B cell isolation, sequencing, and functional testing
Identification of novel molecular targets for immunotherapies, as well as the analysis and diagnosis of cancers, is a crucial step in cancer research. It is vital for developing monoclonal antibody-based drugs, such as immune checkpoint antibodies, but also for other immunotherapies such as CAR T cell therapy.
However, these approaches are often hampered by a lack of technologies that allow for sensitive and comprehensive multiplexed protein analysis. The MACSima™ Imaging Platform enables high-content, cyclic fluorescence–based imaging of individual biological samples in a fully automated fashion.
In this example, the MACSima Imaging Platform was applied for identification of new glioblastoma-specific markers. Screening of 96 antibodies led to the identification of new marker candidates.
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At Miltenyi, we offer a complete workflow for your antibody discovery research.
The MACSQuant® Tyto® Cell Sorter is revolutionizing cell sorting. Our patented microchip-based technology opens up new possibilities with high-speed multiparameter cell sorting in the safety of a fully enclosed and sterile cartridge system, the MACSQuant Tyto Cartridge. The closed cartridge system prevents aerosol and droplet formation, allowing highest safety for the operator. Furthermore, intuitive instrument handling make cell sorting suitable for any lab professional.
The unique features are beneficial for single B cell sequencing and BCR sequencing. The technology enables sorting of rare cell populations with high precision. Cell enrichment of rare target populations improves reads, excludes dead cells and debris avoiding DNA/RNA artefacts and decreases costs.
Read an interview on how the MACSQuant Tyto Cell Sorter is helping a Danish research team to sort antigen-specific B cells here.
Cell separation directly from blood products
Developing antibody-based therapies can be labor intensive and time consuming. With our StraightFrom® MicroBead Kits, your workflow is streamlined and saves valuable hands-on time bringing you closer to your new discovery. StraightFrom MicroBeads allow magnetic cell separation directly from human blood products, such as whole blood, buffy coat, leukoreduction system chambers (LRSCs) and leukopaks. CD19+ and CD138+ B cells can be purified using the autoMACS® Pro Separator, the MultiMACS™ Cell24 Separator Plus, or manual separators.
Maximum timesaving with fully automated cell labeling and separation
Automated cell enrichment using the autoMACS® Pro Separator eliminates time-consuming, hands-on steps usually performed by the operator, including labeling, separation, and washing. After separation, the enriched cells can be directly collected for further analysis. A stable separation of B cells or even Plasma cells can be achieved. This robustness is important as it enables standardized workflows without operational bias for daily lab routines. The system can be used with a variety of surface marker-specific MicroBeads available for other cell types and can be used for other applications, e.g., characterization of B cells (CD19+ or CD20+) or B cell subsets such as Plasma cell (CD138+).
Ex vivo cultivation is a suitable choice to increase B cells in numbers, especially antigen-specific B cells which are very low infrequency. Studying their antibody production, as well as specificity, is a very important step to develop antibody-based drugs. Our B Cell Expansion Kit, human, can streamline your workflow and simplify the process. The formulation offers the perfect solution to expand and activate human B cells from various sources. This enables expansion of B cells that are fully functional and ready for any downstream application.
Reliable activation and expansion of B cells is a clear requirement for effective downstream experiments. The B Cell Expansion Kit, human is optimized for primary B cell expansion in vitro.
Frequency of B cell subsets after 14 day culture
Expanded B cells display an activated phenotype with a distribution of memory and plasma cells similar to the original fraction. The amount of naive B cells decreased under all culture conditions on day 14. Likewise, reports describe that naive B cells have a shorter survival rate.
The most important step in antibody discovery research is the identification of lead candidates. To identify antigen-specific antibodies in tissue culture supernatants, flow cytometry analysis can be used. With the MACSQuant® X Flow Cytometer, these assays can be performed in a fast, standardized, and automated manner. This generates high-content information, which is both reproducible and reliable. The MACSQuant X Instrument is equipped with a syringe needle capable of volumetric pipetting, thereby enabling absolute counting from sample volumes starting at 5 µL. The instrument is fully automated in its calibration and compensation procedures, on top it’s pre-set templates, and instrument settings significantly reduce experiment setup and optimization time.
Testing the mode of action or the functionality of the antibody lead candidate is one of the most vital steps in the therapeutic antibody development process. It is important to assess effects the therapeutic antibody candidates have on body, and how they interact with the human immune system. Cell-based assays have been widely used, not only to discover and develop therapeutic antibodies, but also for immunogenicity testing for neutralizing anti-drug antibodies and in the manufacturing QC process.
The most important cell-based assays are:
Cytokine Secretion Assay: