scholarly journals Generating and characterizing the anti-human CD45 monoclonal antibody

2020 ◽  
Vol 23 (3) ◽  
pp. 665-672
Author(s):  
Giang Huong Ta ◽  
Huy Quoc Nguyen ◽  
Quan Dang Nguyen
Keyword(s):  
Monoclonal Antibody ◽  
Western Blotting ◽  
Jurkat Cells ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
E Coli ◽  
Common Marker ◽  
Sds Page ◽  
Hybridoma Technology ◽  
Mab Production

Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis. Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity. Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen provoked the immune response in Balb/c mice. Hybridoma clones were generated successfully by the fusion of spleen cells from the selected immunized-mouse and myeloma cells. Among these hybridoma clones, one with the highest yield of MAb production was identified. The isotype of the anti-CD45 MAb created in this work is IgG2b, while its the light chain is kappa (k) type. The affinity of this MAb with CD45RO antigen is high with Kd value at the picomolar level. The anti-CD45 MAb can interact with CD45 naturally expressed on the surface of Jurkat cells in Western blotting and fluorescent immuno-staining assay. Conclusion: We have developed successfully an anti-human CD45 MAb using hybridoma technology, which can recognize CD45 in ELISA, Western blotting, and fluorescent immuno-staining analysis. Although further investigations are necessary, obviously, our anti-human CD45 MAb is potential for research and diagnosis applications.

High Expression of Human Amelogenin in E. Coli

1996 ◽  
Vol 10 (2) ◽  
pp. 187-194 ◽  
Author(s):  
D. Deutsch ◽  
E. Chityat ◽  
M. Hekmati ◽  
A. Palmon ◽  
Y. Farkash ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blotting ◽  
Rt Pcr ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Expression Plasmid ◽  
Over Expression ◽  
E Coli ◽  
Sds Page ◽  
Terminal Amino

A human cDNA, encoding for the 175-aminoacid human amelogenin, was prepared by RT PCR from tooth bud mRNA and sub-cloned into pGEX-KG expression plasmid for over-expression in E. coli. The expressed protein was characterized by SDS-PAGE, Western blotting, and N-terminal amino acid sequencing.

scholarly journals TheEscherichia coli-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Xiaolei Wang ◽  
Guihua Yang ◽  
Shanshuang Li ◽  
Meifeng Gao ◽  
Pangfeng Zhao ◽  
...  
Keyword(s):  
Spleen Cells ◽  
New Approach ◽  
E Coli ◽  
Sds Page ◽  
Strain Bl21 ◽  
Healing Wound ◽  
Production Strategy ◽  
Better Than

Thymosinβ4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termedTβ4) was designed based on the Tβ4 protein sequence and used to express a 4 ×Tβ4 concatemer (four tandem copies of Tβ4, termed 4 ×Tβ4) together with a histidine tag (6 × His) inE. coli(strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropylβ-D-thiogalactoside (IPTG). TheE. coli-derived 4 ×Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cellsin vitroandin vivowound healing. The results demonstrate that these activities of theE. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.

scholarly journals A monoclonal antibody to VIII:C produced by a mouse hybridoma

Blood ◽  
1981 ◽  
Vol 58 (5) ◽  
pp. 1000-1006
Author(s):  
HP Muller ◽  
NH van Tilburg ◽  
J Derks ◽  
E Klein-Breteler ◽  
RM Bertina
Keyword(s):  
Monoclonal Antibody ◽  
Myeloma Cell ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Heavy Chains ◽  
Normal Plasma ◽  
Cofactor Activity ◽  
Mouse Myeloma

Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000–10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.

Development of the ELISAs for detection of hormone-disrupting chemicals

2000 ◽  
Vol 42 (7-8) ◽  
pp. 81-88 ◽  
Author(s):  
Y. Goda ◽  
A. Kobayashi ◽  
K. Fukuda ◽  
S. Fujimoto ◽  
M. Ike ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Phthalate Esters ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
Structural Resemblance ◽  
Reaction Test ◽  
Mouse Myeloma

Six kinds of enzyme-linked immunosorbent assay (ELISA) systems were developed for the quantitative analysis of hormone-disrupting chemicals (HDCs), such as estrogen (ES: the total amount of estrone (E1), 17 β-estra (E2) and estriol (E3)), E2, bisphenol A (BPA), alkylphenol (AP), phthalate esters (PE) and chlorophenols (CP). To generate specific monoclonal antibodies against BPA, AP, PE, CP, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with carboxylated derivatives, while anti E2 monoclonal antibody was selected from those available on the market, and anti ES monoclonal antibody was purchased from Teikoku Hormone Mfg Co. Ltd. The detection limits of ES, E2, BPA, AP, PE and CP ELISAs were 0.1, 0.1, 5, 10, 200, 10 μg/L, when E2, E2, BPA, Nonylphenol (NP), Dibutylphthalate (DBP), 2,4-CP were used as standard, respectively, and the specificity of each ELISA was confirmed with the cross-reaction test using several compounds which have structural resemblance to the compounds of interest.

