Monoclonal Antibody and Hybridoma Development

One of our core competency and expertise is in development of new hybrdoma & monoclonal antibody. Our key personals have over 25 years of expertise in this area, making many monoclonals over the years. We are so confident about our expertise and success rate that we guarantee our services with guaranteed clones. The table below gives an outlines of services. Please review the information below and contact us for a specific quote.

We offer hybridoma and monoclonal development in mice, rats and hamsters.

We offer monoclonal antibody development to all range of targets including proteins, peptides, nucleotides, carbohydrates and small molecules. Contact us to discuss your needs.

Our Hybridoma Development Program is divided into the following phases. These phases are optional and client can choose to decide what they want to do. We work with our customers to decide the best approach to meet their requirement.

Phase-0: Project Initiation & Preparation of Immunogen

One of the keys to success of a hybridoma project is the right choice of immunogen.
We guarantee animal immune response and clones if we do sequence analysis and propose the immunogen to be used. And this service is free.

We can make the immunogen for you for additional cost or use the one you have.

Following is a list of immunogen services we offer.

Phase-1: Immunization of animals and IgG titer determination

Time 6-8 weeks

1. Purchase 5 Balb/c mice and maintain for up to 180 days. Optional rat or hamsters.
2. Immunize all animals with the immunogen, provided by client or prepared by ARC, by proprietary immunization technique with adjuvant for DNA, peptide or small molecules. Up to 4 immunization may be performed.
3. Bleed all animals and determine antibody titer by ELISA using provide antigen.
4. Provide serum samples to client for analysis if required.
5. Provide addition 4^th boost if needed, if titer is border line between 1000 – 10,000.
6. Client may choose to give additional boost or terminate the project if titer <1000.
7. Choose a animal for fusion based on data provided, ELISA IgG titer >10,000.
8. Immune sera may be screened by Western Blot, FACS, IHC or other methods using antigen or cell line or protein extract, if needed for additional cost.

Phase-2: Fusion and Generation of Hybridomas

Time 3-4 weeks

1. Final intra venous boost (iv tail vein) of a chosen mice after 4 weeks resting period.
2. Collection of spleen from the chosen mice & preparation of single cell suspension.
3. Preparation of myeloma cells (Ag8) ready for fusion selected to grow in presence of 8-Azaguanine. Client may choose other myeloma such as NS1, NS0 or Sp2/O.
4. Fusion of a mouse spleen cells with myeloma cells.
5. Plating of fused cells in 6 – 10 multi-well plates and HAT selection.
6. Determination of antigen specific antibody producing wells by ELISA after 10-12 days.
7. Provide samples of supernatant to client for screening to test in their specific assays if required.
8. Expansion of 6 positive wells to 24 well plate and back up of cells in liquid Nitrogen storage.
9. Repeat ELISA to reconfirm antibody production of positive wells 16-18 days after fusion and choose wells for subcloning.
10. ELISA and Western Blot data will be provided to client to choose 3 best clones to proceed with subcloning.
11. Up to 2 fusions may be performed without additional cost if required.
12. Additional positive wells may be expanded to freeze for $50 each, store up to a year.
13. Positive wells may be screened by Western Blot, FACS, IHC or other methods using client provided antigen or cell line or protein extract, if needed for additional cost.

Phase-3 Subcloning and Selection of Monoclonals

Time 6-8 weeks

1. 2-3 rounds of limiting dilution cloning of 3 chosen clones to get >95% clonal culture.
2. Determination of clonality and productivity of the subclones by ELISA.
3. Mycoplasma testing and certification of the clone.
4. Isotyping of the subclone.
5. Preparation of 50mL of culture supernatant for client from each subclone.
6. Expansion & preparation of 5 vials each of the 3 subclones for storage at 5E6 cells/mL/vial.
7. Storage of all clones (single isotype and >95% clonality) in liquid Nitrogen for up to 1 year.
8. Provide complete data set to client including ELISA, Mycoplasma, and Isotyping.
9. Additional subcloning for $600 each. Includes selection, cloning and storage of 5 vials for 1 year.
10. Storage of frozen vials for additional years at $25/vial/year.
11. Clones may be test by Western Blot, FACS, IHC or other methods using client provided antigen or cell line or protein extract, if needed for additional cost.

Phase-4: Antibody Production

We offer antibody production in iv vivo mouse asciets or in a variety of in vitro bioreactor production systems. Check out the production file for details.