Identification of an antigenic determinant of clavulanic acid responsible for IgE‐mediated reactions

Background Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin‐CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis. Methods Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD‐I (N‐protein, 3‐oxopropanamide) and AD‐II (N‐protein, 3‐aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches. Results Two synthetic analogs of AD‐I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV‐treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls. Conclusion We have identified AD‐I as one CLV antigenic determinant, which is the drug fragment that remains protein‐bound. The ability of synthetic determinants of clavulanic acid (CLV) to activate basophils from patients with immediate reactions to this drug is influenced by the chemical structure and their reactivity to proteins. Antigenic determinant‐I (AD‐I) has been identified as the fragment of the clavulanic acid structure that remains linked to the protein, which corresponds to the synthetic analogs (Clav2 and Clav3) able to activate basophils. This synthetic determinant analog to AD‐I, with ability to bind proteins, can activate basophils in a higher proportion of patients compared to the native drug, clavulanic acid.