scholarly journals Evaluation of E-rosetting human lymphocytes with OKT11 and other monoclonal antibodies

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 795-799
Author(s):  
SH Ip ◽  
CW Rittershaus ◽  
CC Struzziero ◽  
JA Hoxie ◽  
RA Hoffman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Blood Cells ◽  
Human Lymphocytes ◽  
Immunofluorescent Staining ◽  
Intensity Data ◽  
Blood Samples ◽  
Staining Methods

Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette- positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

scholarly journals Evaluation of E-rosetting human lymphocytes with OKT11 and other monoclonal antibodies

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 795-799 ◽  
Author(s):  
SH Ip ◽  
CW Rittershaus ◽  
CC Struzziero ◽  
JA Hoxie ◽  
RA Hoffman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Blood Cells ◽  
Human Lymphocytes ◽  
Immunofluorescent Staining ◽  
Intensity Data ◽  
Blood Samples ◽  
Staining Methods

Abstract Monoclonal antibody OKT11 was found to compete with sheep red blood cells for binding sites on human lymphocytes. Preincubation of lymphocytes with OKT11 eliminated E-rosette formation. In a study of 142 peripheral blood samples ranging from 1% to over 90% E-rosette- positive cells, comparison to the percent OKT11-positive cells yielded a correlation coefficient of 0.93. In normal donors, subsets of OKT11+ cells were identified using two-color immunofluorescent staining methods with OKT3, OKT4, and OKT8. On the average, approximately 13% of OKT11+ lymphocytes were OKT3- and 13% of OKT11+ lymphocytes were OKT4- and OKT8-. Based on our double antibody fluorescence intensity data, low antigen density OKT11+ lymphocytes were OKT3-. OKT4+ and OKT8+ lymphocytes in normal peripheral lymphocytes have similar OKT11 antigen density.

scholarly journals The Use of Rituximab in Immune-mediated Anaemia

2010 ◽  
Vol 00 (04) ◽  
pp. 11
Author(s):  
Daan Dierickx ◽  
Len Verbeke ◽  
◽  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immune System ◽  
Blood Cells ◽  
Immune Dysfunction ◽  
Chimeric Mouse ◽  
Toxicity Profile ◽  
Immune Mediated ◽  
Haematological Disorders ◽  
Anti Cd20

Immune-mediated anaemia is a collective term describing the occurrence of anaemia due to an immune dysfunction, leading directly or indirectly to the destruction of red blood cells. In recent years, as knowledge of the immune system has progressed, these disorders have also become better understood and their management improved. Monoclonal antibodies have emerged as a powerful tool in the treatment of many different disorders, including both haematological and non-haematological disorders. Most experience has been obtained with the use of rituximab, a chimeric mouse/human anti-CD20 monoclonal antibody, showing high overall response rates with a relatively safe toxicity profile. Here we describe the currently available evidence on the use of rituximab in immune-mediated anaemia. We will also reflect on potential side effects that might hamper the initial enthusiasm for its use in these disorders.

scholarly journals Study on using the hybrid cell a6g11c9 to produce the anti-a monoclonal antibody that agglunating a antigen on the surface of red blood cells

2017 ◽  
Vol 40 (1) ◽  
pp. 25-31
Author(s):  
Nguyen Thi Trung ◽  
Truong Nam Hai
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Red Blood Cells ◽  
Cell Line ◽  
Blood Cells ◽  
Culture Medium ◽  
Hybrid Cell ◽  
In Vitro Method ◽  
Antibody Solution

Almost of the ABO blood grouping reagents is being trading derive from  the monoclonal antibodies. There are two methods to produce the  monoclonal antibodies from hybridoma lines, which were in vitro method (hybridoma cultured in the medium) and in vivo method (hybridoma cultured in the mice intra-abdominal). In Vietnam, Nguyen Thi Trung and co-authors was succesfully screened in hybridoma cell line A6G11C9 which generating of the anti A monoclonal antibody agglutinated A antigen on the surface of red blood cells. The fusion of mouse lymohocyte B generated anti-A antibody with mouse myeloma sp2/0 is formed that hybrid cell lines. The anti-A monoclonal antibody is produced from hybridoma cell line A6G11C9 have been highly intensive confirmed. It is capability of growth and anti B monoclonal antibody producing stability through the generations. In this study, the process to produce large amounts of monoclonal antibodies from B4D10C9 hybridoma by in vitro method are published. Firstly, hybridoma cells are stored in liquid nitrogen to wake by culture in medium. Then, First, hybrid cells are stored frozen in liquid nitrogen to wake cultured cells. Then, they were first inoculated to produce enough biomass to serve a larger scale. Cell biomass continues to be second inoculated into DMEM containing 10% fetal bovin serum for 10 days. The culture medium contained anti-A monoclonal antibodies were collected by centrifugation to remove cells. The anti-A monoclonal antibody levels in culture medium was concentrated and remove phenol red indicator by the precipitation with NH4SO4 50% saturated. The anti-A monoclonal antibody solution at 5 times concentrated have been better agglutinated with erythrocytes containing A antigen than monoclonal antibody solution non-concentration. 150 ml of concentrated antibodies were produced. Antibody titer of the anti-A monoclonal antibodies in the concentrated 5 times solution was 1/512. The intensity of the reaction anti-A monclonal antibody with red blood cell containing A antigen was 4+.   Citation: Nguyen Thi Trung, Truong Nam Hai, 2018. Study on using the hybrid cell a6g11c9 to produce the anti-a monoclonal antibody that agglunating a antigen on the surface of red blood cells. Tap chi Sinh hoc, 40(1): x-xx. DOI: 10.15625/0866-7160/v40n1.9154. *Corresponding author: [email protected] Received 12 January 2017, accepted 20 December 2017 

