Antibody Discovery by Single B Cell Screening on Beacon®
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Amy Sheng, PhD provides an overview of antibody screening platforms and presents applications and case studies using the Beacon® platform for antibody discovery. Single B cell screening is a powerful and efficient strategy for generating antigen-specific monoclonal antibodies. Distinguished with fluorescence-activated B cell sorting, the Beacon® platform is based on plasma cell screening, making it easier to obtain antibody genes. The Beacon® single-cell optofluidic system combines a unique optoelectro positioning (OEP) technology with novel microfluidic technology. It can be used to accurately select single cells on a chip, perform multiple single-cell assays, and export target cells based on specific results. The Beacon® optofluidic platform preserves the diversity of B cells, generating high-quality positive hits at an early stage of discovery and avoiding the loss of “good clones”. Key Topics Include: - B cell differentiation and development - Pros and cons of mainstream antibody screening platforms - Mechanisms, applications, and case studies using the Beacon® platform for antibody screening - Sino Biological’s capacity using the Beacon® platform
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Antibody Discovery by Single B Cell Screening on Beacon®
- 1. Welcome! Technical Account Manager Sino Biological Amy Sheng, PhD Antibody Discovery by Single B Cell Screening on Beacon®
- 2. Sharing of Antibody Discovery of Single B Cell Based on Beacon® Sino Biological Amy Sheng, Ph.D. Technical Account Manager Sino Biological US Inc.
- 3. CONTENT B Cell Differentiation and Development 1 Introduction of Antibody Discovery Platform 2 Introduction of Beacon ® 3 Case of Antibody Screening Based on Beacon® 4 Sino Biological
- 4. B Cell Differentiation and Development 01 Sino Biological
- 5. B Cell Differentiation and Development Central development Peripheral development Immature B Mature B Pro-B Common lymphoid progenitor Germminal center Centroblast Centrocyte Memory B Plasma Pre-B Sino Biological
- 6. Introduction of Antibody Discovery Platform 02 Sino Biological
- 7. Hybridoma Technology Mature and stable Functional and affinity screening can be carried out Low cost Low fusion efficiency：1/1000 Time consuming Species limits Cell fusion Primary B cell (HGPRT pos ) Myeloma B cell (HGPRT neg ) HAT selection Hybridoma Single B cell culture ELISA Sino Biological
- 8. Phage Display Techniques Broad spectrum Full human antibodies are available Low cost Susceptible to antibody repertoire Limited affinity Non-natural antibody pairs Tissue RNA cDNA SCFV Phagemid Transformation Infection Library Sino Biological
- 9. Single B Cell Technology Broad spectrum Natural antibody pair High diversity Short time High affinity Complicated Rely on equipment Laser beam VHDJHCY1 VKJKCK Sino Biological
- 10. Species Sorting——Human, mouse, rabbit… Beacon ——Mouse Cell Sorting ——Memory B cell Beacon ——Plasma cells Functional Assay Sorting——No Beacon——Cell binding, blocking Throughput Sorting——10000cell/s Beacon ——4 chip, 80k cell T 1 2 3 4 Sino Biological Comparison of Fluorescence-activated Cell Sorting and Beacon®
- 11. Introduction of Beacon® 03 Sino Biological
- 13. Flow Diffusion Sino Biological An Example of Cell Culture
- 14. 01 Cell Separation 02 Load 03 Assay 04 Unload Immunization and ASC Enrichment Nanopen Load OEP Gravity In-Channel Fluorescent”Bloom” Assay OEP Nanopen Export VH/VL Recovery, Cloning, Expression 30-60 Minutes 30-60 Minutes 3-4 Minutes per Nanopen Import Export Sino Biological Technical Route
- 15. Cell Separation Load Assay Unload Samples Incubation Antibody Magnetic Particle Target Cell Immunomagnetic Beads Capture Sino Biological
- 16. Cell Separation Load Assay Unload Sino Biological
- 17. Cell Separation Load Assay Unload Antigen Antibody Secondary antibody Avidin beads Cell Sino Biological
- 18. Cell Separation Load Assay Unload Blocking Unblocking Antibody Secondary Antibody Avidin beads Avidin beads Ligand Antigen Sino Biological
- 19. Cell Separation Load Assay Unload Cell Cell Cell Ligand Blocking Unblocking Sino Biological
- 20. Cell Separation Load Assay Unload Sino Biological
- 21. Case of Antibody Screening Based on Beacon® 04 Sino Biological
