Design and Synthesis of Haptens for Ochratoxin A (OTA)

Ochratoxin A (OTA) is one of the most common mycotoxins produced by some species of Aspergillus and Penicillium fungi that often contaminate cereals such as corn and wheat. Chromatography is a preferred analytical approach for OTA determination, however, it’s still complex, expensive, and tedious. As a leading provider in antibody development, Creative Biolabs pays close attention to the current state of anti-hapten antibody research and is committed to mastering the state-of-the-art technologies for antigen production. Notably, our scientists have developed advanced design and synthetic strategies for OTA and other ochratoxins to satisfy clients’ needs.

  • Introduction to OTA
  • OTA is a toxic secondary metabolite of fungal species that can be soluble in alkaline water and organic solvents. The molecule is a pentapeptide biosynthesized from the dihydrocoumarins family coupled to β-phenylalanine. It has shown severe hepatotoxicity and nephrotoxicity to several animal species and can induce toxicological effects, including immune toxicity, genotoxicity, neurotoxicity, and teratogenicity of organisms. The International Agency for Research on Cancer (IARC) has defined OTA as a possible carcinogen (Group 2B) to humans. As an acetate-derived isocoumarin, the structure of OTA displays similarity to another nephrotoxic mycotoxin, citrinin. A methionine (Met)-derived carbon is replaced on the aromatic ring and constitutes an amide bond with the amino group of phenylalanine. Ultimately, the substitution of a chlorine atom converts ochratoxin B (OTB) to OTA and therefore not only protects the nephrotoxin from carboxypeptidases in monogastric digestion, but also seems significant in the expression of its main toxicity, namely inhibition of protein synthesis.

    Chemical structure of OTA. Fig.1 Chemical structure of OTA.

  • Rational Hapten Design Services at Creative Biolabs
  • OTA is structurally composed of a para-chlorophenolic group containing a dihydroisocoumarin moiety-linked to L-phenylalanine. As one of the most frequent mycotoxins, the toxin has two main analogs as OTB and ochratoxin C (OTC). Among them, OTB shares a highly similar structure to OTA with the missing chloridion, and OTC reveals the structural change on the carboxyl group of OTA. Hence, monoclonal antibodies against OTA immunogen normally have better cross-reaction with OTB rather than OTC.

    Like any other small molecule compound, OTA won’t trigger an immune response when treated with model animals. This is to say, OTA can’t be immunogenic by itself. The strategy to produce antibodies to this type of compounds, haptens, consists on the generation of covalent conjugates of the target with an immunogenic macromolecule, for example, a protein. Accordingly, a reactive moiety or activatable chemical group requires to be present in the antigen. In the case of OTA, it holds a carboxyl group that can be easily activated for protein conjugation.

Chemical structures of the synthetic haptens. Fig.2 Chemical structures of the synthetic haptens. (López-Puertollano, 2018)

Our Remarkable Design & Synthesis Strategies

As far as we know, in all published studies reporting the preparation of antibodies to OTA, this mycotoxin itself is covalently linked through its carboxyl group to a protein carrier to form hapten-carrier conjugates for immunization. But this strategy, very likely employed owing to its simplicity, might not be the most inexpensive and convenient from an immunological aspect. At Creative Biolabs, we’re specialized in designing rapid, sensitive haptens and provide cost-effective custom services for OTA antigen development.

Lowest energy conformation of haptens OTAb (a) and OTAe (b). Fig.3 Lowest energy conformation of haptens OTAb (a) and OTAe (b). (López-Puertollano, 2019)

Theoretical calculations have highlighted the great importance of the carboxylic acid in the conformational arrangement of OTA. This chemical group serves as a key antigenic determinant for shaping the binding site during the immunoreaction. Considering these premises, our experts developed haptens with alternative linker tethering sites and the immune response, regarding affinity and specificity, has been assessed in experimental animals. For example, OTAb contains a carboxylated hydrocarbon chain at the C-4 position of the benzene ring. As for OTAd, the carboxylated spacer arm is incorporated at the C-3 position of the dihydroisocoumarin ring. The obtained haptens can further be used to create appropriate conjugates for the generation of monoclonal or polyclonal antibodies to OTA.

OTA is detected worldwide in a variety of food and feed sources and possesses a potential risk to human health. Several methods have been exploited to detect OTA in samples, including mass spectrometry (MS) and high-performance liquid chromatography (HPLC). These methods are reliable but also have several drawbacks. Recently, monoclonal antibodies have been widely applied in the identification of foodborne pathogens because of their lower cost, simplicity, selectivity, and adaptability. With a high reputation in hapten development, Creative Biolabs offers custom services to help clients design and synthesize ideal OTA haptens, as well as produce corresponding antibodies, both monoclonal and polyclonal, specific to this target. If you’re interested in our services, please don’t hesitate to contact us or directly send us an inquiry.

References

  1. López-Puertollano, D.; et al. Novel haptens and monoclonal antibodies with subnanomolar affinity for a classical analytical target, ochratoxin A. Sci Rep. 2018, 8(1): 9761.
  2. López-Puertollano, D.; et al. Study of epitope imprinting for small templates: preparation of nanoMIPs for ochratoxin A. Chem Nano Mat. 2019.
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