In the development of immunoassays for small organic molecules, as well known, hapten design plays a significant role. They have been successfully applied in many fields such as food, medicine, and environmental protection, and have achieved relatively ideal detection results. Haptens are incomplete antigens that do not cause an immune response upon binding because they cannot bind to MHC complexes. Small molecule compound antigens are the most typical haptens. They are usually some organic substances with molecular weight less than 4000 Da, such as most polysaccharides, steroid hormones, fatty amines, lipids, pharmaceutical intermediates, certain small molecular weight drugs, etc.

Unlike antigens, haptens are not immunogenic by themselves. They lack T cell epitopes and cannot directly induce the body to produce corresponding specific antibodies. Therefore, small molecule compounds need to be conjugated with additional molecules to form artificial antigens in order to obtain immunogenicity. The artificial antigens then indirectly induce the proliferation and differentiation of B cells utilizing T cell epitopes, and finally generate specific antibodies. Those additional molecules are called “carriers” and are often proteins that bind tightly to haptens. Although proteins are mostly employed for hapten conjugation, synthetic polypeptides such as Poly-L-glutamic acid, polysaccharides and liposomes can also be used.

Efficient hapten synthesis has been essential to the development of sensitive quantitative and qualitative immunoassays. Many factors affect the yield and quality of artificial antigens, which in turn affect the specific immunity of antibodies to small molecules. In the design of hapten conjugates, consideration must be given to the hapten, the carrier, the coupling strategy, and the hapten density because the amount of hapten attached to the carrier influences the strength of the immune response directed toward the newly created antigenic determinant.

Pharmaceutical drugs are usually small molecules that can be haptens, binding to proteins in the blood. There are two major categories, antibiotics and anesthetics.

※ Antibiotics act as haptens
β-Lactams represent one of the most important groups of antibiotics prescribed for antibacterial treatment today. β-Lactams act as haptens and require conjugation to a carrier molecule to be recognized as a sensitizing molecule and to elicit an allergic response. Through the design and synthesis of β-lactam haptens, there have been many β-lactam antibiotics with different antimicrobial profiles, such as penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems.

Penicillin causes most allergic drug symptoms. The reason is that these drugs are prone to degradation during storage and can lead to the production of benzylpenicilloyl derivatives, which can then bind to other proteins and ultimately elicit an immune response. Cephalosporins, a class of β-lactam antibiotics, also elicit an immune response as a hapten. However, it is not yet clear how cephalosporins become haptens.

※ Anesthetics act as haptens
Halothane was used as an anesthetic around the 1950s. It is metabolized in the body as trifluoroacetyl chloride, which reacts with proteins in the liver to form neoantigens. once the immune response to this neoantigen is elicited then subsequent exposure to halothane can lead to inflammation of the liver through the activation of this immune response.

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