- 22. Screening of Anti-idiotype Antibody（ Anti-ID Abs） An antibody that uniquely binds the idiotype of another antibody Pharmacokinetics and pharmacodynamics analysis Immunogenicity analysis: positive control antibody Antibody drug Anti-ID Abs Target protein Antibody drug Anti-ID Abs Target protein Blocking Unblocking Block antigen antibody binding Detection of free antibody drugs Does not block antigen antibody binding Detection of total antibody drug (Free, half bound, bound) Sino Biological
- 23. 01 02 03 04 Immunization （2weeks） Beacon（1day） Antibody gene amplificat（1week） Expression, purification, detection （2weeks） Antibody drug binding Human IgG binding Target protein competition Sino Biological Technical Route for Anti-ID Abs Development
- 24. Load 11978 Antibody Drug Binding 772 Human IgG Binding 151 Competition Blocking 54 Unblocking 97 Antigen/hIgG Binding Competition Sino Biological Case Study
- 25. Screening of Modified Antibodies Phosphorylated polypeptide Non-phosphorylated polypeptides Target Load Phosphorylated+ Phosphorylated+Non-phosphorylated- SinoA 12400 109 63 Protein modification refers to group modification of protein after translation, including acetylation, ubiquitination, methylation, phosphorylation… Sino Biological
- 26. Screening of CD47 Antibody CD47 Binding Domain of SIRPα Blocks the DON’T EAT ME signal from CD47 but does not bind to red blood cells Delivers an EAT ME signal to macrophages through FcγRs: Differentiated from lgG4 antibodies SIRPαFc triggers activation of FcγRs on macrophages Human lgG1 Fc FcγR SIRPαFc CD47 Sino Biological
- 27. Sample ID IC50(ug/mL) CD47-M0002-1 0.273 CD47-M0065 0.074 CD47-M0137-1 0.198 CD47-M0091-1 0.183 CD47-M0075-1 0.155 CD47-M0120 0.103 CD47-M0126-2 0.134 Control-1 0.033 Control-2 0.204 Sample ID KD (M) kon(1/Ms) kdis(1/s) Control-1 5.97E-10 2.62E+05 1.56E-04 Control-2 1.92E-09 2.50E+05 4.81E-04 CD47-M0002-1 3.68E-10 7.42E+05 2.73E-04 CD47-M0065 1.11E-09 5.45E+05 6.08E-04 CD47-M0091-1 4.52E-10 3.87E+05 1.75E-04 CD47-M0075-1 1.44E-09 4.41E+05 6.34E-03 CD47-M0120 1.60E-09 5.75E+05 9.18E-04 CD47-M0126-2 6.90E-10 4.40E+05 3.04E-04 Control-1 Control-2 M0002-1 M0065 M0091-1 M0075-1 M0120 M0126-2 Blocking Affinity Binding with Cells Target Cell on Beacon® PCR ELISA Blocking Weak Binding with RBC CD47 331 255, 77.04% 108 73 2 Jurkat RBC Sino Biological
- 28. Screening of Monkeypox A29 Antibody Target Load Antigen+ A29 11390 321 Western Blot Assay ELISA Assay The EC50=23.98 ng/ml Sino Biological
- 29. 32,000+ genes 14,000+ antibodies 600+ ELISA kits International Reagent Supplier and Service Provider Citations Recombinant protein expression Recombinant antibody production Monoclonal antibody development Polyclonal antibody development High-quality reagents & one-stop CRO services >10,000 citations till Sep, 2022 6,500+ proteins Company Overview Certification ISO9001:2015 and ISO13485:2016 certified End-Users in 90+ Countries User Distribution
- 30. The Global Leader in Recombinant Technology Advantages Development & Production Capacity of thousand per batch Kg level per batch 5 expression systems Routine high-throughput (HTP) production and screening projects Up to 1,500L bioreactor for scale-up production FucoFree cell line, stable cell Line, insect cells, mammalian cells, and E. coli In-house platforms Proprietary culture media, transfection reagents, and expression vectors Recombinant protein HTP antibody Large-scale antibody Chimeric antibody Fab/scFv/VHH antibody Bispecific antibody Humanized antibody Recombinant Production Sino Biological
- 31. Guangzhou, China PA & TX, USA Frankfurt, Germany Headquarters: Beijing, China Shanghai, China Taizhou & Suzhou, China Kawasaki-shi, Japan Global Presence Sino Biological
- 32. THANK YOU Sino Biological US Inc. (USA) Pennsylvania Office: 1400 Liberty Ridge Drive, Suite 101, Wayne, PA 19087 Texas Office: 10101 Southwest Freeway, Suite 375. Houston, TX 77074 Tel: +1-215-583-7898 Email: order_us@sinobiologicalus.com Sino Biological Europe GmbH (Europe) Address: Düsseldorfer Str. 40, 65760 Eschborn, Germany Tel: +49(0)6196 9678656 Email: order_eu@sinobiologicaleu.com Sino Biological Japan Inc. (Japan) Address: Tech-Pot, 2F, West Wing, KSP, 3-2-1 Sakado, Takatsu-ku, Kawasaki-shi, Kanagawa, 213-0012, Japan Tel: 044-400-1330 Email: order@sinobiological.co.jp Sino Biological, Inc. (Global) Address: Building 9, Jing Dongbei Technology Park, No.18 Kechuang 10th St, BDA, Beijing, 100176, P.R.China Tel: +86-400-890-9989 Email: order@sinobiological.com Custom Antibody Services Beacon® Single B Cell Screening Platform