Antibody research for cancer treatment

B cell isolation, sequencing, and functional testing

Video

MACS Academy: Therapeutic antibody research: Discovery to functional assays

In this webinar we share insights into our complete, comprehensive workflow for therapeutic antibody research. Join us as we discuss our solutions and products available for antibody discovery, functionality analysis and functional assays.

Application tools and data for therapeutic antibody research  by workflow step

Target discovery
 

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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Scientific Poster: 
Characterization and classification of glioblastoma multiforme using the novel MACSima™ Imaging Platform enabling multiparametric cyclic immunofluorescence analysis

Monoclonal antibody development 
 

At Miltenyi, we offer a complete workflow for your antibody discovery research.

MACS Handbook:

B cells (human)

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B cell sorting with the MACSQuant® Tyto® for BCR and single B cell sequencing

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. 

Microchip-based sorting of B cells
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Sorting of highly pure B cells from PBMCs 

The MACSQuant Tyto Cell Sorter enables gentle sorting of CD19+ B cells from PBMCs, resulting in a highly pure and viable target fraction.  
 

Read an interview on how the MACSQuant Tyto Cell Sorter is helping a Danish research team to sort antigen-specific B cells here.

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Magnetic separation and cultivation of B cells to identify therapeutic monoclonal antibody candidates 

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.
 

Isolation of human CD19+ cells from whole blood
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Isolation of human CD19+ cells from whole blood

The StraightFrom LRSC CD19 MicroBead Kits enables isolation of highly pure and viable B cells directly from blood products.

 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+).
 

Manual versus automated pre-enrichment workflows
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Manual versus automated pre-enrichment workflows

Enrichment of PCs directly from whole blood using autoMACS Pro Separator saves hands-on time and minimizes the procedure compared to manual enrichment.

 

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Direct cell isolation from blood or bone marrow on the autoMACS® Pro Separator 

With the autoMACS® Pro Separator, cells from whole blood or bone marrow can be isolated with minimal hands-on time. Take this short instrument tour and see how your target cells are being separated directly from whole blood samples without the need for density gradient centrifugation or red blood cell lysis. 
 

Standardized culture of primary human B cells

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.  

Expansion rate  B  Cell Expansion Kit, human
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Standardized B cell expansion 

Within only 14 days, the B Cell Expansion Kit, human enables a 10-fold expansion of B cells. Furthermore, the kit offers a standardized system for reproducible conditions. 

 

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.  
 

Phenotype of expanded B cells using the B Cell Expansion Kit, human
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Maintained B cell phenotype

The phenotype of the original cell fraction (plasma cells, naive, and memory B cells) are maintained when expanding B cells with the B Cell Expansion Kit, human. The distribution of different phenotypes is similar between expanded and untreated cells, with a reduction of naive B cells after 14 days and a similar amount of memory cells in both setups.
 

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Scientific poster: 
A standardized and reproducible method for expansion and activation of human primary B cells 
Aparajita Singh,  Susanne Bethke, Daniela Vorholt, and Andrzej Dzionek 

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Analyze antigen specificity and find your lead candidate with automated flow cytometry

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. 
 

Fast and reliable high-throughput 96- and 384-well plate processing.
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Reliable high-throughput analysis 


The MACSQuant X Flow Cytometer enables reliable sample analysis even for high-throughput analyses like lead candidate identification. The instrument can analyze a 384-well plate in less than 60 minutes and a 96-well plate in less than 15 minutes. 
 

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Flow cytometry automation for high-throughput screening 

The MACSQuantX can be integrated into a variety of robotic liquid handling systems. 
 

Functional screening of lead candidates 

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: 

ADCC assay:

Immunogenicity testing: 

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Webinar: 
Automation meets cell separation: primary cells for any drug discovery assay 

Primary cell separation and isolation are essential for drug discovery. Conclusive assays can benefit from flexible, automated instruments and reagents. 
 

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