Antibody Production Services

Phase 1. Immunization and Serum Testing

3 Balb/c mice are injected with selected antigen at 0 and 21 days. On day 33, serum is collected and tested for antibody titer by selected assay. The mice with highest titers are used to derive hybridomas.

Antigen type:

  1. Purified Protein Antigen
  2. Whole Cells (Live or Fixed)
  3. Synthetic Peptide Antigen

General Notes:

  • All immunogens should be endotoxin-free and highly pure.
  • Peptides must be conjugated (e.g., to KLH) to be immunogenic.
  • Appropriate adjuvants (e.g., Alum or CPG) will be used to enhance immune response
  • Cell-based immunization is ideal for membrane proteins or native conformation targets.
  • Screening often requires both target and negative control samples.
Sample table

Optional: Subtractive Immunization

  • For complex targets or highly homologous antigens (e.g., self-antigens, tissue-specific isoforms, or tumor-specific markers), subtractive immunization may be employed.
  • This method involves pre-tolerizing the mouse to non-target antigens (e.g., normal tissue or cell lines) prior to immunization with the desired target antigen (e.g., diseased or transformed tissue).

Benefits of Subtractive Immunization:

  • Enhances specificity by reducing the immune response to shared or background antigens.
  • Improves success in generating monoclonal antibodies against:
    • Rare epitopes
    • Disease-specific antigens
    • Minor variants (e.g., isoforms, mutations)
  • Reduces downstream screening workload, as fewer irrelevant or cross-reactive clones are produced.

​​​​Phase 2. Fusion and Hybridoma Screening

One mouse (the best responder) is injected on day 43 or later as a final boost. 4 days later, spleen cells are fused with SP2/0 mouse myeloma cells. Hybridomas are assayed 18-20 days later by selected assay (ELISA, FACS, IHC, western blot, etc.). All antibodies are made available as culture supernatants for client screening.

Phase 3. Hybridoma selection and preservation

Client selects antigen specific hybridomas after screening, all the hybridoma cell lines will be grown up and frozen in liquid nitrogen.

Hybridoma Expansion

  • Provided hybridoma cell lines are thawed or expanded from existing vials
  • Cultured in appropriate media to reach desired volume

Antibody Harvesting

  • Antibodies collected from culture supernatant
  • Optional Purification: Affinity purification using Protein A/G available upon request.