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 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.

scholarly journals Human Rh D monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D+ red blood cells and suppression of Rh D immunization in Rh D- volunteers

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1701-1709 ◽  
Author(s):  
BM Kumpel ◽  
MJ Goodrick ◽  
DH Pamphilon ◽  
ID Fraser ◽  
GD Poole ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Hemolytic Disease ◽  
Barr Virus ◽  
Dependent Cell ◽  
Epstein Barr ◽  
D Cells

The use of prophylactic anti-D to prevent Rh D immunization in Rh D- women and subsequent hemolytic disease in Rh D+ infants is widespread, but has led to shortages of the anti-D Ig. With the aim of substituting monoclonal anti-D for Rh D prophylaxis, we have compared the abilities of monoclonal and polyclonal anti-D to clear Rh D+ red blood cells (RBCs) infused into Rh D- male volunteers and to suppress Rh D immunization. Two human monoclonal antibodies (MoAbs), BRAD-3 (IgG3) and BRAD-5 (IgG1), produced from stable Epstein-Barr virus-transformed B-lymphoblastoid cell lines, were selected because of their proven in vitro activity in promoting RBC lysis in antibody-dependent cell- mediated cytotoxicity assays. RBC clearance was assessed by intravenous injection of 3 mL of 51chromium-labeled D+ RBCs into 27 volunteers 48 hours after intramuscular injection of monoclonal or polyclonal anti-D. Further 3-mL injections of unlabeled D+ cells were administered at 6 and 9 months to induce immunization. Blood samples were taken throughout the 12-month period of study for the serologic detection of anti-D. The mean half-life (t50%) of RBCs in 7 recipients of 300 micrograms BRAD-5 (5.9 hours) was similar to that in 8 recipients of 500 IU polyclonal anti-D (5.0 hours), whereas D+ cells were cleared more slowly in some of the 8 subjects injected with 300 micrograms BRAD- 3 (mean t50% 12.7 hours) and in 1 individual administered 100 micrograms BRAD-3 (t50% 41.0 hours). The rate of RBC clearance in both groups administered 300 micrograms monoclonal anti-D correlated with the amount of antibody bound per cell, determined by flow cytometry. There was no evidence of primary immunization having occurred in any subject after 6 months of follow-up. Five of 24 subjects produced anti- D after one or two further injections of RBCs, confirming that they were responders who had been protected by the monoclonal or polyclonal anti-D administered initially. Four of these responders were recipients of monoclonal anti-D (3 BRAD-3, 1 BRAD-5). One individual who received BRAD-5 produced accelerated clearance of D+ RBCs at the third unprotected RBC challenge but did not seroconvert. This study shows that the human MoAbs BRAD-3 and BRAD-5 can prevent Rh D immunization, and indicates that they may be suitable replacements for the polyclonal anti-D presently used in prophylaxis of Rh D hemolytic disease of the newborn.(ABSTRACT TRUNCATED AT 400 WORDS)

Green Synthesis, Structural, In Vitro and In Vivo Bioactivity Properties of ZnO Nanoparticles for Biomedical Applications

2019 ◽  
Vol 9 (6) ◽  
pp. 731-738 ◽  
Author(s):  
Essam H. Ibrahim ◽  
Obaid Albulym ◽  
Omer Kaygili ◽  
Mona Kilany ◽  
Mohd. Shkir ◽  
...  
Keyword(s):  
Green Synthesis ◽  
Red Blood Cells ◽  
Cell Line ◽  
Zno Nanoparticles ◽  
Blood Cells ◽  
Cytotoxicity Test ◽  
Splenic Cells ◽  
Normal Rat

