Custom Antibodies
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Rabbit Recombinant Monoclonal Antibody Production

  rabbit monoclonal ab generation process_final

    Rabbit monoclonal antibodies are widely recognized and used in research and diagnostics as they are ideal for immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). Traditionally, rabbit monoclonal antibody is made from rabbit hybridoma, a process of fusing antibody-secreting-cell with a rabbit plasmacytoma cell-line. The disadvantage of such process is that it is time-consuming and the success rate is relatively low. Technology advancements have made recombinant rabbit monoclonal antibody production more achievable and these new methods include the use of (1) phage display library, (2) next generation sequencing of B cells and (3) LC-MS/MS of polyclonal antibody. The drawback of these new generation processes is the high cost associated and that the resulting artificial antibodies are often limited to specific applications.  At abm, our process focuses on generating rabbit monoclonal antibody from primary rabbit plasma cell/plasmablast. We utilize single-cell cloning method, memory B cell expansion, and florescence-activated cell-sorting techniques to generate the rabbit monoclonal antibody of interest. Our main strengths are customized antigen preparation, high efficiency (FACS selection of single cell), stable cell-line generation, and humane treatment of animal without having to sacrifice the animal.

abm’s Rabbit Recombinant Monoclonal Antibody Production Service is divided into different phases, with a non-refundable $3,000 to cover the material cost to initiate the project. The balance will be due upon completion of individual phases. We offer three standard protocol packages and several extension options. Our complete package includes antigen production and guaranteed polyclonal Western blot results.

The basic package includes Phase I-IV and the complete package includes generation of recombinant antibody secreting stable cell line. Customer can also supply antigen or immunized whole spleen/PBMC/Bone marrow as the starting material for the project, please refer to the notes section for sample requirements.

Description Cat. No. Price

PHASE I Services: Antigen Production

• Cloning/sub-cloning of gene of interest into a protein-encoding gene expression vector
• Sequence confirmation
• A set of multivariate small scale protein production trials are conducted to test for yield, solubility and optimal growth conditions
• Large scale (>1mg) recombinant protein production and purification (purity ≥90%)

C151 $3,500.00

PHASE II Service: Immunization

• Immunization (2 rabbits) of antigen from PHASE I or customer provided sample*
• Titre testing by ELISA
• Guaranteed rabbit antiserum titre of 1:10000

C152 $1,000.00

PHASE III Service: Single-Cell Cloning

• Isolation of antibody secreting cell from PHASE II or customer provided sample**
• Staining cells with fluorescently labelled reagents
• Single cell sorting by FACS
• Amplification of cognate pair of heavy and light chain variable regions from sorted single cell
• DNA sequencing and single cell cloning of heavy & light chain variable regions
• Cloning/subcloning of heavy and light chain variable regions into a recombinant antibody expression vector
Deliverables: DNA sequencing data of variable regions

C153 $12,000.00

PHASE IV Service: Antibody Production

• Small scale antibody production for validation studies with expression vector from PHASE III
• Expression of full-length and complete recombinant antibody generation
• Guaranteed 3 positive clones (tested via Western Blot)
Deliverables: 25mL of cell culture supernatant of all 3 clones

C154 $3,500.00

PHASE V Service: Stable Cell Line Development

• Cloning/subcloning of heavy and light chain variable regions into a viral vector
• Viral packaging for gene delivery into a producer cell line
• Drug screening and selection of high stable expression
• Validation of recombinant antibody from the producer cell lines via Western Blot
• Expansion and cryopresevation of selected clones
• PCR based mycoplasma testing on selected producer cell lines
Deliverables: 3 stable cell lines generated based on PHASE IV (2 vials each)

C155 $10,000.00
Additional Clone(s): Generated from PHASE III-IV C156 $1,000.00 per clone
Large-Scale production of recombinant antibody and purification of antibody by Protein A/G C157 Inquire  

* Customers can supply the recombinant antigen for immunization:  3-4 mg of purified protein antigen or 2mg of KLH and BSA conjugated antigen (if the antigen is a small molecule).
** Customers can supply immunized whole spleen (in complete growth media) on ice, splenocytes (growth media with 10% DMSO) on dry-ice, fresh whole-blood with heparin on ice, PBMC (growth media with 10% DMSO) on dry-ice, or bone marrow (growth media with 10% DMSO) on dry-ice. Minimal of 10 million cells (splenocytes, PBMC, bone marrow) or 10-30mL of fresh whole-blood is required.
DISCLAIMER: Customers have the option to start or stop the project from any stages of the rabbit monoclonal antibody generation.  Customers will be responsible for the material and labour cost incur if chose to stop the project from any stages of the development.

