Anticorpi ricombinanti per la diagnostica agro-alimentare
Author(s)
Catellani, Marcello
Date Issued
February 26, 2010
Type
Doctoral Thesis
Abstract
Mycotoxins are secondary metabolites produced by several fungi belonging mainly to the genera: Aspergillus, Penicillium and Fusarium. In particular, aflatoxin B1 is a common contaminant occuring in a wide range of important raw food commodities. Consumption of mycotoxin-contaminated food or feed eventually leads to teratogenic, cancerogenic, oestrogenic, neurotoxic, and immunosuppresive effects in humans and/or animals.
Mycotoxin contamination may occur in the field before and/or during harvesting, or in the storage and processing. Although the use of pesticides and good agronomic practices may reduce mycotoxin accumulation, eradication is still a major challenge. Therefore, diagnostics remains a fundamental tool to reduce risks associated to the assumption of contaminated food. To this aim, we concentrated our efforts on the isolation and characterization of new diagnostic immuno-reagents such as recombinant antibodies to be expressed in cost-effective systems as an alternative to classical mammalian cell culture system.
Initially, we attempted to isolate antibodies against aflatoxin B1 and ochratoxin A from two different synthetic antibody phage display libraries (F8 and ETH-2-Gold). In principle, this approach could represent a straightforward solution, as the methodology is simple, inexpensive, enabling simultaneous processing of several antigens. Unfortunatly, no antibody specific to these toxins were isolated from both libraries.Therefore, to isolate antibodies at least against aflatoxin B1, Balb/c mice were immunized with the toxin conjugated to the carrier Keyhole Limpet Hemocyanin (KLH), due to the poorly immunogenic size of this molecule (~300 Da). Three different clones were isolated from hybridomas and all derived monoclonal antibodies were able to recognize free aflatoxin B1 in competitive ELISA.
The genes encoding the antibody heavy and light chains were amplified from the cDNA of the three hybridomas using a set of degenerated primers (as described in literature for the gamma chain - Wang et al., 2000 Journal of Immunological Methods 233: 167-177) and designing Kabat sequence-based primers for the lambda chain (Kabat et al., 1991; Sequences of Proteins of Immunological Interest, 5th edit., US Department of Health and Human Services, US Government Printing Office). Light and heavy chain genes were separately cloned in an Agrobacterium-mediated plant expression vector (pBI) and N. benthamiana plants were co-agroinfiltrated with both constructs for each clone (2D2_G1 and 9E11_D5). Western blot analysis of plant extracts revealed high expression levels of antibody chains, while the correct assembly and functionality of murine IgGs was evaluated by ELISA. The antibodies, purified from infiltrated leaves, bind aflatoxin B1 to the same extent as the cognate antibodies purified from murine hybridomas in ELISA.
Moreover, starting from the variable regions sequences of mAb 2D2_G1, two different recombinant formats were engineered: the scFv and the scFv-Fc. The scFv(2D2_G1) was expressed in E. coli and after purification, antibody specificity to aflatoxin B1 was evaluated by ELISA and SPR (Surface Plasmon Resonance) analysis. In parallel, the scFv-Fc format was cloned in an Agrobacterium-mediated plant expression vector (pBI) and N. benthamiana plants infiltrated. Western blot analysis of plant extracts and ELISA confirmed the correct assembly and functionality of this recombinant antibody format.
In conclusion, two different murine monoclonal antibodies against aflatoxin B1 have been generated and corresponding antibody genes cloned and engineered. All recombinant antibody formats expressed in heterologous systems retain the specificity of the original mAbs reinforcing the idea that plants are convenient bioreactors for the production of IgG alternative to classical mammalian cell culture systems, thus opening new horizons for both agricultural and diagnostic applications.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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