Owing to the fascinating applications of ZnO in modern devices, it is interesting to explore its more aspects for future devices. Hence, herein, we have synthesized the high purity spherical ZnO nanoparticles (SNPs) through a facile green synthesis route and robust structural and biomedical studies are carried out. Hexagonal phase with 93.2% crystallinity was confirmed through XRD analysis. ZnO nanoparticles were tested for their bioactivities both in vivo (acute cytotoxicity test) and in vitro (Anti-cancer activities on the liver (HepG2) and cervical (Hela) cancer cell lines, stimulatory/inhibitory effects on normal rat splenic cells, hemolytic effects on red blood cells and antimicrobial activity). Results showed that ZnO SNPs has no cytotoxic effects on the vital organ like the liver and has no hemolytic action on red blood cells. ZnO SNPs showed inhibitory consequence on normal rat splenic cells growth at all tested concentrations. The ZnO nanoparticles showed a stimulatory effect on Hela cell line. Moreover, ZnO nanoparticles showed an inhibitory effect on HepG2 cell line and microbial cells.

scholarly journals IMMUNOLOGICAL FUNCTIONS OF HUMAN T-LYMPHOID CELL LINE (MOLT)

1974 ◽  
Vol 140 (2) ◽  
pp. 538-548 ◽  
Author(s):  
Akikazu Takada ◽  
Yumiko Takada ◽  
Jun Minowada
Keyword(s):  
Red Blood Cells ◽  
Cell Line ◽  
Blood Cells ◽  
Lymphoid Cells ◽  
Mouse Spleen ◽  
Spleen Cells ◽  
Short Term ◽  
Molt Cell ◽  
Human B Cell

A short term incubation of the mixture of established human T-lymphoid cells (MOLT) and sheep red blood cells (SRBC) resulted in the release of factors which nonspecifically suppressed the response of mouse spleen cells against heterologous erythrocytes in vitro. Neither human B-cell line (RPMI 1788), nor the supernate of MOLT cell suspension in the absence of SRBC had such suppressive effects. The supernate of the mixture of MOLT cells with chicken red blood cells (CRBC) did not suppress either anti-CRBC or anti-SRBC responses of mouse spleen cells. Since CRBC did not form rosettes with MOLT cells, it is suspected that the origin of the production of these factors might be MOLT cells forming SRBC rosettes. Some of these factors are dialysable.

scholarly journals An in vitro method to evaluate hemolysis of human red blood cells (RBCs) treated by airborne particulate matter (PM10)

MethodsX ◽  
2019 ◽  
Vol 6 ◽  
pp. 156-161 ◽  
Author(s):  
Alireza Mesdaghinia ◽  
Zahra Pourpak ◽  
Kazem Naddafi ◽  
Ramin Nabizadeh Nodehi ◽  
Zahra Alizadeh ◽  
...  
Keyword(s):  
Particulate Matter ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Airborne Particulate Matter ◽  
Airborne Particulate ◽  
In Vitro Method ◽  
Human Red Blood Cells

scholarly journals Study on creating the hybridoma for producing a monoclonal antibody that causes agglutination of the red blood cell B group

2018 ◽  
Vol 15 (1) ◽  
pp. 23-29
Author(s):  
Lê Văn Phan ◽  
Nguyễn Thị Trung ◽  
Trương Nam Hải
Keyword(s):  
Monoclonal Antibody ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Serum Sample ◽  
Blood Cells ◽  
Hybrid Cell ◽  
Igg Antibody ◽  
Human Red Blood Cells ◽  
Abo Incompatibility ◽  
Sample Method

