A recombinant approach to enable specificity and reproducibility in antibody-based applications

Download the PDF

Recombinant antibodies are becoming the affinity reagent of choice for use in various laboratory and clinical research applications due to consistency in performance across lots and the ability to express indefinitely in well-defined mammalian expression systems [1–3]. They can be modified at the genetic level for better affinity, specificity, and function, and are typically derived directly from a highly diverse B cell population versus traditional hybridomas that rely on random fusion. Recombinant antibodies also offer the added advantage of being environmentally friendly. 

We use a proprietary Invitrogen ABfinity technology for generation of rabbit recombinant monoclonal antibodies to realize these benefits (Figure 1) combined with a robust development process to generate high-quality proteinbinding reagents.

ABfinity recombinant antibody development process
Figure 1. ABfinity recombinant antibody development process.

Our selection process involves phenotypic screening of antibodies at multiple levels during their development in the relevant assays of interest for specific targets, thereby generating high confidence in sensitivity and specificity of the selected candidates. To support the same confidence for the end-user, the ABfinity recombinant monoclonal antibodies are backed by a performance guarantee* across lots in various applications (Figure 2).

Western blot analysis using three different lots of Invitrogen
Figure 2. Western blot analysis using three different lots of Invitrogen ABfinity Anti-ATG4B Recombinant Rabbit Monoclonal Antibody (0.5–1 μg/mL). The analysis was performed on whole cell extracts (30 μg lysate) of Jurkat (lane 1), PC-3 (lane 2), and MCF-7 cells (lane 3), detected by chemiluminescence using Invitrogen Superclonal goat anti–rabbit IgG (H+L) secondary antibody, HRP conjugate (0.4 μg/mL, 1:2500 dilution), clearly demonstrating lot-to-lot consistency in detection of ATG4B antibody.

ABfinity technology is based on capturing the IgG light chain and heavy chain transcripts from thousands of B cells screened in immunoassays. Therefore, one development cycle yields a rich collection of monoclonal antibodies with a range of sensitivity and diversity in epitope recognition. In addition, the cDNAs of these recombinant antibodies can be accessed even after several years post-development, providing researchers with an option to customize monoclonal antibodies based on assay requirements including immunofluorescence (IF), immunohistochemistry (IHC), western blot, immunoprecipitation (IP), chromatin immunoprecipitation (ChIP), or as capture-detector pairs in sandwich ELISAs. The ABfinity platform is especially powerful for generating antibodies against posttranslational modifications such as phosphorylation, acetylation, and methylation since the process yields the robustness of a polyclonal antibody but with the specificity of a monoclonal antibody (Figure 3).

Currently, rabbits are used as host animals for the ABfinity platform due to an intrinsic set of advantages [4,5]. Because rabbits have one IgG isotype, there is uniformity in recombinant antibody amplification, development, expression, and purification. This also makes it simpler for applying sequence-based modifications relevant to humanization or developing conjugates using various conjugation chemistries. Rabbits have been shown to be more immune-responsive when compared to mice, leading to a higher probability of isolating antibodies to diverse antigens [5]. We envision that in the long term, these features would be highly advantageous for the use of ABfinity recombinant monoclonal antibodies as detection reagents in research and immunodiagnostics over currently available hybridomas and polyclonal antibodies, where variability in lots or genetic drift warrants optimization and hinders data reproducibility.

abfinity antibodies robustnes polyclonal specificity monoclonal antibody
performance of anti-JAK monoclonal antibodies in western blot and fluorescence imaging
Figure 3. ABfinity antibodies: robustness of a polyclonal and specificity of a monoclonal antibody. Using the ABfinity process, we can generate specific antibodies to posttranslational modifications. Here we have generated an antibody specific to the JAK protein, phosphorylated at tyrosines 1007 and 1008. Specificity is shown by competition with peptide and treatment with MG132 + IFN-γ, which leads to localization of phosphorylated JAK in the cytoplasm.

References

  1. Bradbury A,  Plückthun A (2015) Reproducibility: standardize antibodies used in research. Nature 518(7537):27–29.
  2. Bradbury AM, Plückthun A (2015) Antibodies: validate recombinants once. Nature 520(7547):295.
  3. Karu AE, Bell CW, Chin TE (1995) Recombinant antibody technology. ILAR J 37(3):132–141.
  4. Seeber S, Ros F, Thorey I et al. (2014) A robust high-throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood. PLoS One. 9(2):e86184.
  5. Bystryn JC, Jacobsen JS, Liu P et al. (1982) Comparison of cell-surface human melanoma-associated antigens identified by rabbit and murine antibodies. Hybridoma. 1(4):465–472.

For Research Use Only. Not for use in diagnostic procedures.