Custom Monoclonal Antibody Service - Mouse and Rat Monoclonals

SYSY Antibodies offers specialized and fully customizable monoclonal antibody development services designed to meet our customers’ specific research needs. Our complete service portfolio covers every stage — from antigen design and production to monoclonal antibody purification and long-term hybridoma cell storage.

Antigen Design and Production

Designing a suitable protein antigen is one of the most critical and challenging steps in antibody development.
Numerous factors — such as immunogenicity, solubility, proteasomal processing, secondary structure, sequence similarity, and post-translational modifications — must be carefully evaluated. Although the impact of each parameter on antigen quality and immune response can vary, our extensive experience helps us make informed design decisions for optimal results.

To ensure the best possible antigen for immunization, SYSY Antibodies combines advanced computational prediction tools with empirical data accumulated over many years of practical experience.

Antigens may consist of:

• Synthetic peptides (10–20 amino acids)
• Recombinant proteins expressed in bacterial or mammalian systems
• Transfected cells displaying the antigen on their surface, suitable for generating antibodies targeting extracellular epitopes

Our experienced scientific team can assist in identifying and producing the most appropriate antigen for your project, saving you time and effort. Alternatively, an existing antigen can also be used for your customized antibody development.

Hybridoma Generation

Phase I — Immunization

For immunization, the following materials are required:

• 3 mg of purified peptide, or
• 1.5 mg of purified protein

Typically, three Balb/c mice or Lewis rats are immunized with 90–150 µg each. If the antigen’s immunogenicity is limited (e.g., mouse or rat proteins/peptides), alternative animal strains can be used.

We offer two immunization protocols:

• Short Program (17 days)
  Animals are immunized every second day and one day before fusion. This approach yields a broad panel of monoclonal antibodies recognizing multiple epitopes with moderate to high affinity.
• Long Program (3 months)
  Animals are immunized every third week and shortly before fusion. Extended affinity maturation results in a smaller set of antibodies, generally exhibiting higher affinity and lower variability.

Depending on the antigen, different adjuvants are used, including Gerbu or incomplete Freund’s Adjuvant combined with CpG oligonucleotides (Krieg  2002).

Phase II — Fusion

Following immunization, lymph node and spleen cells from all three animals are fused with Ag8 myeloma cells in the presence of PEG 1500. The resulting cells are distributed into 24-well plates, generating 360 oligo clones (each consisting of 5–10 subclones). These cells are cultured for 10 days in selective, conditioned medium to support fused cell growth.

Phase III / Screening and subcloning

A well-planned screening strategy is essential for successful monoclonal antibody generation. Our expert team will collaborate with you to design an optimal screening setup to maximize the success rate of your project.

Within two days, all 360 oligo clone supernatants are screened by ELISA for antibody-positive wells. Depending on your application, we offer four secondary screening options:

• Western blot analysis of up to 72 oligo clone supernatants
• Immunofluorescence testing on GFP-transfected cells (up to 360 supernatants)
• Sandwich ELISA screening (up to 360 supernatants) to identify capture or detector antibodies
• Custom screening by the customer — up to 20 oligo clone supernatants (3–5 ml each) can be shipped for in-house testing

Screening is typically completed within three weeks. After you select the desired oligo clone, it is subcloned to monoclonality by limiting dilution. Each subclone is screened again using the same selected method(s), and further subcloning cycles are performed as needed to obtain a stable monoclonal cell line. The final clone is expanded to produce 100 ml of monoclonal antibody supernatant.

Positive clones can be cryopreserved as backups for up to 12 months, with long-term storage available upon request.

Collaboration and Quality Commitment

Our primary goal is to provide monoclonal antibodies that perform optimally in your specific assays. To achieve this, close collaboration throughout every stage of the project is our highest priority.

For further information please contact us.