ABO incompatibility is a potential lethal barrier in tranfusion therapy. ABO blood cell grouping for all of the donors and potential recipients is the unique way for ensuring the highest safety for potential recipients in blood transfusion. ABO blood cell grouping must be performed by both of the serum sample method and erythrocytes sample method. Nowadays, when applying the serum sample method, the monoclonal antibodies are commonly used to identify the antigens on the surface of the red blood cells. In this study, for the first time in Vietnam, the hybridoma technology was successefully developed and screened hybrid cells for producing monoclonal antibody specifically agglutinated with red blood cells B group. After being isolated from spleen and iliac lympho node of the human red blood cells B group immuned BALB/c mice, the lymphocytes B was fused with myeloma sp2/0. As the results, 10 single hybrid cell lines B4D6C6, B4D6E2, B4D10C9, B4D10B6, B4D10D5, B4D10D6, B4D10E4, B4H6C5, B8F6B6, B8F6D4 have been screened by a specific agglutination with sample red blood cells group B. Among them, the hybrid cell line B4D10C9 was the best secreting anti-B monoclonal antibody into culture, that presented by antibody titer reached 1/256, hence it was selected for further studies. The ELISA method helped to isotype the anti-B monoclonal antibody which was produced from B4D10C9 hybridoma. As a result, the anti-B monoclonal antibody contained IgM heavy chains and kappa light chains. The IgM antibody could be able to agglutinate red blood cells 25 times more than IgG antibody. The success of screening B4D10C9 hybridoma producing IgM monoclonal antibody is the most significant result of this study.

scholarly journals Green Synthesis, Structural, In Vitro and Vivo Bioactivity Properties of ZnO Nanoparticles for Biomedical Applications

2019 ◽  
Author(s):  
Essam H. Ibrahim ◽  
Obaid Albulym ◽  
Omer Kaygili ◽  
Mona Kilany ◽  
Mohd Shkir ◽  
...  
Keyword(s):  
Green Synthesis ◽  
Red Blood Cells ◽  
Cell Line ◽  
Zno Nanoparticles ◽  
Blood Cells ◽  
Hexagonal Phase ◽  
Cytotoxicity Test ◽  
Splenic Cells ◽  
Normal Rat

Owing to fascinating applications of ZnO in modern devices, it is interesting to explore its more features for future devices. Hence, herein, we have synthesized the high quality ZnO spherical nanoparticles (SNPs) through a facile green synthesis route and robust structural and biomedical studies are carried out. Hexagonal phase with 93.2% crystallinity was confirmed through XRD analysis. ZnO nanoparticles were tested for their bioactivities both in vivo (acute cytotoxicity test) and in vitro (Anti-cancer activities on liver (HepG2) and cervical (Hela) cancer cell lines, stimulatory/inhibitory effects on normal rat splenic cells and hemolytic effects on red blood cells). Results showed that ZnO SNPs has no cytotoxic effects on vital organ like liver and has no hemolytic action on red blood cells. ZnO SNPs showed inhibitory consequence on normal rat splenic cells growth at all tested concentrations. ZnO nanoparticles showed an inhibitory effect on HepG2 cell line. While showed stimulatory effect on Hela cell line. Current study presents the synthesized ZnO SNPs as highly applicable in bio-optoelectronics.

scholarly journals Study on some properties of the hybrid cell B4D10C9 producing the anti B monoclonal antibody which was agglutinated the b antigens on the surface of red blood cells

2018 ◽  
Vol 15 (2) ◽  
pp. 243-249
Author(s):  
Nguyễn Thị Trung ◽  
Trương Nam Hải
Keyword(s):  
Monoclonal Antibody ◽  
Red Blood Cells ◽  
Cell Line ◽  
Blood Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Maximum Density ◽  
Hybridoma Cells ◽  
Continuous Growth ◽  
Fetal Bovine

