi, Abdullah M; Zheng, Gengfeng

2017-11-01

Climate change, caused by heavy CO 2 emissions, is driving new demands to alleviate the rising concentration of atmospheric CO 2 levels. Enlightened by the photosynthesis of green plants, photo(electro)chemical catalysis of CO 2 reduction, also known as artificial photosynthesis, is emerged as a promising candidate to address these demands and is widely investigated during the past decade. Among various artificial photosynthetic systems, solar-driven electrochemical CO 2 reduction is widely recognized to possess high efficiencies and potentials for practical application. The efficient and selective electroreduction of CO 2 is the key to the overall solar-to-chemical efficiency of artificial photosynthesis. Recent studies show that various metallic materials possess the capability to play as electrocatalysts for CO 2 reduction. In order to achieve high selectivity for CO 2 reduction products, various efforts are made including studies on electrolytes, crystal facets, oxide-derived catalysts, electronic and geometric structures, nanostructures, and mesoscale phenomena. In this Review, these methods for tuning the selectivity of CO 2 electrochemical reduction of metallic catalysts are summarized. The challenges and perspectives in this field are also discussed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus.

Science.gov (United States)

Nayak, Ajay P; Green, Brett J; Janotka, Erika; Hettick, Justin M; Friend, Sherri; Vesper, Steve J; Schmechel, Detlef; Beezhold, Donald H

2011-09-01

Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

International Nuclear Information System (INIS)

Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

1990-01-01

A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

Energy Technology Data Exchange (ETDEWEB)

Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

1990-08-01

A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb

Science.gov (United States)

Feng, Ningguo; Lawton, Jeffrey A.; Gilbert, Joana; Kuklin, Nelly; Vo, Phuoc; Prasad, B.V. Venkataram; Greenberg, Harry B.

2002-01-01

Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb’s directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle. PMID:11994409

The Relationship of Workplace Culture With Nursing-Sensitive Organizational Factors.

Science.gov (United States)

Hahtela, Nina; McCormack, Brendan; Paavilainen, Eija; Slater, Paul; Helminen, Mika; Suominen, Tarja

2015-01-01

The aim of this study is to explore the relations of workplace culture on nursing-sensitive organizational factors. The need for standardized and valid measures for nursing-sensitive organizational outcomes has already been recognized in the literature. A cross-sectional questionnaire survey of 21 inpatient acute care units in 9 organizations at the municipal primary healthcare level was conducted. Participants included licensed practical nurses, registered nurses, and nurse managers. Workplace culture, especially the overarching factor of stress, correlated with the use of supplemental nursing staff and patients' length of stay. It is essential to find and test workplace-sensitive indicators so that managers will have a wider range of methods to plan and evaluate nursing outcomes.

Basophil Sensitivity Decreases During the Updosing of SCIT in Subjects Allergic to Grass Pollen

DEFF Research Database (Denmark)

Schmid, Johannes Martin; Dahl, Ronald; Hoffmann, Hans Jürgen

).This corresponds to an approximately 14-fold reduction in basophil sensitivity. No significant changes were observed in the baseline, washed cells or control group. CONCLUSIONS: We show that the main mechanism leading to a reduction of basophil sensitivity is humoral. PII: S0091-6749(10)02749-1 doi:10.1016...

Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

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Justas Lazutka

2014-01-01

Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

The use of oligonucleotide probes for meningococcal serotype characterization

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SACCHI Claudio Tavares

1998-01-01

Full Text Available In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs. Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A and 615U (VR3-B used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

Detection of koi herpesvirus (KHV) using a monoclonal antibody against Cyprinus carpio IgM.

Science.gov (United States)

Li, Yingying; Zheng, Shucheng; Wang, Qing; Bergmann, Sven M; Zeng, Weiwei; Wang, Yingying; Liu, Chun; Shi, Cunbin

2017-08-01

Koi herpesvirus disease (KHVD) is associated with high mortality in both common carp and koi carp (Cyprinus carpio L.) worldwide. The indirect detection of fish viruses based on the identification of antibodies has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against carp IgM. By using hybridoma-monoclonal antibody technology, one hybridoma cell line secreting MAbs against IgM from carp was established. In western blot analysis, the secreted MAb from cell line A5-E10 recognized the heavy chain of IgM from common carp or koi but did not react with immunoglobulins from three different fish species: grass carp (Ctenopharyngodon idella), tilapia (Oreochromis mossambicus) and Mandarin fish (Siniperca chuatsi). These results demonstrated that this MAb is highly specific for the IgM of carp and suggested that it can be used for monitoring the immunity level of carp, for example for indirect KHV diagnosis by antibody ELISA. We therefore established an indirect ELISA, which was tested using 200 serum samples from koi from three farms. The final results showed that 147 (73.5%) samples were confirmed to be KHV antibody negative and 53 (26.5%) were definitely positive, containing antibodies against KHV.

