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Enzyme Conjugation with Antibodies
Well established practices of antibody-enzyme conjugates has already been applied in enzyme linked immunosorbent assays (ELISAs), western blotting, immunostaining, and biosensors. For ELISA, there are two main types of enzymes which are commonly used to conjugate with secondary antibodies, namely horseradish peroxidase (HRP) and alkaline phosphatase (AP).
Recently, new antibody-enzyme conjugates (AEC) are also developed as therapeutic agents, presenting precise targeting and high activities that are not matched by traditional antibody-drug conjugates (ADC). Usually, to achieve maximum extravasation and penetration of the target cell, therapeutic antibody-enzyme product is required to be primarily 1:1 antibody/enzyme conjugate. In some cases, the conjugation site needs to be controlled as well.
Antibody-enzyme conjugates are generated by inducing covalent bonds between enzyme and antibody via chemical crosslinkers, enzyme catalysis or fusion proteins. Chemical crosslinking methodologies take advantage of the enormous diversity of available crosslinking reagents that differ in chemical functionality, reactivity, and size. Among them, glutaraldehyde, maleimide groups and aldehyde represent most commonly used crosslinking reagents. Also, fusion proteins are produced, especially for use in directed enzyme prodrug therapy (ADEPT). For example, Ojima-Kato et al. recently reported the development of “Zipbody”, a Fab derivative with leucine zipper (LZ) pair at the C-termini of heavy chain (Hc) and light chain (Lc) of Fab that enhances the association and correct pairing of the Hc and Lc.
Creative Enzymes provides services of antibody-enzyme conjugates generation, including design, preparation, purification and characterization. We are focused on method develop,ent to achieve high yields with required bioactivity and controlled antibody/enzyme ratio. With advanced techniques and instruments, we provide purified conjugates free of residual enzymes and antibodies:
- Antibody fragments production.
- Antibody-enzyme conjugates preparation.
- Fusion protein strategies.
- Purification of conjugates.
- Conjugates validation by mass spectrometry.
References:
- Melton, R.G. (1996) Preparation and purification of antibody-enzyme conjugates for therapeutic applications. Advanced Drug Delivery Reviews. 22(3): 289-301.
- Heck, T., Faccio, G., Richter, M., Thöny-Meyer, L. (2013) Enzyme-catalyzed protein crosslinking. Applied Microbiology and Biotechnology. 97(2): 461-475.
- Xiao, H., Woods, E. C., Vukojicic, P., Bertozzi, C. R. (2016) Precision glycocalyx editing as a strategy for cancer immunotherapy. Proceedings of the National Academy of Sciences. 113(37): 10304-10309.
Related sections
Enzyme Engineering and Modification
Figure 1 Zipbody, a type of antibody-enzyme fusion protein, is used to conjugate luciferase (Luc) or green fluorescent protein (GFP).
(Journal of Bioscience and Bioengineering 2018)