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Single B Cell Antibody Cloning and Sequencing Service

Catalog No. Product Name Size List Price (US$) Quantity
BS068 Single B Cell Antibody Cloning and Sequencing Service 1 package Request
Description

Success rate of single B cell antibody cloning and sequencing is critical for single B cell antibody discovery. Antigen specific single B cells including both memory B cells and plasma cells have been successfully used to develop antibodies which are difficult to obtain using traditional hybridoma, phage or yeast display methods. Three types of single B cells are obtained and used for single B cell antibody cloning:

1) a small library of single B cells normally obtained with B cell isolation by beads.
2) clonal B cells which are cultured from a single B cell.
3) a single B cell.

Pairing of Antibody heavy and light chains is needed after antibody sequencing of single B cell libraries. Next generation sequencing (NGS) with bar coding may be used to solve the problem. However, the cost of gene synthesis and recombinant antibody production before test is high. We do not recommend the strategy for monoclonal antibody development.

Syd Labs provides the single B cell antibody cloning and sequencing services with PCR amplification, DNA subcloning, and Sanger sequencing. After antigen-specific single B cell sorting, if the B cells are cultured for about 2 weeks, the clonal B cells can be treated as hybridoma cell lines for antibody sequencing. The strategy of single B cell culture works very well in our hands for monoclonal antibody development using PBMC or spleen from rabbits, mice, human, Alpaca and Ilama. Supernatant from cultured single B cells is enough for antibody screening so that the number of single B cells for antibody cloning, sequencing, and recombinant production are significantly reduced. In the meantime, Syd Labs can also clone and sequence antibodies from a single memory B cell or plasma cell. Since the materials are invaluable, no one is willing to accept the consequences of failing in cloning and sequecing any single B cell. Our scientists' high success rate make clients no worry in single B cell antibody sequencing.

General procedure of single B cell antibody cloning and sequencing:

* Extract and purify total RNAs from individual B cells;
* Synthesize cDNAs using optimized primer sets;
* Amplify DNA fragments including VH and VL, leader sequences, and partial constant regions (CH and CL) using 5' RACE;
* Subclone and sequence the amplified DNA fragments.

(If interested, please see single B cell antibody cloning and antibody sequencing protocols. Our own protocol is more optimized.)

Required materials:

Single or clonal B cells (contact technical services for details).

Deliverables:

Study report including the number of antibody heavy and light chains identified, the DNA and protein sequences for each chain, and CDR analysis. Leader sequences and the amplified DNA fragments available if requested.

Syd Labs also provides the following antibody cloning and sequencing services:
Cloning and Sequencing Heavy- and Light-chain Variable Regions (VH and VL) of Hybridoma or Clonal B Lines
De novo Antibody Sequencing Service
N-terminal Protein Sequencing Service
Sequence Liability Identification Service

High-throughput next generation sequencing (NGS) or "deep" sequencing of antibody "repertoires" at the single B cell level (b cell repertoire sequencing and b cell repertoire analysis) is also available upon request.

Optional but highly recommended for single B cell antibody cloning and sequencing services:

Without antigen-antibody binding confirmation (the best quality control), it is hard for any supplier to guarantee 100% accuracy of antibody sequencing results.

Production of the recominant IgG antibody: Molecular construction, 1 ml transient production of recombinant IgG in HEK 293 cells, and protein A purification of recombinant IgG.

Antigen-antibody binding confirmation using the recombinant whole IgG antibody: Test whether the recombinant IgG retains the immunological activities of their corresponding parent monoclonal antibodies using ELISA (Please inquire if you prefer to assays such as Biacore, Octet or competitive binding).

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