Reduction of intracellular glutathione content and radiosensitivity

International Nuclear Information System (INIS)

Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

1986-05-01

The intracellular glutathione (GSH) content in HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulfoximine (BSO) or diethyl maleate (DEM). Clonogenicity, single strand DNA breaks (ssb) and double strand DNA breaks (dsb) were used as criteria for radiation induced damage after X- or γ irradiation. In survival experiments DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the OER was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (Auth.)

Reduction of intracellular glutathione content and radiosensitivity

International Nuclear Information System (INIS)

Vos, O.; Schans, G.P. van der; Roos-Verheij, W.S.D.

1986-01-01

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or γ-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb. (author)

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

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Gaëtan Vanier

Full Text Available Monoclonal antibodies (mAbs represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.

Sensitivity of district heating system operation to heat demand reductions and electricity price variations: A Swedish example

International Nuclear Information System (INIS)

Åberg, M.; Widén, J.; Henning, D.

2012-01-01

In the future, district heating companies in Sweden must adapt to energy efficiency measures in buildings and variable fuel and electricity prices. Swedish district heating demands are expected to decrease by 1–2% per year and electricity price variations seem to be more unpredictable in the future. A cost-optimisation model of a Swedish local district heating system is constructed using the optimisation modelling tool MODEST. A scenario for heat demand changes due to increased energy efficiency in buildings, combined with the addition of new buildings, is studied along with a sensitivity analysis for electricity price variations. Despite fears that heat demand reductions will decrease co-generation of clean electricity and cause increased global emissions, the results show that anticipated heat demand changes do not increase the studied system's primary energy use or global CO 2 emissions. The results further indicate that the heat production plants and the fuels used within the system have crucial importance for the environmental impact of district heat use. Results also show that low seasonal variations in electricity price levels with relatively low winter prices promote the use of electric heat pumps. High winter prices on the other hand promote co-generation of heat and electricity in CHP plants. -- Highlights: ► A MODEST optimisation model of the Uppsala district heating system is built. ► The impact of heat demand change on heat and electricity production is examined. ► An electricity price level sensitivity analysis for district heating is performed. ► Heat demand changes do not increase the primary energy use or global CO 2 emissions. ► Low winter prices promote use of electric heat pumps for district heating production.

Enhanced Sensitive Immunoassay: Noncompetitive Phage Anti-Immune Complex Assay for the Determination of Malachite Green and Leucomalachite Green

Science.gov (United States)

2015-01-01

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG–mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2LMG = 0.9841; R2MG = 0.993; R2Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety. PMID:25077381

Gastric intestinal metaplasia as detected by a monoclonal antibody is highly associated with gastric adenocarcinoma.

Science.gov (United States)

Mirza, Z K; Das, K K; Slate, J; Mapitigama, R N; Amenta, P S; Griffel, L H; Ramsundar, L; Watari, J; Yokota, K; Tanabe, H; Sato, T; Kohgo, Y; Das, K M

