ANTIBODY SEQUENCING SERVICE

Entrust experienced professionals for antibody sequencing. Getting your monoclonal antibody sequence is a key starting point :

• to protect you antibody sequence by patent application
• to optimize the potential of your antibody by further engineering
• to prevent the loss of the hybridoma cell line
• for recombinant antibody production.

ProteoGenix offers a fully customizable antibody sequencing service with high guarantees. With 16 years of experience, our services are exhaustive and standardized and featured as follow for optimal performances:

• Length of the sequence: antibody variable domains (VH, VL), full-length antibody (VH, VL, Fc)

Optional:

• Gene synthesis with codon optimization for a chosen expression system: yeast, insect, mammalian
• Recombinant antibody production
• Interaction test against soluble antigen or cell antigen (ELISA, FACS,KD determination by Biacore / SPRi / Octet…)

Deliverables:

• A complete report including the CDR sequence annotated,
• Plasmid containing the sequence (if requested),
• Up to 2mg recombinant antibody (if requested).

Case study: DNA sequencing of variable
regions of hybridoma cell line

Aim Of The Study

Here is an example of a customer requesting to sequence a hybridoma cell line we had previously developed for him.

Materials

• PacBio RS II
• Hybridoma cells provided by ProteoGenix
• TaKaRa MiniBEST Universal RNA Extraction Kit
• PrimeScript RT reagent Kit with gDNA Eraser
• TA-cloning Kit

Hybridoma Sequencing Protocol

Total RNA was extracted from hybridoma cells and cDNA subsequently synthesized. Antibody variable genes were then amplified by isotype-specific PCR, separately sub-cloned into a standard cloning vector and sequenced.

Isotype Determination And Antibody Sequence Analysis

Antibody isotype determination is performed by gel electrophoresis. Details of the protocol are described in the complete report provided in PDF file.

Results of the sequencing are based on the analysis of at least 10 positive clones and include:

• Gene sequence
• Amino acid sequence with annotated CDR

After completion of the antibody sequencing work, we were asked to engineer a chimeric version, synthesize both antibody cDNAs, subclone them into an expression vector, produce them in XtenCHO cells and determine antibody binding affinity (KD determination) of all 3 antibodies. The aim was to confirm that the chimeric recombinant antibody functionality at least remained equivalent to the mouse recombinant antibody and the mouse hybridoma antibody.

After confirmation, we were then able to develop humanized antibodies as well as bispecific antibodies for human therapeutic purposes.

Click on the button below to get the complete report “Parental Antibody Sequencing from Hybridoma”