Hybridoma Cell Line Development

Hybridoma cell line development consists of four (4) phases including immunization, fusion, cloning and In vitro antibody production. Typically, 4 - 6 mg of soluble antigen is required to complete a protein or peptide based project. Upon successfully developing a panel of stable hybridoma cell lines, further characterization of secreted monoclonal antibodies and small-scale production is included at no additional cost. Complete documentation of all phases and a development summary is provided to clients at the conclusion of each project.

Status Reports

Communication with clients is of essential importance to Leinco Technologies’ quality development process. Project status reports are generated in real time and are available to clients on our secure internet project management system. Teleconferences are scheduled to discuss data and the next steps in each project.

Monoclonal Antibody Development
in Mice or Hamsters

Leinco Technologies scientific staff has extensive monoclonal antibody development experience and offers clients the opportunity to collaborate on the development of antibodies against soluble proteins, cell surface molecules, or haptens for use in detection, neutralization or in vivo depletion. Through the use of proprietary development technologies, we have demonstrated success in developing monoclonal antibodies against single amino acid substitutions, splice variants and for neutralization. Host animals include mice or hamsters.

Leinco Technologies is the only biotechnology company that offers development of monoclonal antibodies in hamsters. The use of hamster as the host animal offers the unique advantage of an antibody that can be used in vivo in the mouse model without eliciting an immune response against the injected antibody. The applications of antibodies developed at Leinco Technologies include early discovery research, in vivo preclinical therapeutic investigations, and in vitro diagnostics.

Phases of hybridoma cell line development:

Phase I Immunization and Serum Screening (6-10 Weeks)

To expedite our client’s development projects and enhance the immunization process, up to ten (10) mice or hamsters may be immunized for each antigen. Immune responses are elicited using immunogens such as transfected cell lines, DNA, recombinant proteins, native proteins, peptides or haptens conjugated to carrier molecules. A variety of adjuvants are available for non cellular immunogens. Test bleeds are taken for purposes of monitoring antibody titer and/or neutralization activity, usually by ELISA. Screening data from immunized animals is presented to clients in graphical form to determine the best animal(s) suited for B-cell fusions.

Phase II Splenocyte Fusion and Screening of
Parental Cultures (4-6 Weeks)

A splenocyte fusion is performed on one or more animals selected from phase I. Splenocytes are isolated and fused with one or more myeloma cell lines (P3X63Ag8.653) using polyethylene glycol. Fused cells are plated at specific densities and grown under selection in HAT media. The parental cultures from the fusion are screened by ELISA or an appropriate application based method and stable positive cultures are subcloned and expanded for cryopreservation.

Phase III Subcloning and Screening (4-6 Weeks)

Cloning I: Strongly positive parental cultures exhibiting the desired functions derived from fusion cultures are selected for first round cloning using the limiting dilution method. 96 well panels are screened and positive clones are selected for round two limiting dilution cloning.
Cloning II: Positive clones from first round cloning will be subjected to a second round of limiting dilution cloning. Stable clones will be expanded into 24 well plates and then into T- flasks for further characterization. At this time, supernatant from each clone may be sent to clients for further testing and characterization.

Phase IV In vitro Monoclonal Antibody Production (6-8 Weeks)

At no additional cost, clients may select one clone for in vitro production of approximately one liter of supernatant. The supernatant is then purified using protein A chromatography and provided to clients for further characterization.
Leinco Technologies offers a full range of custom biotechnology services and contract manufacturing, including In vitro Antibody and Protein Production in bioreactors and large-scale Antibody Purification.

800.538.1145 ® Copyright Leinco Technologies, Inc. 2006 – 2012