Design and Synthesis of Haptens for Melamine

Melamine is a high nitrogen compound that may be illegally added to inflate the apparent protein content of food products. The adulteration with melamine has led to an urgent requirement for sensitive, specific, rapid, and reliable quantitative/screening methods. Based on rational hapten modification and antibody development for melamine, the immunoassay detection of melamine in milk, milk powder, and feed samples has been increasingly prosperous. To promote the development of screening methods for melamine analysis, Creative Biolabs provides comprehensive hapten services, including but not limited to melamine hapten design, modification, and synthesis, as well as subsequent antibody discovery, labeling, and characterization.

Background of Melamine

Melamine (2,4,6-Triamino-1,3,5-triazine, C3H6N6) is a colorless crystalline substance belonging to the family of heterocyclic organic compounds. The high nitrogen content of melamine has led to its being used as a non-protein nitrogen additive artificially and illegally. In addition, melamine has been shown to cause a variety of toxic effects, including nephrolithiasis, chronic kidney inflammation, and bladder carcinoma, etc. Therefore, the determination of melamine is of biological, clinical, and food industry importance. Driven by this, various methods have been developed for melamine detection, amongst, immunoassays are the most widely used method for food and environmental analyses. Melamine immunoassay is mainly based on the specific interaction between an antibody and a corresponding melamine hapten antigen. So it is important to design and synthesize melamine hapten and discover anti-melamine antibodies for highly sensitive and specific melamine detection.

Structure of melamine. Fig.1 Structure of melamine.

Design and Synthesis of Melamine Hapten

Because the amino groups on the triazine ring having the same reactivity, the direct use of melamine itself as a reactant to derive a hapten may lead to extremely complicated hapten products with one, two, or all three amino be substituted. Therefore, 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) was chosen to produce hapten because its active chloride group is capable of reacting relatively easily with the aliphatic primary amino or the sulphydryl group of a spacer arm reagent such as 6-aminocaproic acid or 3-mercaptopropionic acid, respectively. In terms of this, scientists use CAAT to react with 6-aminocaproic acid or 3-mercaptopropionic acid to produce two haptens of melamine: hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. These two melamine haptens introduce spacer arms of different lengths and structures on the triazine ring of the CAAT.

Synthesis of melamine haptens. Fig.2 Synthesis of melamine haptens. (Lei, 2010)

Preparation of Hapten-protein Conjugates as Immunogens

To be immunogenic, the synthesized haptens 1 and 2 are usually coupled with a carrier protein, such as BSA (bovine serum albumin) or OVA (ovalbumin) using the active ester method. The structures of these re-designed haptens can be identified by nuclear magnetic resonance spectrometry, mass spectrometry, and infrared spectrometry.

Production of anti-Melamine Hapten Antibodies

The monoclonal antibody was produced by hybridoma technology. Usually, BALB/c mice were immunized with the synthesized immunogens and the positive hybridomas were subcloned for monoclonal antibody production. The specificity of the produced monoclonal antibody was calculated by cross-reactivity (CR) experiments using several compounds structurally related to melamine as competitors.

Featured Services at Creative Biolabs

Anti-hapten antibodies play important roles in food safety and environmental monitoring. As a specialist in small molecule antibody development, Creative Biolabs offers custom services for hapten molecules design, modification, and synthesis, as well as anti-hapten antibody development.

  • Hapten Design
  • Based on the immuno-chemical properties of the target molecule (chemical structure and composition, immunogenicity, competitors), we choose the appropriate active group, linker site, carrier protein, conjugation ratio, conjugation protocol, etc. to re-design the hapten.

  • Hapten Modification
  • Introducing active groups of hapten derivatives, such as -COOH, -NH, -OH, etc. Our services extend from simple modification of a portion of the existing hapten to total de novo synthesis of the entire modified hapten.

  • Antibody Production
  • Immunization with the designed hapten-carrier conjugates to either produce monoclonal or polyclonal antibodies, that are tailored for research or industrial applications, ensuring high reproducibility, availability, and scalability.

If you are interested in hapten services for melamine, please do not hesitate to contact us for more suggestions and scientific support.


  1. Lei, H.; et al. Hapten synthesis and antibody production for the development of a melamine immunoassay. Analytica chimica acta. 2010, 665(1): 84-90.
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