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Rabbit Recombinant Monoclonal Antibody Production

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 hybridomas, a process of fusing an antibody-secreting-cell with a rabbit plasmacytoma cell-line. The disadvantage of such a 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 cells/plasmablasts. We utilize a single-cell cloning method, memory B cell expansion, and florescence-activated cell-sorting techniques to generate the rabbit monoclonal antibody of interest. Our monoclonal antibody production service offers customized antigen preparation, high efficiency (FACS selection of single cell), stable cell-line generation, and humane treatment of the animal without having to sacrifice the animal.

We offer three standard protocol packages and several extension options. Our complete package includes antigen production and guaranteed polyclonal Western Blot results.

 

“I am a fan of abm: great product quality, great price, great rewards and great experience.”

Dr. Vincent Emond, Centre de recherche du CHU de Québec CHUL

Additional Resources:

Service Details

 

abm’s Rabbit Recombinant Monoclonal Antibody Production Service is divided into different phases, with a non-refundable $4500.00 deposit to cover the material cost to initiate the project. The balance will be due upon completion of individual phases.

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.

Core Services

SERVICE DESCRIPTION CAT. NO. UNIT
PHASE I Services: Antigen Production
  1. Cloning/sub-cloning of gene of interest into a protein-encoding gene expression vector
  2. Sequence confirmation
  3. A set of multivariate small scale protein production trials are conducted to test for yield, solubility and optimal growth conditions
  4. Large scale (>1mg) recombinant protein production and purification (purity ≥90%)
 C151 1 Service
PHASE II Service: Immunization
  1. Immunization (2 rabbits) of antigen from PHASE I or customer provided sample*
  2. Titre testing by ELISA
  3. Guaranteed rabbit antiserum titre of 1:10000
 C152 1 Service
PHASE III Service: Single-Cell Cloning
  1. Isolation of antibody secreting cell from PHASE II or customer provided sample**
  2. Staining cells with fluorescently labelled reagents
  3. Single cell sorting by FACS
  4. Amplification of cognate pair of heavy and light chain variable regions from sorted single cell
  5. DNA sequencing and single cell cloning of heavy & light chain variable regions
  6. Cloning/subcloning of heavy and light chain variable regions into a recombinant antibody expression vector

Deliverables: DNA sequencing data of variable regions

 C153 1 Service
PHASE IV Service: Antibody Production
  1. Small scale antibody production for validation studies with expression vector from PHASE III
  2. Expression of full-length and complete recombinant antibody generation
  3. Guaranteed 3 positive clones (tested via Western Blot)

Deliverables: 25mL of cell culture supernatant of all 3 clones

 C154 1 Service
PHASE V Service: Stable Cell Line Development
  1. Cloning/subcloning of heavy and light chain variable regions into a viral vector
  2. Viral packaging for gene delivery into a producer cell line
  3. Drug screening and selection of high stable expression
  4. Validation of recombinant antibody from the producer cell lines via Western Blot
  5. Expansion and cryopresevation of selected clones
  6. PCR based mycoplasma testing on selected producer cell lines

Deliverables: 25mL of cell culture supernatant of all 3 clones

 C155 1 Service
 

Add-On Services

DESCRIPTION CAT. NO. UNIT
Additional Clone(s): Generated from PHASE III-IV C156 1 Clone
Large-Scale production of recombinant antibody and purification of antibody by Protein A/G C157 1 Service

Note:

  1. * 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).
  2. ** 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.
  3. Customers have the option to start or stop the project from any stages of the mouse monoclonal antibody generation service. Customers will be responsible for the material and labour cost incurred thus far should early termination be requested.

Additional Info

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 USD > 40,000 USD
Disadvantages
  • Low fusion rate
  • Low survival rate
  • Instability
  • Cell-line unavailable
  • Insufficient mRNA
  • Technical difficulty
  • 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 Antibody Advantages

  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

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FAQs

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.

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 us for more information.

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 with us 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.

To produce an antibody that has specificity and affinity at acceptable levels, we require a minimum of 10 amino acids.

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.

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.

No, the customer will only receive the custom antibody and the variable domains sequences. The customer will not receive any antibody expression vectors.

Please provide the antigen design or gene accession number and the service you are looking for (i.e. basic, complete or partial) to quotes@abmgood.com.

You can collect blood either 7 days after or 14-days after boost.

Citations

01 Ko, FC et al. “PKA-induced dimerization of the RhoGAP DLC1 promotes its inhibition of tumorigenesis and metastasis.” Nat Commun 4:1618 (2013). PubMed: 23511482