Cdhybridoma media, by Thermo Fisher Scientific - Product details

1

Chimeric Immunogen Antibody Generation

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Example 7

A synthetic gene coding for the chimeric immunogen hSortilin-FC, (human Sortilin AA (78-756) from SEQ ID NO:169) and human IgG1-FC AA (104-330) from SEQ ID NO:169 was cloned into pcDNA3.1 and used for expression using the freestyle system from Invitrogen. The antigen was purified from cell culture supernatants by protein-A affinity chromatography using standard procedures for antibody purification as described above for human antibodies.

Hybridoma Generation

hSortilin-FC was used as immunogen and 5 BALB/c mice were immunized. A mouse with satisfactory immune response was selected for cell fusion and hybridoma generation. Hybridoma supernatants were screened by ELISA using hSortilin-ECD as coating antigen. A total of eighteen hybridoma cell lines derived from nine parental clones were generated.

Expression

Hybridomas were initially grown in complete growth medium, DMEM with 10% FBS+ antibiotics, and subsequently adapted to CDhybridoma media (Invitrogen) for expression experiments.

Purification

Mouse monoclonal antibodies were purified from hybridoma cell culture supernatants by protein-G sepharose according to standard procedures recommended by the supplier (GE healthcare).

US11548950B2. Agent, uses and methods for treatment (2023-01-10). H. Lundbeck A/S [DK]. Inventors: Lars Christian Biilmann Rønn [DK], Ibrahim John Malik [DK], Jeffrey B. Stavenhagen [DK], Søren Christensen [DK], Jan Egebjerg [DK], Arnout Gerritsen [NL], Edward van den Brink [NL], Paul Parren [NL], Rob de Jong [NL].

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2

Chimeric hSortilin-FC Immunogen and Antibody Generation

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Example 7

A synthetic gene coding for the chimeric immunogen hSortilin-FC, (human Sortilin AA (78-756) from SEQ ID NO:169) and human IgG1-FC AA (104-330) from SEQ ID NO:169 was cloned into pcDNA3.1 and used for expression using the freestyle system from Invitrogen. The antigen was purified from cell culture supernatants by protein-A affinity chromatography using standard procedures for antibody purification as described above for human antibodies.

Hybridoma Generation

hSortilin-FC was used as immunogen and 5 BALB/c mice were immunized. A mouse with satisfactory immune response was selected for cell fusion and hybridoma generation. Hybridoma supernatants were screened by ELISA using hSortilin-ECD as coating antigen. A total of eighteen hybridoma cell lines derived from nine parental clones were generated.

Expression

Hybridomas were initially grown in complete growth medium, DMEM with 10% FBS+ antibiotics, and subsequently adapted to CDhybridoma media (Invitrogen) for expression experiments.

Purification

Mouse monoclonal antibodies were purified from hybridoma cell culture supernatants by protein-G sepharose according to standard procedures recommended by the supplier (GE healthcare).

US10428147B2. Anti-sortilin antibodies, uses and methods for treatment (2019-10-01). H. Lundbeck A/S [DK]. Inventors: Lars Christian Biilmann Rønn [DK], Ibrahim John Malik [DK], Søren Christensen [DK], Jan Egebjerg [DK], Jeffrey B. Stavenhagen [DK], Arnout Gerritsen [NL], Edward van den Brink [NL], Paul Parren [NL], Rob de Jong [NL].

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3

Generation and Purification of Monoclonal Antibodies

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Example 7

Immunogen

A synthetic gene coding for the chimeric immunogen hSortilin-FC, (human Sortilin AA (78-756) from SEQ ID NO:169) and human IgG1-FC AA (104-330) from SEQ ID NO:169 was cloned into pcDNA3.1 and used for expression using the freestyle system from Invitrogen. The antigen was purified from cell culture supernatants by protein-A affinity chromatography using standard procedures for antibody purification as described above for human antibodies.

