It is critical for single B cell antibody generation technologies to clone and sequence the antibody variable region (VH and VL) from antigen specific single or clonal B cells after detection and isolation of antigen specific B cells by flow cytometry-based single B cell sorting. Syd Labs has successfully developed human, monkey, rabbit, mouse, and rat monoclonal antibodies by cloning and sequencing the variable antibody regions of single antigen-specific mouse memory B cells or clonal B cells grown in culture.
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; * Sequence the amplified DNA fragments.
(If interested, please see single B cell antibody cloning and antibody sequencing protocols. Our own protocol is more optimized.)
Single or clonal B cells (contact technical services for details).
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).