Similar to process of hybridoma or clonal B cell antibody cloning and sequencing, the work flow of single B cell antibody cloning and sequencing is: separation of peripheral blood mononuclear cells (PBMC) from blood, staining of PBMC with the B cell selective marker (CD19 antibody), sorting of stained B cells by flow cytometry, total RNA extraction, cDNA synthesis, two-step PCR amplification, sequencing of the PCR products, and sequence analysis with V region germline database and the Genbank database. Single B cell antibody cloning and antibody sequencing protocols from public literatures:

– Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.
A two-step amplification protocol.

– Systematic design and testing of nested RT-PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells.
112 nested primers to amplify the rearranged / expressed VH and VL genes from any mouse immunoglobulin V gene family from a single or a small number of B cells.

– Human immunoglobulin variable region gene analysis by single cell RT-PCR.
A detailed protocol for single B cell antibody cloning and antibody squencing.

– Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.
A simple FACS-based single cell RT-PCR protocol for the amplification of correctly paired IgG Fab from single human B cells or plasma cells.

The technique of single B cell antibody cloning and antibody sequencing is attractive but challenging for most academia and industrial clients. Experienced scientists at Syd Labs provide single B cell antibody cloning service and antibody sequencing service if the project is difficult for you.

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.