2003-06-01

Some forms of gastric intestinal metaplasia (GIM) may be precancerous but the cellular phenotype that predisposes to gastric carcinogenesis is not well characterised. Mucin staining, as a means of differentiating GIM, is difficult. A monoclonal antibody, mAb Das-1 (initially called 7E(12)H(12)), whose staining is phenotypically specific to colon epithelium, was used to investigate this issue. Using mAb Das-1, by a sensitive immunoperoxidase assay, we examined histologically confirmed GIM specimens from two countries, the USA and Japan. A total of 150 patients comprised three groups: group A, GIM (fields away from the cancer area) from patients with gastric carcinoma (n=60); group B, GIM with chronic gastritis (without gastric carcinoma) (n=72); and group C, chronic gastritis without GIM (n=18). Fifty six of 60 (93%) patients with GIM (both goblet and non-goblet metaplastic cells) from group A reacted intensely with mAb Das-1. Cancer areas from the same 56 patients also reacted. In contrast, 25/72 (35%) samples of GIM from patients in group B reacted with mAb Das-1 (group A v B, p<0.0001). None of the samples from group C reacted with the mAb. Reactivity of mAb Das-1 is clinically useful to simplify and differentiate the phenotypes of GIM. The colonic phenotype of GIM, as identified by mAb Das-1, is strongly associated with gastric carcinoma.

Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

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Bipulendu Jena

Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

Science.gov (United States)

Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

2016-03-01

Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

Multi-UAV joint target recognizing based on binocular vision theory

Directory of Open Access Journals (Sweden)

Yuan Zhang

2017-01-01

Full Text Available Target recognizing of unmanned aerial vehicle (UAV based on image processing take the advantage of 2D information containing in the image for identifying the target. Compare to single UAV with electrical optical tracking system (EOTS, multi-UAV with EOTS is able to take a group of image focused on the suspected target from multiple view point. Benefit from matching each couple of image in this group, points set constituted by matched feature points implicates the depth of each point. Coordinate of target feature points could be computing from depth of feature points. This depth information makes up a cloud of points and reconstructed an exclusive 3D model to recognizing system. Considering the target recognizing do not require precise target model, the cloud of feature points was regrouped into n subsets and reconstructed to a semi-3D model. Casting these subsets in a Cartesian coordinate and applying these projections in convolutional neural networks (CNN respectively, the integrated output of networks is the improved result of recognizing.

Mining Emerging Patterns for Recognizing Activities of Multiple Users in Pervasive Computing

DEFF Research Database (Denmark)

Gu, Tao; Wu, Zhanqing; Wang, Liang

2009-01-01

Understanding and recognizing human activities from sensor readings is an important task in pervasive computing. Existing work on activity recognition mainly focuses on recognizing activities for a single user in a smart home environment. However, in real life, there are often multiple inhabitants...... activity models, and propose an Emerging Pattern based Multi-user Activity Recognizer (epMAR) to recognize both single-user and multiuser activities. We conduct our empirical studies by collecting real-world activity traces done by two volunteers over a period of two weeks in a smart home environment...... sensor readings in a home environment, and propose a novel pattern mining approach to recognize both single-user and multi-user activities in a unified solution. We exploit Emerging Pattern – a type of knowledge pattern that describes significant changes between classes of data – for constructing our...

Sensitivity Analysis of the Critical Speed in Railway Vehicle Dynamics

DEFF Research Database (Denmark)

Bigoni, Daniele; True, Hans; Engsig-Karup, Allan Peter

2014-01-01

We present an approach to global sensitivity analysis aiming at the reduction of its computational cost without compromising the results. The method is based on sampling methods, cubature rules, High-Dimensional Model Representation and Total Sensitivity Indices. The approach has a general applic...

Radon in water - fast, simple and sensitive measurement

International Nuclear Information System (INIS)

Philipsborn, H. von

1996-01-01

Glass fiber filters of a certain brand were found to be very efficient (retention >95%) for adsorption of short-lived radon decay products during filtration of water. Limit of detection is 2 Bq/L in 10 min total time if the dried filters are evaluated in the Living Level Monitor mab LLM 500. Locally high radon concentrations in drinking water have activated country-wide surveys and proposals for compulsory action levels. (orig.) [de

The Fragile X Mental Retardation Protein, FMRP, Recognizes G-Quartets

Science.gov (United States)

Darnell, Jennifer C.; Warren, Stephen T.; Darnell, Robert B.

2004-01-01

Fragile X mental retardation is a disease caused by the loss of function of a single RNA-binding protein, FMRP. Identifying the RNA targets recognized by FMRP is likely to reveal much about its functions in controlling some aspects of memory and behavior. Recent evidence suggests that one of the predominant RNA motifs recognized by the FMRP…

Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species.

