Custom Hybridoma Development and Monoclonal Antibody Production


Leveraging our world-class peptide synthesis expertise and antibody production experience, AnaSpec, EGT Group is pleased to offer high quality custom monoclonal antibody services to the research community and pharmaceutical industry. We have the capabilities to raise monoclonal antibodies to difficult antigens, including peptide sequences that contain post-translational modifications.

• Consultation in peptide antigen design

• Peptide antigens with post-translational modifications can be made

• Flexible protocol and schedule based on customer’s needs

• Immunization time frame of four to eight weeks

• Up-to-date report and customer validation following each project step

• Accessible technical support with expertise in antibody production

• Resources and technical proficiency to work with customer on unconventional projects

Our routine production of monoclonal antibodies is comprised of three phases (Phase I – III). Customers only pay for each phase that is successfully completed. Phase IV, large-scale production of monoclonal antibodies, is optional and available as a stand-alone purchase. Details of each phase are shown below.

Phase I: Antigen Preparation and Immunization (~8-9 weeks)

  • Design and production of synthetic peptide antigens with or without modifications
  • Site-directed conjugation of peptide antigens to carrier proteins (e.g., KLH and BSA) for flexibility in antigen presentation
  • Preparation of peptide and protein antigens with adjuvants for immunization
  • Initial and boost immunizations of at least four animals
  • Serum separation, ELISA testing and customer consultation before proceeding to Phase II

Phase II: Fusion and Selection of Parental Clones (~3 weeks)

  • Splenocytes are harvested for cell fusion from mice with the highest titer
  • Clones of fused cells are expanded and screened by ELISA.
  • Supernatants, from up to ten ELISA-positive parental clones, are shipped to customer for further antibody testing in immunoassays
  • The best parental clones (up to three) are chosen for Phase III after customer consultation

Phase III: Subcloning and selection of Single Clones (~3 weeks)

  • Parental clones are sub-cloned by limiting dilution to isolate single clones
  • Isotype determination of single clones – IgG subtype guaranteed single clones are expanded and screened by ELISA
  • Single clones with high specificity (e.g., phospho vs. non-phospho) and antibody titer will be expanded and cryopreserved
  • Three hybridomas, and their supernatants, will be shipped to customer

Phase IV: Large-Scale Production of Monoclonal Antibodies (optional) (~3-6weeks)

  • In vivo ascites or in vitro Bioreactor antibody production is available
  • Purification of monoclonal antibodies by affinity chromatography using Protein G or with the immunizing peptide antigen
  • ELISA and SDS-PAGE are done to validate the purity of the antibody
  • AnaSpec offers a range of production scales with flexible cost adjustment

In addition to our custom services, AnaSpec, EGT Group offers popular catalog monoclonal antibodies such as the anti-amyloid (1-40) and (1-42) antibodies.

Product

Size

Catalog #

Anti-β-Amyloid (1 - 40), mouse monoclonal NEW

50 µg

55921

Anti-β-Amyloid (1 - 42), mouse monoclonal NEW

50 µg

55922


Figure 1. IHC of brain tissue from APP Tg2576 transgenic mouse probed with mouse anti-β-amyloid (1-40) antibody (Cat# 55921). Paraformaldehyde fixed sections were pretreated with 70% formic acid for 5 minutes followed by steaming for 30 minutes and blocked with normal goat serum (no need for antigen retrieval if fresh or frozen sections are used). Sections were incubated with anti-β-amyloid (1-40) antibody at 0.5 mg/ml overnight. Immunoreactivity was visualized using DAKO Envision HRP System (1:10,000 dilution). This antibody stains cored plaques only. Image courtesy of Mayo Clinic, Florida.


Figure 2. IHC of brain tissue fromAPP Tg2576 transgenic mouse probed with mouse anti-β-amyloid (1-42) antibody (Cat# 55922). Paraformaldehyde fixed sections were pretreated with 70% formic acid for 5 minutes followed by steaming for 30 minutes and blocked with normal goat serum (no need for antigen retrieval if fresh or frozen sections are used). Sections were incubated with anti-β–amyloid (1-42) antibody at 0.05 mg/ml concentration overnight. Immunoreactivity was visualized using DAKO Envision HRP System (1:10,000 dilution). This antibody stains both cored and diffused plaques. Image courtesy of Mayo Clinic, Florida.