Hybridoma Generation

hSortilin-FC was used as immunogen and 5 BALB/c mice were immunized. A mouse with satisfactory immune response was selected for cell fusion and hybridoma generation. Hybridoma supernatants were screened by ELISA using hSortilin-ECD as coating antigen. A total of eighteen hybridoma cell lines derived from nine parental clones were generated.

Expression

Hybridomas were initially grown in complete growth medium, DMEM with 10% FBS+antibiotics, and subsequently adapted to CDhybridoma media (Invitrogen) for expression experiments.

Purification

Mouse monoclonal antibodies were purified from hybridoma cell culture supernatants by protein-G sepharose according to standard procedures recommended by the supplier (GE healthcare).

US10889650B2. Agent, uses and methods for treatment (2021-01-12). H. Lundbeck A/S [DK]. Inventors: Lars Christian Biilmann Rønn [DK], Ibrahim John Malik [DK], Jeffrey B. Stavenhagen [DK], Søren Christensen [DK], Jan Egebjerg [DK], Arnout Gerritsen [NL], Edward van den Brink [NL], Paul Parren [NL], Rob de Jong [NL].

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4

Generation of Anti-Sortilin Monoclonal Antibodies

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Example 7

Immunogen

A synthetic gene coding for the chimeric immunogen hSortilin-FC, (human Sortilin AA (78-756) from SEQ ID NO:169) and human IgG1-FC AA (104-330) from SEQ ID NO:169 was cloned into pcDNA3.1 and used for expression using the freestyle system from Invitrogen. The antigen was purified from cell culture supernatants by protein-A affinity chromatography using standard procedures for antibody purification as described above for human antibodies.

Hybridoma Generation

hSortilin-FC was used as immunogen and 5 BALB/c mice were immunized. A mouse with satisfactory immune response was selected for cell fusion and hybridoma generation. Hybridoma supernatants were screened by ELISA using hSortilin-ECD as coating antigen. A total of eighteen hybridoma cell lines derived from nine parental clones were generated.

Expression

Hybridomas were initially grown in complete growth medium, DMEM with 10% FBS+ antibiotics, and subsequently adapted to CDhybridoma media (Invitrogen) for expression experiments.

Purification

Mouse monoclonal antibodies were purified from hybridoma cell culture supernatants by protein-G sepharose according to standard procedures recommended by the supplier (GE healthcare).

US10479835B2. Agent, uses and methods for treatment (2019-11-19). H. Lundbeck A/S [DK]. Inventors: Lars Christian Biilmann Rønn [DK], Ibrahim John Malik [DK], Jeffrey B. Stavenhagen [DK], Søren Christensen [DK], Jan Egebjerg [DK], Arnout Gerritsen [NL], Edward Van Den Brink [NL], Paul Parren [NL], Rob De Jong [NL].

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5

Purification and In Vivo Application of Anti-CD4/CD8 Antibodies

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TIB-207 (anti-CD4) and TIB-105 (anti-CD8) hybridoma cell lines were obtained from ATCC for the collection of T-cell depletion antibodies. CD hybridoma media (Gibco®) supplemented with 4% GlutaMAX™ was used for hybridoma cell culture. Conditioned media from >90% confluent 10 cm plates of hybridoma cells was collected, spun down to remove cell debris, and filtered through a 0.2 μm filter set before purification. The HiTrap Protein G HP column (GE Healthcare Life Sciences) was used to purify antibody using binding buffer (20mM sodium phosphate pH 7.0) elution buffer (0.1 M glycine-HCl pH2.7) and neutralization buffer (1 M Tris-HCl pH 9.0) following the manufacturer’s instructions. Purified antibodies were then concentrated using Amicon ultra-15ml (3K) spin tubes (Millipore) at 4000 rpm for 45 min at 20°C. Concentrated antibody was dialyzed against PBS at 4°C overnight and then antibody concentration was quantified by standard Bradford assay. For in vivo T-cell depletion experiments, animals were injected with either 125 μg/mouse of antibody or same amount of isotype control in PBS by i.p. injection three days prior to tumor cell injection and then every three days post tumor cell injection.

