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Development of sophisticated dendrimeric nanostructural materials with potential applications in drug allergy diagnosis: Towards immunoassays and cellular tests (basophil activation tests)

Final Report Summary - DENDRIMMUNEASSAYS (Development of sophisticated dendrimeric nanostructural materials with potential applications in drug allergy diagnosis: Towards immunoassays and cellular tests (basophil activation tests).)

Project objectives
The goal of Dendrimmuneassays is to understand the mechanisms by which different model drugs are able to induce allergic reactions. This will help us to know how to detect and quantify the specific Immunoglobulin E (IgE) produced in these allergic reactions. The proposal mainly focuses on the use of hapten-dendrimer structures for the design of versatile materials with applications to the in vitro diagnosis of allergy to different drugs. In order to do that, we proposed four specific objectives:


Aim 1. Identification of the antigenic determinants of different drug models.
Antigenic determinants are the structures derived from drugs that after binding to proteins are able to induce the production of IgE.
Work performed: Several structures corresponding to different proposed antigenic determinants of clavulanic acid, aminocephalosporins (cefaclor and cephadroxil) and diclofenac have been synthesized, purified and chemically characterized. To study their IgE molecular recognition, these structures are being clinically evaluated using sera of patients by radioimmunoassay (RIA) and basophile activation tests (BAT). Other studies involving the stability of penicillin antigenic determinant structures and their implications in diagnosis were carried out.

Aim 2. Design and synthesis of dendritic molecules displaying multiple presentations of relevant drug antigenic determinants: hapten-dendrimer conjugates.
Work performed: The synthesized determinants of clavulanic acid have been coupled to the periphery of several carrier molecules: dendrimers, polymers and several proteins. Other well-identified antigenic determinants as those of benzylpenicillin (Benzylpenicilloyl, BPO) and Amoxicillin (Amoxicilloyl, AXO) have been attached to dendrimers, by the inclusion of one penicillin determinant or two different determinants at the same carrier molecules, yielding either monohaptenic or bihaptenic Dendrimeric Antigens. These hapten-carrier conjugates have been evaluated by RIA using sera from patients to these drugs. On the other hand, the formation of real hapten-carrier conjugates with amoxicillin in sera was studied by the use of a biotinilated amoxicillin.

Aim 3. Development of methodologies to anchor antigenic determinants and/or nanoconjugates to solid phases.
Work performed: Cellulose and silica particles surfaces have been appropriately functionalized to be employed as solid phases. The antigenic determinants and/or the hapten-carrier conjugates developed in previous objectives were anchored to these solid phases. The resulting materials have been clinically evaluated by RIA employing sera of patients allergic to these drugs and tolerant controls.

Aim 4. Study of the optimal distance in crosslink-IgE to produce the basophil activation.
Work performed: Different architectures consisting of silica particles (micro or nanoparticles) decorated with antigenic determinants of amoxicillin and with amoxicillin-dendrimer conjugates have been prepared to yield homogeny particles of different sizes. These have been chemically characterized and clinically evaluated through basophil activation test (BAT) employing blood samples of patients allergic to amoxicillin and tolerant controls.

Main Results and Potential Impact and Use:

A number of synthetic antigenic determinants of betalactams and diclofenac have been evaluated for IgE recognition for first time. This is providing relevant information about the structure recognized by specific IgE antibodies, which is difficult to investigate using other approaches. The structures that demonstrated to be recognized by IgE antibodies are likely responsible of the induced allergy. Therefore these could be included in drug allergy diagnostic tests. Moreover, the study of their stability has shown important implications in their use in diagnostic tests.
Novel bi-epitope Dendrimeric Antigens including two kind of haptens (Benzylpenicillin and Amoxicillin) in the same carrier molecule have showed important outcomes: (i) a deep study of the bidirectional biomolecular recognition behavior of carriers possessing two different epitopes in the context of immune system interactions, which had never been studied before; (ii) information about the structural chemical requirements to develop a universal in vitro test, being the described mixed-dendrimers potential candidates to progress in allergy diagnosis to betalactams.
New candidate proteins in sera for amoxicillin haptenization have been identified. This methodology is a good approach for identifying haptenated proteins and studying real conjugates that can be included in diagnostic tests.
Regarding the development of new solid phases, silica particles have been successfully used for the in vitro diagnosis of allergy to Amoxicillin. Dendrimeric silica particles containing the antigenic determinant of amoxicillin (AXO) proved effective in detecting and quantifying IgE in sera from patients allergic to amoxicillin, in a specific and selective way. This new material is thus a promising candidate for improving in vitro clinical diagnostic practice. These results have been submitted to be patented. Other solid phases consist of cellulose surfaces functionalized with hapten-carrier conjugates including two different haptens (amoxicillin and benzylpenicillin). These materials proved to be helpful to recognize IgE antibodies against either of these determinants and useful for screening sera with different specificities.
With regard to the study of the optimal distance in crosslink-IgE, silica nanoparticles functionalized with conjugates have been employed for studying architectures consisting of two or more hapten-dendrimeric conjugates separated each other with a fixed distance. Preliminary results indicate that the size of the nanoparticles and distance between conjugates are relevant for the stimulation of the basophil. The use of these nano/microstructures will have a potential impact in the increase of the sensitivity of BAT employed for the diagnosis of allergic patients.
All these results are improving existing diagnostic tests for drug allergy, and have open new research questions that would need to be study with further projects.