Custom Monoclonal Antibody Development Process:
Target Analysis / Project Strategy
Immunization using an appropriate immunogen is critical in the custom monoclonal antibody development of application-specific antibodies. Proper selection and validation of the antigen is important for developing a specific immune response and for selecting antigen and utility-specific hybridoma clones. To determine optimal project strategy, Kemp Proteins recommends discussing custom monoclonal antibody development goals and details—including final application of the mAbs, nature of the antigen in the sample of the intended assay, desired sensitivity, and other factors—prior to project initiation.
Antigen Design
As your gene-to-protein partner, Kemp Proteins can help you express your protein, design your peptide, and discuss with you the advantages and disadvantages of different antigen options.
Mouse Immunization
Kemp Proteins typically immunizes 3 to 5 mice per project using our proprietary Repetitive Immunization at Multiple Sites (RIMMS) protocol, which generates a robust immune response to a variety of immunogens in an accelerated timeframe. Kemp Proteins can obtain a high titer of antigen-specific hyperimmune serum in about 3 to 5 weeks. We typically generate a strong, specific Ab titer sufficient for fusion (A620 > 0.5 at serum dilution of 1:100,000) by the initial serum collection. If needed, we will perform additional boosts to increase the specific Ab titer.
Electrofusion
Lymphocytes are harvested from mouse/mice exhibiting the highest titer for fusion. Lymphocytes are fused using electrofusion technology. Electrofusion provides a superior fusion efficiency (>10-fold) compared to traditional PEG-mediated fusion. From a typical electrofusion, up to 10,000 hybridoma cells can be generated consistently, providing ample hybridoma clones to be screened from a single fusion.
Direct Cloning & Imaging
Kemp Proteins operates a full suite of cutting-edge molecular device equipment: Clonepix2 (florescent robotic picking) & Imager (clonality assurance). Post-fusion cells are plated in a methocellulose-based, semi-solid cloning culture medium containing HAT, a selection reagent that prevents B-Cells and Myeloma cells from growing while allowing hybridoma cells to grow. Colonies of clonal hybridoma cells emerge in the semi-solid media. Our clone-picking process integrates imaging, robotics, and digital image analysis software for selecting clonal hybridomas and uses stringent picking clonality parameters, IgG expression, and/or antigen specificity to optimize the selection of stable hybridoma clones for screening. The use of the CloneSelect Imager provides a series of images to provide clonality assurance.
High-Throughput Screening (HTS)
Hybridoma clones generated as part of custom monoclonal antibody development are screened using an application-compatible method. Kemp Proteins can utilize a variety of screening methods to identify target antigen-specific clones that will be useful in the intended assay including ELISA-based, fluorescence-based, Octet-based, or a combination of methods. If the antibodies are used for binding to the antigen in solution, an in-solution screening method such as Octet based screening is available. If the antibodies are used for cell-surface antigen recognition, flow cytometry screening is incorporated.
Culture Supernatant
Kemp Proteins can provide clients supernatant from all confirmed positive clones for testing and selection of final clones. Approximately 3-5 ml of hybridoma supernatant is harvested and delivered.
Antibody Characterization
Additional antibody characterization is an important step in identifying top mAb candidates. Kemp Proteins offers scouting affinity of mAb candidates by Octet to characterize mAbs for candidate ranking. Epitope binning in a label-free sandwich immunoassay is another critical characterization we include in custom monoclonal antibody development strategies to help identify mAb pairs.
Cell Banking
Kemp Proteins expands and freezes at least 5 vials of frozen hybridoma cells per selected clone. The vials are cryopreserved in a 10% DMSO freezing media. Use of serum-free freezing media is available on request.
Production & Purification
Many Kemp clients choose to jump-start their research projects with a small-scale (5 mg) production as part of the initial project. Once they have determined the antibody (ies) of choice, our team will scale production from 10 mg to gram levels using various modes of in vitro systems production. Purification is performed on a Bio-Rad NGC system or AKTA Pilot purification system using pre-packed protein G or protein A columns. Additional antibody characterization steps can be incorporated on request, such as analytical SEC, IEF, and SDS-PAGE testing for purity, ELISA testing (if applicable) for binding, and any additional characterizations that were undertaken. A signed Certificate of Analysis is provided for each production.
Converting Hybridoma to Recombinant
Some clients decide to sequence and convert the hybridoma to recombinant. For details, please visit our Hybridoma-to-Recombinant Conversion.
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