Moreover, conjugation of CLV with human serum albumin (HSA) was studied by liquid chromatography mass-spectrometry (LC/MS-MS) and a biotinylated derivative of CLV was used as tool for identification of serum proteins target of modification by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) and peptide mass fingerprinting. By the other hand, we pursued the amplification of immunoassays detection signal. With this objective, fluorescent dendrons allowing site-specific conjugation to biomolecules and bearing 1, 2 or 8 fluorescent units were designed and synthesized, and their suitability for labeling antibodies and fluorescence amplification capacity evaluated [6].
The main conclusions of this work have been that synthesized cyclized structures derived from cefaclor and cefadroxil (Cef1 and Cef2, respectively) are specifically recognised by sIgE from patients allergic to the corresponding cephalosporin or to penicillins containing the same R1 side chain, shedding light into the mechanism involved in allergy to α-aminocephalosporins and complementing studies previously reported [7, 8]. Also, the ability of our synthetic determinants of CLV to activate basophils from patients with immediate reactions to this drug has been demonstrated to be influenced by the chemical structure and their reactivity to proteins. Clav2, coming from AD-I, activates basophils in a higher proportion of patients compared to CLV itself, and thus represents a promising structure to improve the sensitivity of in vitro diagnosis. Furthermore, a 70 Da mass structure was found to modify Lys 195 and Lys 475 of HSA, being this mass compatible with that of AD-I, which confirms that Clav2 is part of the drug-protein conjugate. Moreover, a water soluble biotinylated derivative of CLV (CLV-TEG-B) showed concentration-dependent protein haptenation capacity in vitro, and made possible the identification of HSA, haptoglobin and heavy and light chains of immunoglobulins as potential serum proteins target of modification by CLV. Finally, synthesized fluorescent dendrons G0-Cy5, G1-Cy5 and G3-Cy5 showed an increment in fluorescence with the number of fluorescent units and they were proved to be suitable for site-specific labeling of a model antibody. The labeling with G1-Cy5 dendron (2 fluorescent units) resulted to be the most suitable for immunoassays signal amplification applications, since it was found to enhance fluorescence by one order of magnitude when compared with the antibody labeled with the monovalent probe G0-Cy5 [6].
Fecha de lectura Tesis Doctoral: 30 octubre 2018.Drug hypersensitivity reactions are a significant public health problem with important consequences on patient health and healthcare costs. It has been reported that only a low percentage of initial cases suspected of allergy to antibiotics are finally confirmed [1, 2], and betalactam antibiotics (BLs) are the drugs most frequently involved [3, 4]. In vivo tests (skin test and drug provocation test) are often the first and only option for diagnosis, nevertheless they could be risky for patients. Thus, in vitro tests are a more convenient and safer alternative for diagnosis, however, a sensitive and specific detection of specific IgE (sIgE), crucial for in vitro allergy diagnosis, is difficult to achieve in cases of allergy to drugs due to the extremely low sIgE concentration present in patients serum [5].
The sensitivity of in vitro tests for diagnosing allergy to BLs depends, among other factors, on: (i) the similarity between the structure used in the assay as emulator of the antigenic determinant (AD) formed in vivo and the structure actually formed after the BL intake, which is related to the mechanisms involved in the allergic process, and (ii) the intensity of the detection signal (radioactivity, enzimatic proccess or fluorescence) at low concentrations of sIgE to drugs.
The general objective of this thesis is to carry out studies directed to improve current in vitro tests for diagnosing immediate allergic reactions to BLs. More specifically, by one hand, we aimed to research, from a chemical approach, the structures recognized by sIgE for cephalosporins and clavulanic acid (CLV) since there has been an increase of cases of allergy to these BLs in the last years, due to the change in prescription patterns [1]. For this purpose, structures derived from these BLs were designed and synthesized and their immunological evaluation performed using RadioAllergoSorbent Test (RAST) or Basophil Activation Test (BAT)
