Design and Synthesis of Class-specific Haptens for Aflatoxins

Aflatoxins are a group of highly toxic and carcinogenic compounds produced by Aspergillus flavus and Aspergillus parasiticus. The main species include aflatoxins B1, B2, G1, G2, and M1. These aflatoxins can seriously endanger the storage safety of food and threaten human health. However, the analysis of aflatoxin is a difficult task, and methods such as enzyme-linked immunosorbent assay (ELISA) are currently very suitable for rapid routine diagnostic applications of aflatoxin detection. The use of these aflatoxin immunochemical analysis procedures requires sufficient and cheap antibodies. Creative Biolabs provides haptens design and synthesis services for aflatoxin to help customers ensure the production process of antibodies.

Reaction Scheme of AFB1-CMO Hapten Synthesis. Fig.1 Reaction Scheme of AFB1-CMO Hapten Synthesis. (Wicaksono, 2015)

Why Haptens are Needed?

Aflatoxin is one of the main causes of agricultural product contamination and has high carcinogenicity. Until now, various test methods have been developed for the detection of aflatoxin, such as high-performance liquid chromatography (HPLC) or liquid chromatography tandem mass spectrometry (LC-MS). Although these methods have high sensitivity and specificity, they require special expertise, long time analysis and very expensive instruments, thus cannot be widely used. Immunoassays and related immunochemical analysis procedures provide many advantages for the rapid and easy screening of aflatoxins, they are highly dependent on the quality of their detection agents, namely antibodies. Therefore, the ability to generate sensitive and specific antibodies against aflatoxin has become very important.

Aflatoxins are mostly low molecular weight non-protein compounds. Therefore, when it enters the living system, it is not recognized as an antigen and therefore does not produce antibodies. To be able to act as an antigen, it needs to be bound to a larger protein. However, since aflatoxins generally do not have reactive groups that can be coupled to proteins, we usually design a linker to help build the hapten.

Class-specific Hapten Aflatoxins Design and Synthesis Service

To help the customer achieve stable and efficient antibody production, Creative Biolabs provides class-specific haptens design and synthesis services for aflatoxins. Structurally, aflatoxins are difuranocournarins/difurocoumarins synthesized via the polyketide pathway, and they consist of a coumarin nucleus to which are attached a difuran moiety on one side and either a pentene ring or a six-membered lactone ring on the other side. We used the carbodiimide method and carboxymethyl hydroxylamine hemihydrochloride (CMO) as a linker to couple aflatoxin molecules with bovine serum albumin (BSA) to synthesize haptens. CMO has three atoms as long arm space, and can generally retain specific groups of aflatoxin, so it has high antigenicity.

Many AF antibodies immunized with AFB1 conjugates showed cross-reactivity with AFB2, AFG1 and AFG2. We observed that almost all of these antibodies had the lowest cross-reactivity with AFG2. Therefore, it is speculated that antibodies with high affinity to AFG2 are most likely to become AF-specific antibodies. Using AFG2 at 10 ng/ml as a competitor to screen for antibodies with high affinity to AFG2 can increase the likelihood of obtaining class-specific antibodies against AF because aflatoxin B1, B2, G1 and G2 have only the same moiety as the common of aflatoxin G2 and the immunizing hapten. Aflatoxin G2 and immune haptens are common. There are three types of specific monoclonal antibodies: 8E11, 10C9 and 8F6, and their ELISA results prove that they exhibit a high degree of inhibitory effect on AFG2.

If you want to inquire more about our class-specific aflatoxins hapten design and synthesis services, please contact us.

Reference

  1. Wicaksono, W, P.; et al. Synthesis of Polyclonal Antibodies against Aflatoxin B1[J]. Makara Journal of Science. 2015: 91-95.
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