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

12 p.-6 fig.-1 graf. abst.
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 present study has been supported by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF): PI12/02529, PI15/01206, CP15/00103, RETICs RIRAAF RD12/0013/0001, RD12/0013/0003 and RD12/0013/0008, RETIC ARADYAL RD16/0006/0001, RD16/0006/0012 and RD16/0006/0021, Biobank Network RD09/0076/00112, and Biobank platform PT13/0010/0006) and by State Secretariat for Research, Development and Innovation of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF), “Una manera de hacer Europa”: MINECO SAF2012-36519, SAF2015-68590R, CTQ2013-41339P, and CTQ2016-75870P). Andalusian Regional Ministry of Economy and Knowledge (grants cofunded by European Regional Development Fund (ERDF), “Andalucía se mueve con Europa”: CTS-06603); Andalusian Regional Ministry Health (grants: PI-0699-2011, PI-0159-2013, PI-0179-2014, PI-0241-2016, and PI-0250-2016), Merck-Serono Research Grant from Fundación Salud 2000 and “Premio UNICAJA a la innovación en biomedicina y salud.” CM holds a “Nicolas Monardes” research contract by Andalusian Regional Ministry Health: C-0044-2012 SAS 2013. MIM holds a “Miguel Servet I” and GB holds a “Rio Hortega,” both research contracts by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Social Fund (ESF)): CP15/00103 and CM16/00067. TDF hold a “Ramon y Cajal” research contract from Ministry of Economy and Competitiveness (RYC-2013-13138).