 

rabbit-monoclonal-ab-production-timeline_1-01

 

Comparison of Rabbit Monoclonal Antibody Generation Methods

Traditional Fusion Method

abm Single-cell Method

Phage Display Method

Lead Time

> 7-8 months

< 6 months

>8-10 months

Cost

>15,000 USD

~20,000 CAD

>40,000 USD

Disadvantages

Low fusion rate
Low survival rate
Instability
Cell-line unavailable

Insufficient mRNA
Technical

Random pairing
Slow process
Instability
Very high cost

Advantages

Mass production
Established method

Fast process
No cell storage
Antibody engineering

Antibody library
Antibody engineering

 

 

Rabbit Monoclonal Antibodies Advantages
Rabbit monoclonal antibodies have distinct advantages over traditional mouse monoclonal antibodies (hybridoma), including higher specificity, affinity (KD10-12M to 10-14M) and ability to react to epitopes that are non-immunogenic in mice or rat. The rabbit monoclonal antibodies advantages are summarized as follow:

Mouse

Rabbit

Antigen recognition

Limited immune response
Limited response to rodent/human
Inability to recognize small molecules/peptides
Recognize limited epitopes

Recognize small molecule/peptides
Response to rodent/human
Recognizes several epitopes
Recognize structural variations

Affinities

Nanomolar (~10-9KD M)

Picomolar (10-12KD M) to 10-14M

Specificity

Medium to High

High

Applications

Westerns, ELISA, Flow Cytometry, IP, may not be suitable for IHC, ICC, diagnostic kits

Western, ELISA, Flow Cytometry, IP, excellent IHC, ICC, diagnostic kits

Product Documents
Product References
FAQs
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Why abm?
We work closely with you to design the process and provide regular updates on the project. You have the option to start or stop the project from any stages of the rabbit monoclonal antibody generation. Customers will be responsible for the material and labour cost incur if chose to stop the project from any stages of the development.
What process do you use to generate rabbit monoclonal antibody?
We use FACS to isolate plasma cell, plasmablast or memory B cells. We then clone out and sequence the single antibody secreting cell. In the case of memory B cells, the single memory B cell will be expanded using our proprietary method. We will test the expanded supernatant and clone out any positive clone. After sequence analysis, we will perform transient transfection and collect the culture supernatant to test the antibody’s functionality.
What will be delivered at the end of the project?
For the basic package, we will deliver ~25 mL of cell culture supernatant and the monoclonal antibody sequences. Stable cell-line will be supplied with the complete package. We do offer additional services, including large-scale antibody production, please contact our customer service for more detail.
What assay do you use to confirm the functionality of the produced antibody?
For rabbit monoclonal antibody service, we use Western blotting to ensure the produced antibody have affinity and specificity towards the antigen. Immunohistochemistry (C095) and Immunoprecipition tests (C094) are also available, however are NOT included in the production service. Please inquire customer service for further antibody characterization. For rabbit polyclonal antibody services, we guarantee the antibodies will work against the designed antigens in ELISA format. Due to experimental variations, we do not guarantee downstream applications. It is important to understand that the customer takes full responsibility for the antigen designs or delivery.
What is the shortest length peptide that you can produce an antibody against?
To produce an antibody that has specificity and affinity at acceptable levels, we require a minimum of 10 amino acids.
Can you produce an antibody against a native protein?
Yes, however, in order to guarantee the antibody is effective against the native protein, purified native protein must be used for immunization. Recombinant protein or peptide extracts can also be used to immunize, but we cannot guarantee these will be effective against the native protein due to folding and other conformational concerns. Our service guarantees that the immunized protein (native or recombinant) or peptide will be recognized by the produced antibody.
What concentration and amount of antigen is required for production if I am supplying the protein/peptide?
For peptide or small molecule antigens we require at least 2 mg conjugated to carrier proteins such as KLH, OVA or BSA, while if it is a protein, 3-4 mg with concentrations of 0.5-1.0 mg/ml are acceptable.
Do you deliver the cloned expression vectors along with the custom antibody?
No, the customer will only receive the custom antibody and the variable domains sequences. The customer will not receive any antibody expression vectors.
What information do I need to provide to start the project?
Please provide the antigen design or gene accession number and the service you are looking for (i.e. basic, complete or partial) to technical@abmgood.com.
If I want to deliver fresh whole-blood,when I should bleed rabbit and collect sample after re-boost?
You can collect blood either 7 days after or 14-days after boost.