Design and Synthesis of Haptens for Dibutyl Phthalate

Haptens are small xenobiotic (exogenous) chemical molecules with a low molecular weight that need to be bound to a carrier to be immunogenic. There are numerous haptens in native foods, contaminants, or additives (i.e. dibutyl phthalate, DBP). Hapten design is a key step for the successful preparation of an immunoassay antibody against food additives. Based on the advances of chemical analyses, theoretical studies, and molecular modeling assisted hapten design, Creative Biolabs provides customized services for the design, transformation, and synthesis of food additives haptens, and helps to develop associated anti-hapten antibodies.

Introduction of DBP

DBP is an organic compound commonly used in a range of households, consumer goods, and food packaging. DBP is a phthalate ester that is the diester obtained by the formal condensation of the carboxy groups of phthalic acid with two molecules of butan-1-ol (butyl alcohol). The chemical formula for DBP is C₆H₄(CO₂C₄H₉)₂. Although used extensively as a plasticizer, DBP is a ubiquitous environmental endocrine disruptor, with reproductive toxicity, developmental toxicity, and potential carcinogenic effects. Several analytical methods for DBP detection have been established, amongst, immunoassay has been a useful tool due to its high sensitivity, high throughput, rapid turnaround time, and portability. Therefore, it is urgent to synthesize DBP hapten and discover anti-DBP antibodies to benefit immunoassay development.

Fig.1 Structure of DBP.

Design and Synthesis of DBP Haptens

In designing hapten, it is necessary to retain or introduce a suitable functional group to be coupled with carrier proteins. Because DBP has no functional groups, when used as a hapten, we usually re-design and synthesize the hapten with an amine group so as to directly couple to the carrier for the preparation of artificial antigen. Usually, there are two main DBP-hapten redesigned formats, aminated DBP hapten (DBAP) and carboxylated DBP hapten (DBCP). DBAP is synthesized via esterification of 4-nitrophthalic acid (4-NPA) with butanol followed by reduction with purified zinc dust and concentrated hydrochloric acid. While DBCP is commonly prepared by the reaction of DBAP with succinic anhydride. Then, the modified DBP haptens can be coupled with a carrier protein, such as BSA (bovine serum albumin) or OVA (ovalbumin), by a diazotization method to generate artificial immunogen DBP-BSA or DBP-OVA. The hapten-protein conjugate can be then used as an immunogen for antibody production.

Fig.2 Reactions used to synthesize the aminated DBP hapten (DBAP). (Xu, 2014)

With rich experience and expertise in antibody development, Creative Biolabs has accomplished several cases in designing & preparing DBP (or analogs) haptens, and developing small molecule antibodies with the desired specificity. Our featured services extend from hapten design, modification, and synthesis, to subsequent antibody production, labeling, and characterization.

If you are doing hapten redesign and antibody development for food additive immunoassays, please do not hesitate to contact us for more suggestions and scientific support.

Reference

1. Xu, F.; et al. Indirect competitive enzyme-linked immunosorbent assay for the detection of dibutyl phthalate in white wine, compared with GC-MS. Food analytical methods. 2014, 7(8): 1619-1626.