scholarly journals A monoclonal antibody to VIII:C produced by a mouse hybridoma

Blood ◽  
1981 ◽  
Vol 58 (5) ◽  
pp. 1000-1006 ◽  
Author(s):  
HP Muller ◽  
NH van Tilburg ◽  
J Derks ◽  
E Klein-Breteler ◽  
RM Bertina
Keyword(s):  
Monoclonal Antibody ◽  
Myeloma Cell ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Heavy Chains ◽  
Normal Plasma ◽  
Cofactor Activity ◽  
Mouse Myeloma

Abstract Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000–10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.

scholarly journals A monoclonal antibody, specific for human fibrinogen, fibrinopeptide A- containing fragments and not reacting with free fibrinopeptide A

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 503-507 ◽  
Author(s):  
PW Koppert ◽  
CM Huijsmans ◽  
W Nieuwenhuizen
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Genetic Variants ◽  
Mammalian Species ◽  
Fibrinopeptide A ◽  
Spleen Cells ◽  
Human Fibrinogen ◽  
Myeloma Cells ◽  
Normal Human

Abstract Spleen cells of BALB/c mice, immunized with fragments Y of normal human fibrinogen, were fused with P3 X 63 Ag 8653 myeloma cells. A clone was found which produces monoclonal antibodies (Mab-Y18) of the IgM kappa type. Mab-Y18 is immunoreactive with normal human fibrinogen, and its fragments X, Y, N-terminal disulphide knot, A alpha-chain, and A alpha stretch 1–51. The immunoreactivity with these same fragments disappears upon treatment with thrombin or arvin. This strongly suggests that fibrinopeptide A is an essential component of the Mab-Y18 epitope. This is supported by the finding that Mab-Y18 prolongs the thrombin and arvin clotting times of human fibrinogen by inhibition of the fibrinopeptide A release. More detailed information about the nature of the Mab-Y18 epitope was obtained from studies with genetic variants of human fibrinogen (especially fibrinogen Metz) and with fibrinogens from other mammalian species. These studies show that amino acid residue A alpha 16 (arginine) of fibrinopeptide A is essential for the Mab-Y18 epitope. Mab-Y18 does not react with free fibrinopeptide A.

scholarly journals An antifibrin monoclonal antibody useful in immunoscintigraphic detection of thrombi

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 708-714 ◽  
Author(s):  
MN Wasser ◽  
PW Koppert ◽  
JW Arndt ◽  
JJ Emeis ◽  
RI Feitsma ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Degradation Product ◽  
Spleen Cells ◽  
Fibrinogen Degradation Product ◽  
Myeloma Cells ◽  
High Concentrations ◽  
Preformed Plasma

Abstract Balb/c mice were immunized with human plasmin-generated fibrinogen degradation product Y. Spleen cells were fused with P3X63-Ag8.653 myeloma cells. A clone (Y22) was found that produces monoclonal antibodies (MoAbs) with a strong reactivity with human fibrin and only a weak reactivity with fibrinogen in an enzyme immunoassay (EIA). Y22 also reacts with fibrin of rabbits, rats, sheep, and dogs. The antibodies are of the IgG1 kappa-type and appear to be directed against a conformation-dependent epitope in the D-domain of fibrin. Experiments with 99mTc-labeled Y22 in vitro show that Y22 binds rapidly to forming clots. 99mTc-Y22 also binds to preformed plasma clots in a plasma milieu, even in the presence of high concentrations of heparin. Clot localization experiments in rabbits and rats confirm the high fibrin specificity and the potential of 99mTc-Y22 for thrombus imaging in vivo.

scholarly journals Application of a unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissue.

