scholarly journals Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 874-880 ◽  
Author(s):  
AD D'Andrea ◽  
PJ Szklut ◽  
HF Lodish ◽  
EM Alderman
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Sodium Dodecyl ◽  
Dependent Manner ◽  
Epo Receptor ◽  
High Affinity ◽  
Group I ◽  
Dependent Cell ◽  
Group Ii ◽  
Murine Erythroleukemia

Abstract We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N- terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO- dependent cell line. Only Group II MoAbs inhibit a newly developed EPO- dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

scholarly journals Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 874-880
Author(s):  
AD D'Andrea ◽  
PJ Szklut ◽  
HF Lodish ◽  
EM Alderman
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Sodium Dodecyl ◽  
Dependent Manner ◽  
Epo Receptor ◽  
High Affinity ◽  
Group I ◽  
Dependent Cell ◽  
Group Ii ◽  
Murine Erythroleukemia

We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO). All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L. Two MoAbs, designated Group I MoAbs, bind to an epitope within the N- terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO. Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO. We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding. MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L). To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO. Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner. We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO- dependent cell line. Only Group II MoAbs inhibit a newly developed EPO- dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3675-3683 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Toshiaki Aoki ◽  
Masamichi Shiraga ◽  
Yoshiyuki Kurata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Binding Sites ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Fibrinogen Binding ◽  
Group I ◽  
Group Ii ◽  
Inhibition Studies

Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

scholarly journals Proteomics Analysis of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Identified Two New Occlusion-Derived Virus-Associated Proteins, HA44 and HA100

2007 ◽  
Vol 81 (17) ◽  
pp. 9377-9385 ◽  
Author(s):  
Fei Deng ◽  
Ranran Wang ◽  
Minggang Fang ◽  
Yue Jiang ◽  
Xushi Xu ◽  
...  
Keyword(s):  
Helicoverpa Armigera ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Proteins ◽  
Proteomics Analysis ◽  
Group I ◽  
Associated Proteins ◽  
Helicoverpa Armígera ◽  
Group Ii ◽  
First Time

ABSTRACT Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3675-3683 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Toshiaki Aoki ◽  
Masamichi Shiraga ◽  
Yoshiyuki Kurata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Binding Sites ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Fibrinogen Binding ◽  
Group I ◽  
Group Ii ◽  
Inhibition Studies

Abstract Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

scholarly journals Isolation and characterization of β- and γ-caseins from horse milk

1982 ◽  
Vol 203 (1) ◽  
pp. 131-139 ◽  
Author(s):  
S Visser ◽  
R Jenness ◽  
R J Mullin
Keyword(s):  
Room Temperature ◽  
Sodium Dodecyl ◽  
Sulphate Group ◽  
Group Iii ◽  
Isolation And Characterization ◽  
Group I ◽  
Beta Casein ◽  
Group Ii ◽  
Phosphate Groups

Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the action of a plasmin-like enzyme present in horse milk. It does not contain phosphate or carbohydrate. Homology of this group with bovine gamma-caseins is demonstrated. Both beta- and gamma-like caseins are more soluble at 4 degrees C than at room temperature.

scholarly journals Monoclonal Antibodies against Extracellular Domains of Claudin-1 Block Hepatitis C Virus Infection in a Mouse Model

2015 ◽  
Vol 89 (9) ◽  
pp. 4866-4879 ◽  
Author(s):  
Masayoshi Fukasawa ◽  
Shotaro Nagase ◽  
Yoshitaka Shirasago ◽  
Manami Iida ◽  
Mayo Yamashita ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Infection ◽  
Dependent Manner ◽  
Entry Inhibitors ◽  
High Affinity ◽  
Extracellular Domains ◽  
Very High

ABSTRACTHepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1). Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infectionin vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents.IMPORTANCESafe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibitedin vitroandin vivoHCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV.

scholarly journals Murine Monoclonal Antibodies against the Receptor Binding Domain of SARS-CoV-2 Neutralize Authentic Wild-Type SARS-CoV-2 as Well as B.1.1.7 and B.1.351 Viruses and Protect In Vivo in a Mouse Model in a Neutralization-Dependent Manner

mBio ◽  
2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B. Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Mouse Model ◽  
Protective Effect ◽  
Receptor Binding ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Cross Neutralization ◽  
Murine Monoclonal Antibodies

Cross-neutralization of SARS-CoV-2 variants by RBD-targeting antibodies is still not well understood, and very little is known about the potential protective effect of nonneutralizing antibodies in vivo . Using a panel of mouse monoclonal antibodies, we investigate both of these points.