Shen M., Smith H.A., Wei Y., Jiang Y.Z., Zhao S., Wang N., Rowicki M., Tang Y., Hang X., Wu S., Wan L., Shao Z.M, & Kang Y. (2021). Pharmacological Disruption of the MTDH-SND1 Complex Enhances Tumor Antigen Presentation and Synergizes with Anti-PD-1 Therapy in Metastatic Breast Cancer. Nature cancer, 3(1), 60-74.

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6

Purification and In Vivo Application of Anti-CD4/CD8 Antibodies

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TIB-207 (anti-CD4) and TIB-105 (anti-CD8) hybridoma cell lines were obtained from ATCC for the collection of T-cell depletion antibodies. CD hybridoma media (Gibco®) supplemented with 4% GlutaMAX™ was used for hybridoma cell culture. Conditioned media from >90% confluent 10 cm plates of hybridoma cells was collected, spun down to remove cell debris, and filtered through a 0.2 μm filter set before purification. The HiTrap Protein G HP column (GE Healthcare Life Sciences) was used to purify antibody using binding buffer (20mM sodium phosphate pH 7.0) elution buffer (0.1 M glycine-HCl pH2.7) and neutralization buffer (1 M Tris-HCl pH 9.0) following the manufacturer’s instructions. Purified antibodies were then concentrated using Amicon ultra-15ml (3K) spin tubes (Millipore) at 4000 rpm for 45 min at 20°C. Concentrated antibody was dialyzed against PBS at 4°C overnight and then antibody concentration was quantified by standard Bradford assay. For in vivo T-cell depletion experiments, animals were injected with either 125 μg/mouse of antibody or same amount of isotype control in PBS by i.p. injection three days prior to tumor cell injection and then every three days post tumor cell injection.

Shen M., Smith H.A., Wei Y., Jiang Y.Z., Zhao S., Wang N., Rowicki M., Tang Y., Hang X., Wu S., Wan L., Shao Z.M, & Kang Y. (2021). Pharmacological Disruption of the MTDH-SND1 Complex Enhances Tumor Antigen Presentation and Synergizes with Anti-PD-1 Therapy in Metastatic Breast Cancer. Nature cancer, 3(1), 60-74.

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7

Cell Culture of SH-SY5Y Neurons

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Wild-type SH-SY5Y cells and SH-SY5Y transfected with full-length hTau with a C-terminal GFP tag (Tau-GFP cells) were cultured in Dulbecco’s modified Eagle’s media (Gibco), supplemented with 10% (v/v) foetal calf serum (Gibco) and 1% (v/v) penicillin–streptomycin (Gibco) at 37°C with 5% CO₂. Tau-GFP cells were selected with 6-mg/ml blasticidin (Thermo Fisher Scientific). For expression of the RNJ1 IgG, wild-type SH-SY5Y cells were maintained in 90% (v/v) CD Hybridoma Media (Gibco), 10% (v/v) Dulbecco’s modified Eagle’s media (Gibco) and supplemented with 1% (v/v) foetal calf serum and 0.1% (v/v) penicillin–streptomycin.

Wongsodirdjo P., Caruso A.C., Yong A.K., Lester M.A., Vella L.J., Hung Y.H, & Nisbet R.M. (2024). Messenger RNA-encoded antibody approach for targeting extracellular and intracellular tau. Brain Communications, 6(2), fcae100.

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Cdhybridoma media

Lab products found in correlation

7 protocols using cdhybridoma media

Chimeric Immunogen Antibody Generation

Chimeric hSortilin-FC Immunogen and Antibody Generation

Generation and Purification of Monoclonal Antibodies

Generation of Anti-Sortilin Monoclonal Antibodies

Purification and In Vivo Application of Anti-CD4/CD8 Antibodies

Purification and In Vivo Application of Anti-CD4/CD8 Antibodies

Cell Culture of SH-SY5Y Neurons

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