The B4D10C9 hybridoma cell line producing the monoclonal antibody which was specifically agglutinated red blood cells B group, belongs to the IgM class and kappa light chains that have been created in our previous study. This study presents some biological charateristics of B4D10C9 such as the capability of growth, anti B monoclonal antibody producing, the stability of growth and cell line and antibody secreting of this cell line. The hybridoma cells grew well in the DMEM medium added with fetal bovine serum (FBS) at the final concentration of 1% or 10%. However, the maximum density of cells reached 9,9 x 105 cells per ml after 50 hours post incubation only when it was cultured in the medium containing 10% of FBS. The maximum antibody titers in the supernatant that collected after 150 hours of culture reached 1/512. In contrast to that, the maximum density of cells only reached 3,4 x 105 cells/ml after 72 hours post incubation when was cultured in DMEM medium containing 1% FBS. The maximum antibody titer was 1/64 after 50 hours post incubation. These cells were cultured through 3 different generations and the ability of antibody production in each generation was evaluated. As a result, this cell line had continuous growth stability and antibody secretion through 3 generations. The quantity of monoclonal antibody obtained from the supenantant of the culture medium at 150 hours of incubation was about 100 micrograms per ml. Thus, the growth of the cell line B4D10C9 and its ability of secretion of anti B monoclonal antibody were better when was cultured in the DMEM medium containing 10% fetal bovine serum. Preservation, storage and recovery does not affect to the growth and antibody producing of this cell line.

scholarly journals Sickle Human Umbilical Cord Derived Erythroid Progenitor Cells (S-HUDEP2): An Ideal in-Vitro System for Screening Anti-Sickling Compounds for Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3675-3675
Author(s):  
Alicia Chang ◽  
So Hyun Park ◽  
Ciaran M Lee ◽  
Alireza Paikari ◽  
Gang Bao ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Cell Line ◽  
Blood Cells ◽  
Human Umbilical Cord ◽  
Cell Disease ◽  
Erythroid Progenitor ◽  
In Vitro System ◽  
Sickle Hemoglobin ◽  
Hypoxic Conditions

Abstract Background: Sickle Cell Disease (SCD) is a devastating inherited disease, characterized by polymerization of sickle hemoglobin under deoxygenated conditions that can lead to acute pain crises, ischemia, and chronic organ damage. Pharmacologic anti-sickling agents that decrease polymerization are currently under investigation, however there is no consistent in vitro system to study these compounds; whole patient blood, subject to clinical variability and limited supply, is most often used. Human Umbilical Cord Derived Erythroid Progenitor 2 cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell (HSC) derived erythroid precursor cell line that can differentiate into red blood cells. We have engineered S-HUDEP2 cells to express sickle hemoglobin (HbS) via CRISPR/Cas9 gene editing. We hypothesized that this cell line will sickle under hypoxic conditions, produce dense red blood cells (DRBC, red cells with a density>1.11 mg/mL that are dehydrated and prone to sickling. If these intrinsic, essential SCD RBC properties are found, we propose to use this novel cell line to screen drug compounds for anti-sickling capabilities. Methods: S-HUDEP cells were cultured as previously described (Kurita et al, 2013). %HbS and %HbA produced by parent HUDEP and S-HUDEP2 cells were measured by high performance liquid chromatography (HPLC). Hypoxia was induced by placing the cells at 2% O2 for four hours. Parent HUDEP and S-HUDEP2 cells were then fixed with glutaraldehyde and Giemsa stained. % sickling estimated at 40x magnification by a pathologist blinded to cell group counting sickle forms out of 1000 cells. The percentage of dense red blood cells (DRBCs) was quantified by an ADVIA hematology analyzer (Siemens). S-HUDEP2 cells were dosed with 0, 2.5 and 5 µM 5-hydroxymethylfurfural (5-HMF) and 75, 150, and 300 µM GBT440, two known anti-sickling agents, on day ten and day 14 of culture for one hour, subjected to hypoxic conditions and % sickling quantified as described above. Results: S-HUDEP2 cells express 98% HbS. Under hypoxia, 20% of S-HUDEPs sickle at day 10 of differentiation; 30% of S-HUDEP2 cells sickle at Day +14 of differentiation. Parent HUDEP-2 cells, which produce 98% HbA, did not sickle under hypoxic conditions at any stage of differentiation. 70% of S-HUDEP2 cells were determined to be DRBC under hypoxia at Day 10 and 14 time points; parent HUDEP-2 cells did not produce DRBC under hypoxia. Treatment of S-HUDEP2 cells with 5μM of 5-HMF and 150 μM of GBT440 reduced sickling by 40-50% under hypoxic conditions compared with untreated S-HUDEP2 cells (p<0.01), and reduced %DRBC by 30% (p<0.01). Conclusions: S-HUDEP2 cells express HbS, form DRBC, and sickle under hypoxic conditions, just like erythroid precursors and mature red blood cells from individuals with SCD. Exposure to two anti-sickling agents, one of which is currently in Phase III clinical trials, significantly decreased S-HUDEP2 sickling under hypoxic conditions, and reduced %DRBC, a marker of disease severity. Advantages of S-HUDEPs over patient samples include genetic and phenotypic uniformity, no human pathogens, and availability to groups without access to patient samples. We therefore conclude that S-HUDEP2s have utility in clinical research, may be used to screen anti-sickling and anti-dense cell compounds in vitro, and may lead to identification of new therapeutic options for SCD patients. Disclosures No relevant conflicts of interest to declare.