KIRs Expression and Cytotoxic Activities of Cord Blood Natural Killer Cells Against Acute Lymphoblastic Leukemia with MLL Gene Rearrangement.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2803-2803
Author(s):  
Hiroko Honna ◽  
Kumiko Goi ◽  
Kinuko Hirose ◽  
Itaru Kuroda ◽  
Takeshi Inukai ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Cell Lines ◽  
Cord Blood Transplantation ◽  
Cytotoxic Activities ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Group I ◽  
Significant Difference ◽  
Group Ii

Abstract MLL-rearranged ALL is associated with an extremely poor prognosis despite intensive chemotherapy and hematopoietic stem cell transplantation (SCT). We have reported that MLL-rearranged ALL is resistant to death-inducing ligands TRAIL and FasL expressed on cytotoxic T lymphocytes, and therefore T-cell mediating graft-versus-leukemia (GVL) effect is not expected post allogeneic SCT. We recently demonstrated that MLL-rearranged ALL cells were effectively killed by allogeneic NK cells from adult peripheral blood (PB) in a perforin-dependent manner when KIR (killer cell Ig-like receptor) ligand incompatibility exists between ALL cells and NK cells. This KIR ligand incompatibility has been reported to possibly reduce the relapse rate of acute myeloid leukemia post SCT. To pursue the clinical implication in KIR ligand incompatible cord blood transplantation (CBT) for the treatment of MLL-rearranged ALL, we examined the KIRs expression on allogeneic NK cells from umbilical cord blood (CB) by flow cytometry and their in vitro cytotoxic activities against MLL-rearranged ALL cell lines by a standard Cr-release assay at an effector-to-target ratio of 20 to 40. The results were compared between NK cells from adult PB and CB, and between KIR ligand compatible and incompatible NK cells. NK cells from CB were enriched by negative selection and classified into 2 groups based on HLA-C alleles; C1/C1 type (n=5) both alleles belonging to group I (Cw1, Cw3 et. al.) and C1/C2 type (n=4) each of alleles belonging to group I and group II (Cw2, Cw4, et. al.). NK cells from adult PB were similarly classified into C1/C1 type (n=4) and C1/C2 type (n=5). All of the MLL-rearranged ALL cell lines established in our laboratory (n=10) were C1/C1 type, and two cell lines with MLL-ENL (KOPN1, KOCL50) were used as targets. K562 lacking HLA class I expression was used as a positive control target. Although there was no significant difference in the HLA-C group II receptor (KIR2DL1, CD158a) expression between CB- and adult PB-NK cells (17.8±6.3% vs. 26.5±15.2%), the HLA-C group I receptor (KIR2DL2/L3, CD158b) expression on CB-NK cells was significantly lower than on adult PB-NK cells (24.3±10.2% vs. 47.4±19.2%, p=0.009). The CD158b expression showed no difference between C1/C1 and C1/C2 types of CB-NK cells, but it expressed higher on C1/C1 type of adult PB-NK cells than on C1/C2 type (58.9±17.4% vs. 35.9±14.0%. p=0.047), suggesting that the CD158b expression on NK cells increases as getting older particularly in C1/C1 type individuals. In the cytotoxic assay, CB-NK cells irrespective of C1/C1 and C1/C2 types exhibited a lower cytotoxicity against K562 compared with adult PB-NK cells (42.0±19.2% vs. 63.6±9.5%, p=0.009). Of importance, although both C1/C1 and C1/C2 CB-NK cells showed a similar cytotoxicity against K562, C1/C2 CB-NK cells exhibited a significantly higher cytotoxicity against C1/C1 MLL-rearranged ALL cell lines than did C1/C1 CB-NK cells when assessed by a relative cytotoxicity to K562 (KOPN1, 0.84±0.19 vs. 0.47±0.13, p=0.028; KOCL-50, 0.87±0.27 vs. 0.40±0.14, p=0.028), suggesting that a loss of inhibitory signal to CD158a on NK cells from leukemia cells can specifically enhance their alloreactivity. Taken together, MLL-rearranged ALL cells are sensitive to killing by KIR ligand incompatible allogeneic CB-NK cells, and therefore the maximal GVL effect against this leukemia could be expected if the specific CB whose NK cells can exert their alloreactivity is selected for CBT.

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