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Home > Services > Antibody Services > Antibody Engineering Services > Single B Cell Antibody Cloning and Sequencing Service

Single B Cell Antibody Cloning and Sequencing Service

Catalog No. Product Name Size List Price (US$) Quantity
BS068A Plate-based Single B Cell Antibody VH and VL Sequencing Service by Nanopore Sequencing 1 package Request
BS068B Plate-based Single B Cell Full Length Antibody Sequencing Service by Nanopore Sequencing 1 package Request
BS068C Droplet-based Single B Cell Antibody VH and VL Sequencing Service by Nanopore Sequencing 1 package Request
BS068D Droplet-based Single B Cell Full Length Antibody Sequencing Service by Nanopore Sequencing 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 sequencing:

1) clonal B cells which are cultured from a single B cell
2) .a single B cell per well on plates
3) a small library of single B cells normally obtained with B cell isolation using magnetic beads or cell sorting.

Syd Labs provides the single B cell antibody sequencing services with NGS nanopore sequencing.

It is the easiest to do single B cell antibody sequencing from cultured or clonal single B cells. 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, and also the cost of antibody discovery.

Sequences, both varialbe regions and full length, of antibodies from a single memory B cell or plasma cell can be obtained by nanopore sequencing. Since the experimental materials are invaluable, the consequences of failing in cloning and sequecing any single B cell antibody are not acceptable. Our scientists' high success rate make clients no worry in single B cell antibody sequencing. Nanopore sequencing-based 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 used for a single B cell per well on 96-well or 384-well plates, and a small library of single B cells normally obtained with B cell isolation using magnetic beads or cell sorting.

General procedure of plate-based single B cell antibody sequencing by nanopore sequencing, antibody variable regions (BS068A: CDR analysis included; Leader sequences available if requested):

1) Synthesize cDNAs from single B cells;
2) Amplify DNA fragments including VH and VL, leader sequences, and partial constant regions (CH and CL);
3) Do amplicon sequencing by nanopore;
4) Perform CDR analysis using sequencing data.

General procedure of plate-based single B cell antibody sequencing by nanopore sequencing, full length antibody (BS068B: CDR analysis included; Leader sequences available if requested):

1) Synthesize cDNAs from single B cells;
2) Amplify DNA fragments of full length antibody;
3) Do amplicon sequencing by nanopore;
4) Perform CDR analysis using sequencing data.

General procedure of droplet-based single B cell antibody sequencing by nanopore sequencing, antibody variable regions (BS068C: CDR analysis included; Leader sequences available if requested):

1) Synthesize cDNAs from single B cells;
2) Amplify DNA fragments including VH and VL, leader sequences, and partial constant regions (CH and CL);
3) Do amplicon sequencing by nanopore;
4) Perform CDR analysis using sequencing data.

General procedure of droplet-based single B cell antibody sequencing by nanopore sequencing, full length antibody (BS068D: CDR analysis included; Leader sequences available if requested):

1) Synthesize cDNAs from single B cells;
2) Amplify DNA fragments of full length antibodies;
3) Do amplicon sequencing by nanopore;
4) Perform CDR analysis using sequencing data.

Timeline:

2-3 weeks for sequencing antibody variable regions and full length antibodies from single B cells by NGS nanopore sequencing. A record for the single B cell antibody sequencing service by nanopore sequencing: 2 days.

Required materials:

Single B cells including both memory B cells and plasma 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 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) or full length antibodies from Hybridoma or Clonal B Cells
De novo Antibody Sequencing Service
N-terminal Protein Sequencing Service
Sequence Liability Identification Service

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 CHO 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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