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Antibody Sequencing, Hybridoma Sequencing

Cloning Hybridoma Antibody Heavy- and Light-chain Variable Regions, Antibody Cloning
Catalog No. Product Name Size List Price (US$) Quantity
BS045A 5' RACE-based Cloning and Sequencing Antibody VH and VL (CDR Analysis Included; Leader Sequences Available) 1 hybridoma 1200.00
BS045B 5' RACE-based Cloning and Sequencing of Antibody VH and VL + 0.03 Liter Production and Purification of Reconstructed scFv-Fc 1 package 3400.00
BS045C 5' RACE-based Cloning and Sequencing of Antibody VH and VL + 0.03 Liter Production and Purification of Reconstructed Whole IgG 1 package 3400.00
BS045F 5' RACE-based Cloning and Sequencing of Antibody VH and VL + 0.03 Liter Production and Purification of Reconstructed scFv-Fc + Antigen-antibody Binding Confirmation using scFv-Fc 1 package 4900.00
BS045G 5' RACE-based Cloning and Sequencing of Antibody VH and VL + 0.03 Liter Production and Purification of Reconstructed Whole IgG + Antigen-antibody Binding Confirmation using Whole IgG 1 package 4900.00
Description

Comprehensive monoclonal antibody cloning and antibody sequencing services are provided: The IgG, IgM, IgA, or IgE antibody variable regions of the heavy chain (VH) and the light chain (VL) from mouse, rat, hamster, rabbit, monkey, or human hybridomas or clonal B cells are PCR amplified and sequenced. If requested, the service of cloning and sequencing antibody constant regions of the heavy chain (CH) and the light chain (CL) from hybridomas or clonal B cells is also available.

100000 cells per hybridoma are sufficient for cloning and sequencing antibody genes. Welcome to challenge us with smaller number of clonal B cells or non-viable cells for hybridoma rescue.

Case study 1: A mouse IgG hybridoma stored for 30 years could not grow. We cloned and sequenced its VH and VL, produced a chimeric IgG antibody by transient expression in HEK 293 cells, and confirmed its binding activity. 

Case study 2: After one round of antibody cloning, only a truncated antibody sequence was repeatedly observed. However, the hybridoma secreted functional antibody. It means that noise in the sample was so significant that it was difficult to obtain the correct antibody sequence. The case is not unusual and occurs in 10-20% samples from clients and in house. Most service suppliers asked clients to sequence the N-terminal 10-15 residues of the purified protein so that sets of specific PCR primers can be designed to amplify desired antibody genes. After trying various methods, we optimized the process to obtain the antibody sequences without protein sequencing.

$1200 per sample is for cloning and sequencing IgG VH and VL from mouse hybridomas or clonal B cells. Prices are higher for rat, hamster, moneky, and human IgG antibody samples as well as IgM, IgA, and IgE samples for all species.

General procedure of 5' RACE-based cloning and sequencing antibody variable regions of hybridoma or clonal B cells (BS045A: CDR analysis included; Leader sequences available if requested):

1) Extract and purify total RNAs from hybridoma or clonal B cells (Please see how to prepare samples for antibody sequencing);
2) Synthesize cDNAs;
3) Amplify DNA fragments including VH and VL, leader sequences, and partial constant regions (CH and CL) using 5' RACE;
4) Subclone PCR positive bands;
5) Sequence up to 20 clones in total;
6) Perform CDR analysis using sequencing data.
(To know more about how to clone and sequence antibody genes, please see hybridoma antibody cloning and antibody sequencing protocols that we collect from public literature. Our own antibody cloning protocol is more optimized.)

Fast turnaround: 2-3 weeks for cloning and sequencing monoclonal antibody variable regions. A record for the antibody cloning and sequencing service: 4 days.

Deliverable:

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.

We also provide the following antibody cloning and antibody sequencing services:
Single B Cell Antibody Cloning and Sequencing Service
De novo Antibody Sequencing Service
N-terminal Protein Sequencing Service
Sequence Liability Identification Service

The method of high-throughput next generation sequencing (NGS) or "deep" sequencing of binding populations can also be applied to understand natural antibody repertoires (the "antibodyome") and develop synthetic antibody repertoires for antibody engineering. (If you want to know more information about how to clone and sequence antibody repertoires, please see high-throughput antibody repertoire sequencing protocols that we collect from public literature. Our own antibody repertoire cloning protocol is more optimized.)

Optional but highly recommended for monoclonal antibody cloning and antibody sequencing services:

Compared with the mass spectrometry (MS)-based de novo antibody (protein) sequencing method, the PCR-based antibody (mRNA/cDNA) sequencing method is much more cost effective if cells rather than the protein only is available. Without antigen-antibody binding confirmation (the best quality control), it is hard for any supplier to guarantee 100% accuracy of antibody sequencing results.

Molecular construction of recombinant single chain antibodies (scFv-Fc), 0.03 liter transient production of recombinant scFv-Fc in HEK 293 cells, and protein A purification of recombinant scFv-Fc (+ BS045A = BS045B)

Molecular construction of recombinant IgG, 0.03 liter transient production of recombinant IgG in HEK 293 cells, and protein A purification of recombinant IgG (+ BS045A = BS045C)

Antigen-antibody binding confirmation using the recombinant scFv-Fc or whole IgG: Test whether the recombinant scFv-Fc or whole 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). (+ BS045B = BS045F) and (+ BS045C = BS045G)

References:

1. "The variable regions of the heavy and light chains of the MAbs were sequenced at the DNA level (Syd Labs, Malden, MA)." Neutralizing capacity of monoclonal antibodies that recognize peptide sequences underlying the carbohydrates on gp41 of simian immunodeficiency virus. Martinez-Navio JM, Desrosiers RC. 2012 J Virol. 86(23): 12484-93.

2. "The 2H2 108 antibody amino acid sequences of the light and heavy chain variable domains were determined by Syd Labs, Inc." Obstruction of dengue virus maturation by Fab fragments of the 2H2 antibody. Wang Z, Li L, Pennington JG, Sheng J, Yap ML, Plevka P, Meng G, Sun L, Jiang W, Rossmann MG. 2013 J Virol. 87(16):8909-15.

3. "the heavy and light chains of MAbs A6.1 and A6.2 were sequenced by Syd Labs, Inc. (Boston, MA), and found to be identical." Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization. Kolawole AO, Li M, Xia C, Fischer AE, Giacobbi NS, Rippinger CM, Proescher JB, Wu SK, Bessling SL, Gamez M, Yu C, Zhang R, Mehoke TS, Pipas JM, Wolfe JT, Lin JS, Feldman AB, Smith TJ, Wobus CE. 2014 J Virol. 88(8):4543-57.

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