Single B Cell Antibody Engineering

Engineering custom monoclonal antibodies (mAbs) doesn’t have to be a months-long process. Single B cell isolation offers a fast, high-throughput method to generate a cell line producing your antibody of interest. B cells are isolated from an immunized animal, sorted into single wells, lysed, and their genomes sequenced. Genes encoding desired proteins are cloned into a vector and transiently transfected into mammalian cells. Utilize a single B cell workflow to leapfrog many of the traditional antibody engineering challenges, such as long turnaround time, extensive hands-on inputs, and multiple rounds of cell culture.

Isolate B cells

Challenge: Separate only viable B cells from large, heterogeneous cell mixture.

Maximizing the sample size and diversity of B cells isolated is critical for generating antibody hits downstream. Donor animals, typically mice, are injected with an antigen to create an immune response. Immunized animals are bled to test for appropriate antibodies. If the test bleed is positive for an immune response, spleens are harvested and agitated to create a cell suspension. B cells are roughly separated by density centrifugation and sorted from other cell types using a cell sorter. B cells are mortal and do not tolerate culture. They must be quickly used or cryopreserved.

Genome extraction and vector creation

Challenge: Maintain quality and consistency while handling large libraries of samples in parallel.

Sequencing DNA “preserves” the antigen-specific antibody indefinitely. With a large and diverse pool of B cells sorted and individually plated, lyse cells and extract DNA. In large populations, preparing whole-genome sequencing libraries can be burdensome. We can help you automate this process for maximum quality, yield, and short turnaround times. Once sequences of interest have been selected, order sequence-verified oligonucleotides and assemble into a plasmid vector of choice. Transform the vector into bacterial cells to create a strain producing your plasmid for later transfection into mammalian cells.

Transfection and Verification

Challenge: Develop new cell lines while capturing data on viability and production before plasmids are lost.

After plasmid vectors are prepped and cleaned up, transiently transfect into animal cells. Transfection is performed chemically or by electroporating cells. After allowing cells to recover, grow your transfected cells in culture. Harvest antibodies from supernatant to initially evaluate product quality and titer before cells degrade the transfected plasmid. Select cell lines that produce your antibody, using plasmids for stable transfection and future cell line development.

Summary

• Single B cell isolation offers a potent solution for custom antibody engineering.
• Use cell sorting to quickly sort B cells after harvesting to preserve sample size and sample diversity.
• Easily create large vector libraries for transfection by automating liquid handling and data logging functions.

Products and methods described are not intended for use in diagnostic procedures.