SEAL™: better technology, better antibodies



SEAL™ overview

Surface Epitope Antibody Library is a proprietary technology developed at Abmart for generating a library of high affinity monoclonal antibodies against a library of antigens representative of a protein surface. Both linear peptides and structural epitopes on the protein surface are targeted. A tyical SEAL™ library
  • consists of 6-12 high affinity monoclonal antibodies
  • specifcally targets 4-8 independent protein surface epitopes.
  • greatly increases the chance to discover successful antibodies compared to traditional methods in which antibodies only target 1-3 epitopes against short peptides or soluble protein antigens.
Based on application results from >100 SEAL™ libraries generated for membrane proteins, soluble factors, transcription factors and kinases, on average, at least 2 times more successful antibodies for Western and IP experiments are discovered from SEAL™ antibodies than from antibodies generated by conventional methods. For difficult targets such as membrane receptors and transcription factors, SEAL™ antibodies are 3-4 times more likely to be successful. This is not surprising since the numbers of independent antibodies and epitopes targeted are multiple times larger in SEAL™ antibodies and for low abundance proteins, this seems to be an even more critical factor for generating successful antibodies. SEAL™ antibody library generation has significant advantages over traditional monoclonal and polyclonal antibody generation in time, cost, and most importantly, the quality of antibodies.

SEAL™ library generation

A proprietary computational model is used to identify ten-residue-long stretches that are exposed on the protein surface. By combining secondary structure prediction, phylogenetic analysis and structural modeling, this method strikes a 73% accuracy (67% sensitivity and 82% specificity) in identifying exposed linear peptide epitopes (SEAL™ epitopes).
Synthetic genes encoding multiple SEAL™ epitopes are inserted into a proprietary DNA vector consisting of Immunogenicity Enhancement Factors (IEF, for example, multiple T epitopes) and DNA sequences that will result in particulate, highly immunogenic recombinant proteins that sample the surface of the target protein when expressed in E.Coli. 5

Structural domains are produced as soluble proteins in E.Coli or mammalian cells in parallel to linear SEAL™ epitopes. A proprietary bacterial expression/secretion system is used to produce soluble proteins in the culture medium.

Mice immunized with structural immunogens will produce antibodies that may recognize a few dominant conformational epitopes of the target proteins8. This type of antibody is often more successful for applications such as IP, flow cytometry or functional studies.

Mice immunized with IEF-SEAL™ epitope antigens using a proprietary, optimized immunization with only a small quantity of antigens produce high affinity, IgG immune responses within three weeks6, 7. Spleen cells of the sacrificed mice are fused with SP2/0 cells and screened with quantitative competitive ELISA to select hybridoma cell lines that secrete antibodies that specifically recognize epitope sequences with at least nano molar affinity. Selected hybridomas are then subjected to multiple rounds of sub-cloning to establish stable cell lines.

SEAL™ library characteristics

  • An antigen library consisting of 6 — 9 surface peptide and soluble protein antigens representative of a protein is constructed for immunization.
  • A monoclonal antibody library made from an antigen library typically consists of tens, up to a hundred, protein-specific antibodies.
  • >70% of the input antigens successfully produce antibodies.
  • >70% of the peptide antigens have ≥2 unique antibody responses and therefore likely consist of ≥2 independent epitopes.
  • Antibody-binding epitopes, usually in the range of 4 — 8, include a diverse set of linear epitopes, as well as conformational epitopes.
  • >95% one-pass project success rate for antibody library generation.
  • 98 days of development time from protein sequence antibody library delivery.
  • Antigens of mouse self-proteins can generate strong antibody responses and epitope-specific monoclonal hybridoma cell lines.

SEAL™ is a general approach to produce a large library of monoclonal antibodies to target multiple, independent protein surface epitopes. SEAL™ is fast, with consistently high success rate of antibody generation and at a fraction of cost of current approaches. The multi-antigen strategy, coupled with fundamental methodology improvements in antigen production, immunization and library screening, have transformed laborious and unpredictable monoclonal research process into a robust and industrialized production process. SEAL™ will dramatically change today’s monoclonal antibody generation practices and will bring customers unprecedented savings in time and cost, and more importantly, successes in discovering valuable monoclonal antibodies.

 

Key references

1. Appel J.R., et al. Elucidation of discontinuous linear determinants in peptides. J. Immuonology. 144, 976–983 (1990)
2. Thornton, J.M., et al. Location of ‘continuous’ antigenic determinants in the protruding regions of proteins. EMBO J. 5, 409–413 (1986)
3. Radivojac, P., et al. Intrinsic Disorder and Functional Proteomics. Biophysical Journal 92, 1439–1456 (2007)
4. Jones DT. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195-202 (1999)
5. Ludwig C and Wagner R. Virus-like particles—universal molecular toolboxes. Current Opinion in Biotechnology. 18, 537–545 (2007)
6. Kenny JS, et al. Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J Immunol Methods. 121, 157–66 (1989)
7. Caterson, B., et al. Identification of a Monoclonal Antibody That Specifically Recognizes Corneal and Skeletal Keratan Sulfate. JBC, 258, 8848–8854 (1983)
8. Miller, A., et al. The primary and secondary antibody response to lysozyme: Each show distinctive idiotypy and restricted epitope recognition. In The Immune Response to Structurally Defined Proteins: The Lysozyme Model. Smith-Gill and E. Sercarz, eds. Adenine Press, New York, p. 341 (1989)