Rapid discovery of lead molecules is critical for accelerating potential biotherapeutics to the clinic but finding those desirable candidates can be challenging. The Opto™ Plasma B Discovery 4.0 workflow enables rapid screening of up to 100,000 B cells to identify 1000s of hits, down-selection of lead molecules by profiling antibody function, and then allows sequencing and re-expression of up to 1,000 functionally characterized antibodies. This key last step in the discovery process — sequence recovery and re-expression — can be costly, time consuming, and laborious. Here our in-house experts discuss some of the major challenges encountered with sequencing and re-expression, and how the Opto Plasma B Discovery 4.0 workflow can help.

What are some challenges with sequence recovery and re-expression of lead molecules?

Popular methods used for sequencing like Sanger sequencing are cost prohibitive and can only be performed on a limited number of antibodies, while NGS sequencing is less expensive, but is not as accurate for full-length sequences. Once sequences are finally obtained, costly gene synthesis and laborious bacterial cloning can limit the number of lead molecules that are re-expressed.

How do kits like OptoSeq™ Barcoded BCR and OptoSeq™ Rapid Re-expression help?

OptoSeq™ Barcoded BCR offers a less expensive and more efficient method for rapid, accurate sequencing of paired heavy/light chain antibody genes by DNA fragmentation, NGS sequencing, and bioinformatics analysis. This gives you the best of both worlds — the accuracy and full-length sequence information of Sanger at the speed and cost of NGS.

The OptoSeq BCR Rapid Re-expression kit enables re-expression of >1,000 antibodies without gene synthesis and cloning in just one week.

Expert tips

• Barcoded RNA capture beads make the multiplex export possible and increase the number of cells being sequenced up to 1,000 within one 96-well plate.
• A quality check of the amplified cDNA is the first step to verify the on-chip workflow and to ensure successful antibody sequence recovery and re-expression. No cDNA or degraded cDNA almost guarantee failure of both.
• Random fragmentation followed by NGS not only allows for high throughput sequencing recovery but also provides higher sequencing accuracy compared to transitional Sanger sequencing and direct full length variable region NGS sequencing. 
• Amplification of variable region from cDNA as the first validation of antibody function makes the overall process of identifying the candidates faster and easier. 
• Cross contamination can be avoided with a designated working place and instrumentation, regular thorough cleaning, and of course accurate pipetting.

Show me how it works!

OptoSeq Barcoded BCR enables rapid, accurate sequencing of paired heavy/light chain antibody genes by DNA fragmentation, NGS sequencing, and bioinformatics analysis. Bioinformatic assembly of full-length antibody variable regions from short reads (150 x 150) enables accurate sequencing of thousands of antibodies from a single run on standard Illumina sequencing platforms. Function of re-expressed antibodies can then be confirmed using plate-based ELISA assays. 

Re-expression with OptoSeq BCR Rapid Re-expression

The OptoSeq BCR Rapid Re-expression kit can convert antibody sequences to molecules in just 1 week for plate-based confirmation assays. Amplified cDNA is cloned into expression constructs in one step, and up to 1,000 antibodies can be expressed for rapid functional confirmation.

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Show me more data…

Read our Opto Plasma B Discovery 4.0 datasheet to learn more about how OptoSeq Barcoded BCR and OptoSeq BCR Rapid Re-expression kits can be used to sequence and re-express up to 1,000 functionally characterized antibodies in less than 1 week.

This story originally appeared in the Spring/Summer 2021 edition of BLI News. To read more, check out our latest edition.