Establishment of a procedure for producing the anti-b monoclonal antibody from B4C10D9 hybridoma cell line

2017 ◽  
Vol 39 (3) ◽  
pp. 342-348
Author(s):  
Nguyen Thi Trung ◽  
Truong Nam Hai
Keyword(s):  
Monoclonal Antibody ◽  
Blood Transfusion ◽  
Red Blood Cells ◽  
Cell Line ◽  
Antibody Titer ◽  
Blood Cells ◽  
Hybridoma Cells ◽  
Post Inoculation ◽  
Reaction Intensity ◽  
Blood Grouping

There are two the most important blood group system in blood transfusion, that is the ABO system and the Rh system. The anti-A or anti-B antibodies in the blood of recipient cause strongly agglutination the red blood cells of donor bearing A or B antigens on their surface respectively. So, all donated blood and received blood must be typed group for the ABO system to be safe in blood transfusion. To blood grouping, used of know antisera reacts with the whole blood samples to determine antigen on the surface of red blood cell or use of know red blood cells reacts with the individual's serum to determine antibody. The serology method is widely used, antisera are usually monoclonal antibodies secreted by hybridoma cells. Our previous studies showed that the B4C10D9 hybridoma cell line generated anti-B monoclonal antibody was produced by hybridoma technology. This antibody causes specificity agglutination the red blood cells caring B antigen on their surface. It is  classed as IgM antibody with it heavy chains are µ and κ light chains. The aim of the study was to produce the anti-B monoclonal antibody form B4C10D9 hybridoma cells line serve as a part of ABO blood grouping reagent. Based on results of experiments, procedure for producing the anti-B monoclonal antibody from B4C10D9 hybridoma cell line has also been established with following steps. At first, refresh the frozen B4C10D9 hybridoma cells and the density of B4C10D9 hybridoma cells is 4.0 x 104 cells/ml at 24 hours post-inoculation. Then the B4C10D9 hybridoma cells are splitted 1:5 every 48 hours for 2 times to obtain the cell biomass. In the next step, the hybridoma cells are splitted into 125 cm2 flasks with the initial cell density of 105 cells/ml. The B4C10D9 hybridoma cells density reach 9.9 x 106 cells/ml at 48 hours post-inoculation. After 144 hours of post-inoculation, collect culture medium and concentrate the anti-B monoclonal antibody by precipitation with saturated ammonium sulfate at a final concentration of 50%. If resuspending volume equals initial volume, then the anti-B monoclonal antibody titer reaches 1/256 and the antibody reaction intensity will be 3+. And if the obtained antibody solution volume equals one fifth of the original volume, then the anti-B monoclonal antibody titer reaches 1/1024, and the antibody reaction intensity will be 4+.   Citation: Establishment of a procedure for producing the anti-b monoclonal antibody from B4C10D9 hybridoma cell line. Tap chi Sinh hoc, 39(3): 342-348. DOI: 10.15625/0866-7160/v39n3.10765. *Corresponding author: [email protected] Received 20 August 2017, accepted 20 September 2017

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