Science.gov (United States)

Yu, Meng; Stevens, Vicky; Berry, Jody D; Crameri, Gary; McEachern, Jennifer; Tu, Changchun; Shi, Zhengli; Liang, Guodong; Weingartl, Hana; Cardosa, Jane; Eaton, Bryan T; Wang, Lin-Fa

2008-02-29

Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.

New avenues in hypoxic cell sensitization

International Nuclear Information System (INIS)

Huilgol, N.G.; Chatterjee, N.A.; Singh, B.B.

1995-01-01

Hypoxic cells in tumors represent a population of cells that are resistant to radiotherapy. Bio-reductive agents like RSU 1069, RBU 6145 and EOg and vasoactive drugs in conjunction with hypoxic cell sensitizers are being evaluated as hypoxic cell cytotoxins. Chlorpromazine a membrane active drug and AK-2123- a nitrotriazole with a potential to deplete intracellular thiols induced vasoconstriction and sensitize hypoxic cells have stretched the boundaries of innovation. A preliminary experience with these drugs is discussed. 8 refs., 2 tabs., 2 figs

Natural IgM antibodies that bind neoepitopes exposed as a result of spinal cord injury , drive secondary injury by activating complement.

Science.gov (United States)

Narang, Aarti; Qiao, Fei; Atkinson, Carl; Zhu, Hong; Yang, Xiaofeng; Kulik, Liudmila; Holers, V Michael; Tomlinson, Stephen

2017-06-19

Natural IgM antibodies (Abs) function as innate immune sensors of injury via recognition of neoepitopes expressed on damaged cells, although how this recognition systems function following spinal cord injury (SCI) exposes various neoepitopes and their precise nature remains largely unknown. Here, we investigated the role of two natural IgM monoclonal Abs (mAbs), B4 and C2, that recognize post-ischemic neoepitopes following ischemia and reperfusion in other tissues. Identification of post-SCI expressed neoepitopes was examined using previously characterized monoclonal Abs (B4 and C2 mAbs). The role of post-SCI neoepitopes and their recognition by natural IgM Abs in propagating secondary injury was examined in Ab-deficient Rag1-/- or wild type C57BL/6 mice using Ab reconstitution experiments and neoepitope-targeted therapeutic studies, respectively. Administration of B4 or C2 mAb following murine SCI increased lesion size and worsened functional outcome in otherwise protected Ab-deficient Rag1-/- mice. Injury correlated with colocalized deposition of IgM and C3d in injured spinal cords from both mAb reconstituted Rag1-/- mice and untreated wild-type mice. Depletion of peritoneal B1 B cells, a source of natural Abs, reduced circulating levels of IgM with B4 (annexin-IV) and C2 (subset of phospholipids) reactivity, reduced IgM and complement deposition in the spinal cord, and protected against SCI. We therefore investigated whether the B4 neoepitope represents a therapeutic target for complement inhibition. B4-Crry, a fusion protein consisting of a single-chain Ab derived from B4 mAb, linked to the complement inhibitor Crry, significantly protected against SCI. B4-Crry exhibited a dual function in that it inhibited both the binding of pathogenic IgM and blocked complement activation in the spinal cord. This study identifies important neoepitopes expressed within the spinal cord after injury. These neoepitopes are recognized by clonally specific natural IgM Abs that

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

Science.gov (United States)

Zhang, Cathy X. Y.; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

2016-01-01

ABSTRACT The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. IMPORTANCE L. monocytogenes is

Kinetic Modeling of Methionine Oxidation in Monoclonal Antibodies from Hydrogen Peroxide Spiking Studies.

Science.gov (United States)