scholarly journals Identification of Residues within Human Glycoprotein VI Involved in the Binding to Collagen

2004 ◽  
Vol 279 (50) ◽  
pp. 52293-52299 ◽  
Author(s):  
Christelle Lecut ◽  
Véronique Arocas ◽  
Hans Ulrichts ◽  
Anthony Elbaz ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Molecular Modeling ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Assays ◽  
Glycoprotein Vi ◽  
Ligand Binding Sites ◽  
Human Receptor

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

scholarly journals MuSK myasthenia gravis monoclonal antibodies

2019 ◽  
Vol 6 (3) ◽  
pp. e547 ◽  
Author(s):  
Maartje G. Huijbers ◽  
Dana L. Vergoossen ◽  
Yvonne E. Fillié-Grijpma ◽  
Inge E. van Es ◽  
Marvyn T. Koning ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Myasthenia Gravis ◽  
Binding Sites ◽  
Neuromuscular Junctions ◽  
Affinity Maturation ◽  
Fab Fragments ◽  
Serum Igg4 ◽  
Muscle Specific Kinase ◽  
Achr Clustering

ObjectiveTo isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.MethodsWe generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.ResultsThe isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.ConclusionsPatient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic.

scholarly journals Preferential Binding of Anti-Neutrophil Cytoplasmic Antibodies to an Unexpected Epitope of a Chimeric Proteinase 3 Mutant

2019 ◽  
Author(s):  
Marta Casal Moura ◽  
Gwen E. Thompson ◽  
Darlene A. Nelson ◽  
Lynn A. Fussner ◽  
Amber M. Hummel ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Granulomatosis With Polyangiitis ◽  
Antigen Binding ◽  
Proteinase 3 ◽  
Necrotizing Vasculitis ◽  
Novel Treatment ◽  
Preferential Binding ◽  
Major Antigen

AbstractProteinase 3 (PR3) is the major antigen for anti-neutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3-targeting ANCAs (PR3-ANCAs) recognize different epitopes on PR3 and are thought to be pathogenic for the development of the necrotizing vasculitis. To identify epitopes recognized by PR3-ANCAs, we pursued a strategy based on human-murine chimeric PR3 mutants. Interestingly, rather than observing reduced binding of PR3-ANCAs to Epitope 5 on a PR3 mutant (iHm5-Val103) with chimeric mutations in Epitope 5, we found substantially increased binding of the majority of PR3-ANCAs to iHm5-Val103 compared with the PR3 mutant (iPR3-Val103) clinically used to detect PR3-ANCAs. More interestingly, using iHm5-Val103 we identified a monoclonal antibody (moANCA518) from a patient with GPA that bound selectively to iHm5-Val103. Inhibition experiments using epitope-specific monoclonal antibodies and their antigen-binding fragments mapped the binding sites of moANCA518 and PR3-ANCAs (from patients displaying preferential binding to iHm5-Val103 over iPR3-Val103) to Epitope 3 on iHm5-Val103, a mutation-free epitope located far from the mutation sites in Epitope 5. These results demonstrate that the selective binding of moANCA518 (and likely the preferential binding of PR3-ANCAs from patients) to iHm5-Val103 is conferred by increased antigenicity of Epitope 3 on iHm5-Val103 caused by distal mutations, indicating that PR3-ANCAs bind to epitopes of a folded antigen conducive to allosteric effects of mutations—a previously unrecognized characteristic with implications for studying antibody-mediated autoimmune diseases and novel treatment approaches.

scholarly journals Creating the hybridoma to produce monoclonal antibodies causing the agglutination of the human red blood cells containing A antigen

2016 ◽  
Vol 14 (3) ◽  
pp. 411-417
Author(s):  
Nguyễn Thị Trung ◽  
Nguyễn Thị Hằng ◽  
Vũ Thị Thu Hằng ◽  
Lê Văn Phan ◽  
Trương Nam Hải
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Red Blood Cells ◽  
Blood Group ◽  
Blood Cells ◽  
Hybrid Cell ◽  
Selective Medium ◽  
Standard Group ◽  
Abo Blood Group ◽  
Group A

