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Bispecific Antibody Production with Guaranteed Stability

$4,950.00

Item Cat No.: BCFTBS

Antibody: Monoclonal Antibody

Concentration: 1 mg/ml purified IgG

Application: In Vitro and In Vivo

Reactivity: Human, Mouse, Rat

BiCell Scientific’s bispecific antibody production service provides customized recombinant bispecific antibody expression and purification in lentivirus transfected B cell hybridomas.

The bispecific monoclonal antibody (BsMAb, BsAb) is an artificial antibody protein that can simultaneously bind to two different types of antigen or two different epitopes on the same antigen. Naturally occurring antibodies typically only target one antigen. BsAbs can be designed to recruit and activate immune cells, to interfere with receptor signaling, and to stimulate interactions of protein complexes. BsAbs have shown great promises in cancer immunotherapy, drug delivery, and slowing the development of the Alzheimer’s disease.

Recombinant bispecific monoclonal antibodies were previously cloned into mammalian expression vectors, such as pcDNA3.1  and transiently transfected into HEK293 or COS cells for transgenic expression. This approach has three disadvantages – (1) HEK293 cells and COS cells are surrogate cell models that lack endogenous antibody production and secretion machinery as in native B cells; (2) Transient transfection lacks the ability to make stable cell lines that can continuously secrete IgG molecules over many generations of cell culture; (3) Transient transfection is difficult to simultaneously introduce two monoclonal antibody molecules into the same cell to facilitate bispecific antibody assembly.

BiCell Scientific’s bispecific antibody production service harnesses the advantageous lentiviral transduction ability to sequentially introduce two monoclonal antibodies into naive B cell hybridomas. The resultant B cell hybridomas can produce recombinant antibodies at a similar level to native B cell-myeloma fused hybridomas. The two monoclonal antibody molecules carry the “knobs-into-holes” mutations in the Fc domain, which will facilitate their heterodimeric assembly into a functional bispecific antibody.

  • Molecular cloning of IgG (A and B, corresponding to the two binding sites in bispecific antibody) heavy chain and light chain sequences into lentiviral backbone vectors respectively
  • Mutagenesis of IgG (A) heavy chain Fc domain residue T366Y and IgG (B) heavy chain Fc domain residue Y407T
  • Transfection of HEK293T cells with lentiviral backbone vectors as well as vectors expressing gagpol, rev and vsvg
  • Harvesting and purification of lentivirus from cell culture medium
  • Transducing naive Sp2/0-Ag14 hybridoma cells with lentivirus encoding IgG (A)
  • Clonal selection and stable cell line establishment in 96-well plate
  • Validation of recombinant IgG (A) antibody expression in 96-well plate against antigen A and selection of the highest expressing clone
  • Transducing IgG (A) clone with lentivirus encoding IgG (B)
  • Clonal selection and stable cell line establishment in 96-well plate
  • Validation of recombinant bispecific antibody expression against antigen A and B in 96-well plate and selection of the highest expressing clone against both antigens
  • Clonal expansion and supernatant collection
  • 3-step affinity purification (against protein A/G, antigen A and antigen B columns sequentially)

Customers will receive the following deliverables at the end of each project:

  • One stable Sp2/0-Ag14 hybridoma cell line expressing recombinant bispecific monoclonal antibody with the highest yield
  • 1 mg of triple-affinity column purified bispecific monoclonal antibody

For Research Use Only. Not for use in clinical diagnostics or therapeutics.

  • IgG heavy chain and light chain cloning into lentiviral vector – 2-3 weeks
  • Lentivirus packaging for 1st IgG molecule  – 1-2 weeks
  • Naive Sp2/0-Ag14 hybridoma cell transduction – 1-2 weeks
  • Clonal selection and stable cell line establishment – 3-4 weeks
  • Recombinant antibody expression validation – 2-3 weeks
  • Lentivirus packaging for 2nd IgG molecule  – 1-2 weeks
  • Transduction of stable cells expressing 1st IgG molecule with lentivirus encoding 2nd IgG molecule – 1-2 weeks
  • Clonal selection and stable cell line establishment – 3-4 weeks
  • Recombinant antibody expression validation – 2-3 weeks
  • Cell culture expansion and affinity purification – 2-3 weeks

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"I am really impressed with your approach. We tried multiple times previously to create monoclonal and polyclonal antibodies to claudin-2 and MLCK1. We have had limited success generating polyclonals and no success generating monoclonals. You have generated outstanding monoclonals to both. I look forward to continuing to work with you."

Jerrold R. Turner, M.D., Ph.D.

Brigham and Women’s Hospital | Harvard Medical School

"The polyclonal antibody you generated for KIAA0408 is stunning! KIAA0408 is a novel cilium molecule that has never been studied. So, clearly there will be a lot of demand for it as we have discovered a very interesting finding and the story will be published in a high impact journal. I am strongly inclined to generate monoclonal antibody for this protein too and we should think about patenting it."

Univ.-Prof. Jay Gopalakrishnan PhD

Heinrich-Heine-Universität | Universitätsklinikum Düsseldorf

"Your ARL13B antibody works beautifully!!! We’re so happy to have a cilia-specific antibody made in rat! I can send you high resolution images to be posted on your website."

Julie Craft Van De Weghe, PhD

School of Medicine | University of Washington

"The assay is a homophilic interaction mediated cell adhesion on purified protein (in this case, immobilized purified Pcdhga9 to Pcdhga9 expressed on cell surface). Compared to control, cell adhesion is reduced in the presence of Pcdhga9 monoclonal antibody supernatants!"

Divyesh Joshi, PhD

School of Medicine | Yale University

"The rabbit hybridoma supernatants of anti-APOBEC3 project are tested positive by ELISA, and we are very happy about it! We previously tried a company, Abclone. Their Project "A" has immune response that is <10,000 titer in antiserum, which would explain why there is no positive mAb after fusion. Their project "B" didn't have any immune response in rabbit."

Harshita B Gupta, PhD.

School of Medicine | UT Health San Antonio

"We have tested anti mouse T cell antiserum samples from both rabbits you sent to us.

They worked very well! Thank you!"

Victoria Gorbacheva, PhD.

School of Medicine | Cleveland Clinic

Contact us for questions or custom requests!