1985 ◽  
Vol 33 (6) ◽  
pp. 587-594 ◽  
Author(s):  
E Wang ◽  
J G Krueger
Keyword(s):  
Monoclonal Antibody ◽  
Basal Layer ◽  
Positive Staining ◽  
Staining Reaction ◽  
Spleen Cells ◽  
Cultured Fibroblasts ◽  
Myeloma Cells ◽  
Culturing Conditions ◽  
Mouse Myeloma

A monoclonal antibody (clone S-30), directed to a protein of 57,000 daltons, was developed from the fusion of mouse myeloma cells and the spleen cells of mice injected with cytoskeletal extracts of fibroblasts that have been aged in in vitro culturing conditions according to a schedule of serial passaging (Cristofalo VJ, Charpentier R: J Tissue Culture Meth 6:117, 1981; Wang E: J Cell Biol, submitted). The staining activity of S-30 antibody was observed exclusively in the nuclei of nonproliferating senescent fibroblasts, but not in their young counterparts. Immunolocalization of S-30 antibody in frozen tissues from various sites reveals the positive staining reaction in the nuclear envelope region in those cells that are at the final stage of differentiation and are no longer replicating. These tissue sites include epithelial cells of the suprabasal layer of epidermis, hair sheath, and tongue, a subpopulation of fibroblasts in the dermis, chondrocytes, hepatocytes, and cells of cardiac muscle. The absence of S-30 staining activity was noted in tissues such as simple epithelium located in the gastrointestinal tract and kidney, and keratinocytes in the basal layer. These results suggest that the S-30 antibody can be used as a marker for nonproliferating cells both in cultured fibroblasts and in some tissues. It seems that the mechanism that controls the cessation of cell proliferation is related, in part, to the postmitotic expression of the 57,000 dalton protein.

A monoclonal antibody-derived antigen ofBabesia divergens: characterization and investigation of its ability to protect gerbils against virulent homologous challenge

Parasitology ◽  
1989 ◽  
Vol 99 (3) ◽  
pp. 341-348 ◽  
Author(s):  
C. M. Winger ◽  
E. U. Canning ◽  
J. D. Culverhouse
Keyword(s):  
Monoclonal Antibody ◽  
Western Blotting ◽  
Affinity Purification ◽  
Host Protein ◽  
Peptide Analysis ◽  
Babesia Divergens ◽  
Sds Page ◽  
Homologous Strain ◽  
Purified Antigen ◽  
Homologous Challenge

SummaryABabesia divergensmerozoite antigen was purified by affinity chromatography using a monoclonal antibody. Silver staining of SDS-PAGE gels revealed 2 bands of Mr50–60 kDa and Mr24–29. It is proposed that the Mr24–29 kDa band represents the native protein and that it is complexed, perhaps as a dimer or perhaps to host protein to give the Mr50–60 kDa band. Peptide analysis of the antigen followed by Western blotting with the monoclonal antibody revealed only an Mr20–24 kDa band. After affinity purification the antigen was precipitated with acetone and inoculated subcutaneously, together with complete Freund's adjuvant, into gerbils. Two inoculations were given 3 weeks apart, after which the immunized animals and a group of control animals were challenged with 5 × parasitized red cells from a virulent microaerophilus stationary phase (MASP) culture of an homologous strain ofB. divergens. Parasitaemias rose rapidly in control animals and all were dead by the fifth day. Two of the vaccinated animals survived the challenge. Parasitaemias in the remaining two rose slowly but both animals succumbed on the seventh day. Western blotting of merozoites and of the purified antigen, using sera from the surviving animals showed strong reactivity with the Mr24–29 kDa band.

scholarly journals Interspecies spleen-myeloma hybrid producing monoclonal antibodies against mouse lymphocyte surface glycoprotein, T200.

1978 ◽  
Vol 148 (1) ◽  
pp. 313-323 ◽  
Author(s):  
I S Trowbridge
Keyword(s):  
Monoclonal Antibody ◽  
Surface Glycoprotein ◽  
Binding Assays ◽  
Spleen Cells ◽  
Mouse Lymphocyte ◽  
Lymphocyte Surface ◽  
Sds Page ◽  
A Cell ◽  
B And T Lymphocytes ◽  
Mouse Lymphoma

A cell hybrid has been selected from fusion of a mouse myeloma and rat spleen cells immunized against mouse lymphoma cells that produces monoclonal antibody against the mouse lymphocyte surface glycoprotein, T200. Antibody binding assays employing the monoclonal antibody show that there are about 50,000-100,000 molecules of T200 glycoprotein on mouse thymocytes and that similar antigens are present on spleen and bone marrow but not detected on nonlymphoid tissues. Examination of the labeled molecules precipitated from detergent extracts of spleen cells and thymocytes iodinated by the lactoperoxidase technique by SDS-PAGE confirm that there are structural differences between the antigens found on B and T lymphocytes. The B-cell glycoprotein consists of at least one component of apparent mol wt 220,000 on SDS-PAGE, while the T-cell glycoprotein has an apparent mol wt of about 190,000.

Sign in / Sign up

Export Citation Format

Share Document