The determination of ABO blood group is obliged in many cases especially before blood transfusion, that is indicated at Point a, Clause 4, Article 14 Circular 26/2013/TT-BYT - Vietnam, date 09.16.2013. For this purpose, both standard sera (monoclonal antibodies) and standard red blood cells are common used but monoclonal antibodies are prefered. In Vietnam, monoclonal antibodies against ABO blood group are not available in domestic production. In this study, we succeeded in the generation of hybidoma cells secreting anti-A monoclonal antibody. Firstly, Balb/c mice were injected with Vietnamese human group A red blood cells to evoke B lymphocyte cells against A antigen present on the surface of the red blood cells. Afterward the lymphocytes were fused with sp2/0 myeloma cells in the presence of polyethylene glycol (PEG) to gain hybrid cells that were identified through ability to expand cells in a selective medium (hypoxanthine aminopterine thymine - HAT) at 37°C and 5% CO2. During screening and isolation process, the positive clones were identified by agglutination test with standard group A red blood cells. Of the 1440 wells, 12 monoclonal hybrid clones were selected. The hybrid cell line (designated A6G11C9) was the best one secreting the highest anti-A monoclonal antibody into culture with the antibody titer of 512. The antibody showed good intensity (+++), and the agglutination was visible by 10 seconds. This antibody is the promising for ABO-grouping kit development.

1-Desamino-8-D-Arginine Vasopressin (DDAVP) Infusion in Type IIB von Willebrand’s Disease: Shortening of Bleeding Time and Induction of a Variable Pseudothrombocytopenia

1990 ◽  
Vol 64 (01) ◽  
pp. 117-120 ◽  
Author(s):  
Alessandra Casonato ◽  
M Teresa Sartori ◽  
Luigi de Marco ◽  
Antonio Girolami
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Count ◽  
Arginine Vasopressin ◽  
Calcium Concentration ◽  
Sodium Citrate ◽  
Bleeding Time ◽  
Blood Collection ◽  
Von Willebrand's Disease ◽  
Blood Samples ◽  
Von Willebrand’S Disease

SummaryWe have investigated the effects of 1-desamino-8-D-arginine vasopressin (DDAVP) infusion on platelet count and bleeding time in 4 patients with type IIB von Willebrand’s disease (vWd). Three of four patients showed a normalization of the bleeding time within 1 h after the infusion, while bleeding time was not modified in the fourth. In accordance with the literature, thrombocytopenia was observed after DDAVP infusion, but this thrombocytopenia was due to the anticoagulants used for blood collection. In two patients (F. I., G. F.) no thrombocytopenia was observed when platelets were counted by fingerstick method but there was a 20% platelet decrease in blood samples collected in sodium citrate and a 50% decrease in samples collected in EDTA. Dramatic falls in platelet counts (70–95%) were observed in the additional two patients (C. A., D.Z.) after DDAVP infusion, when both sodium citrate or EDTA were used as anticoagulants. In the latter two patients there was also a 50% decrease in platelet count when the fingerstick method was used. The decrease in the patient’s platelet count in EDTA samples after DDAVP infusion could be prevented, in part, by the previous additions of an anti GPIb monoclonal antibody and an anti GPIIb-IIIa monoclonal antibody.Thus, the thrombocytopenia observed in the four IIB vWd patients studied after DDAVP infusion seems to be, at least partially, a pseudothrombocytopenia depending on the calcium concentration in the blood samples and the availability of GPIb and GPIIb-IIIa receptors. These findings and the normalization of the bleeding time observed in three of the four patients has led us to reconsider the possible use of DDAVP in the treatment of our IIB vWd patients.

Receptor Mediated Binding of the Fibrinolytic Components, Plasminogen and Urokinase, to Peripheral Blood Cells

1987 ◽  
Vol 58 (03) ◽  
pp. 936-942 ◽  
Author(s):  
Lindsey A Miles ◽  
Edward F Plow
Keyword(s):  
T Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Binding Sites ◽  
Blood Cells ◽  
High Capacity ◽  
Red Cells ◽  
Peripheral Blood Cells ◽  
Cellular Functions ◽  
Fibrinolytic Proteins

SummaryGlu-plasminogen binds to platelets; the monocytoid line, U937, and the human fetal fibroblast line, GM1380 bind both plasminogen and its activator, urokinase. This study assesses the interaction of these fibrinolytic proteins with circulating human blood cells. Plasminogen bound minimally to red cells but bound saturably and reversibly to monocytes, granulocytes and lymphocytes with apparent Kd values of 0.9-1.4 μM. The interactions were of high capacity with 1.6 to 49 × 105 sites/cell and involved the lysine binding sites of plasminogen. Both T cells and non-rosetting lymphocytes and two B cell lines saturably bound plasminogen. Urokinase bound saturably to gianulocytes, monocytes, non-rosetting lymphocytes and a B cell line, but minimally to T cells, platelets and red cells. Therefore, plasminogen binding sites of high capacity, of similar affinities, and with common recognition specificities are expressed by many peripheral blood cells. Urokinase receptors are also widely distributed, but less so than plasminogen binding sites. The binding ol plasminogen and/ or urokinase to these cells may lead to generation of cell- associated proteolytic activity which contributes to a variety of cellular functions.

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