Hui, Ada; Lam, Xanthe M; Kuehl, Christopher; Grauschopf, Ulla; Wang, Y John

2015-01-01

When isolator technology is applied to biotechnology drug product fill-finish process, hydrogen peroxide (H2O2) spiking studies for the determination of the sensitivity of protein to residual peroxide in the isolator can be useful for assessing a maximum vapor phase hydrogen peroxide (VPHP) level. When monoclonal antibody (mAb) drug products were spiked with H2O2, an increase in methionine (Met 252 and Met 428) oxidation in the Fc region of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Fc-Met and H2O2 was stoichiometric (i.e., 1:1 molar ratio), and the reaction rate was dependent on the concentrations of mAb and H2O2. The consumption of H2O2 by Fc-Met oxidation in the mAb followed pseudo first-order kinetics, and the rate was proportional to mAb concentration. The extent of Met 428 oxidation was half of that of Met 252, supporting that Met 252 is twice as reactive as Met 428. Similar results were observed for free L-methionine when spiked with H2O2. However, mAb formulation excipients may affect the rate of H2O2 consumption. mAb formulations containing trehalose or sucrose had faster H2O2 consumption rates than formulations without the sugars, which could be the result of impurities (e.g., metal ions) present in the excipients that may act as catalysts. Based on the H2O2 spiking study results, we can predict the amount Fc-Met oxidation for a given protein concentration and H2O2 level. Our kinetic modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study to support the use of VPHP isolator for antibody drug product manufacture. Isolator technology is increasing used in drug product manufacturing of biotherapeutics. In order to understand the impact of residual vapor phase hydrogen peroxide (VPHP) levels on protein product quality, hydrogen peroxide (H2O2) spiking studies may be performed to determine the sensitivity of monoclonal antibody

A sensitivity analysis of timing and costs of greenhouse gas emission reductions

NARCIS (Netherlands)

Gerlagh, R.; van der Zwaan, B.C.C.

2004-01-01

This paper analyses the optimal timing and macro-economic costs of carbon emission reductions that mitigate the global average atmospheric temperature increase. We use a macro-economic model in which there are two competing energy sources, fossil-fuelled and non-fossil-fuelled. Technological change

Anti-Podocalyxin Monoclonal Antibody 47-mG2a Detects Lung Cancers by Immunohistochemistry.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances in lung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers. We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ), both of which were suitable for immunohistochemical analyses of oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG 2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG 2a stained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting PODXL in lung cancers.

Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

Science.gov (United States)

Wang, Sheng-Yu; Li, Zhe; Wang, Xian-Jiang; Lv, Sha; Yang, Yun; Zeng, Lian-Qiang; Luo, Fang-Hong; Yan, Jiang-Hua; Liang, Da-Feng

2014-10-01

Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

Evaluation and Development of Chemical Kinetic Mechanism Reduction Scheme for Biodiesel and Diesel Fuel Surrogates

DEFF Research Database (Denmark)

Poon, Hiew Mun; Ng, Hoon Kiat; Gan, Suyin

2013-01-01

The aim of this study is to evaluate the existing chemical kinetic mechanism reduction techniques. From here, an appropriate reduction scheme was developed to create compact yet comprehensive surrogate models for both diesel and biodiesel fuels for diesel engine applications. The reduction...... techniques applied here were Directed Relation Graph (DRG), DRG with Error Propagation, DRG-aided Sensitivity Analysis, and DRG with Error Propagation and Sensitivity Analysis. Nonetheless, the reduced mechanisms generated via these techniques were not sufficiently small for application in multi......-dimensional computational fluid dynamics (CFD) study. A new reduction scheme was therefore formulated. A 68-species mechanism for biodiesel surrogate and a 49-species mechanism for diesel surrogate were successfully derived from the respective detailed mechanisms. An overall 97% reduction in species number...

Framework for benchmarking FA-based string recognizers

CSIR Research Space (South Africa)

Ngassam, EK

2010-10-01

Full Text Available of suggested algorithms by domain-specific FA-implementers requires prior knowledge of the behaviour (performance-wise) of each algorithm in order to make an informed choice. The authors propose a based string recognizers such that FA-implementers could capture...

Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

Science.gov (United States)

Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

2001-09-15

Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

Science.gov (United States)

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

Generation of neutralizing monoclonal antibodies against a conformational epitope of human adenovirus type 7 (HAdv-7 incorporated in capsid encoded in a HAdv-3-based vector.

Directory of Open Access Journals (Sweden)

Minglong Liu

Full Text Available The generation of monoclonal antibodies (MAbs by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5 of adenovirus type 7 (HAdv-7 was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses.

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

International Nuclear Information System (INIS)

Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S.

2014-01-01

Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

Energy Technology Data Exchange (ETDEWEB)

Pan, Yang [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Sasaki, Tadahiro [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Inoue, Yuji [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yasugi, Mayo [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Du, Anariwa [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Boonsathorn, Naphatsawan [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Ibrahim, Madiha S. [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour (Egypt); and others

2014-07-18

Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan