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Pomezia, Italy

Adornetto G.,University of Rome Tor Vergata | Fabiani L.,University of Rome Tor Vergata | Volpe G.,University of Rome Tor Vergata | De Stefano A.,University of Rome Tor Vergata | And 6 more authors.
Analytical and Bioanalytical Chemistry

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95 % clinical sensitivity and 96 % clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age. [Figure not available: see fulltext.] © 2015 Springer-Verlag Berlin Heidelberg. Source

De Stefano A.,University of Rome Tor Vergata | Volpe G.,University of Rome Tor Vergata | Adornetto G.,University of Rome Tor Vergata | Bernardini S.,University of Rome Tor Vergata | And 4 more authors.

This work describes the development of an ELIME (Enzyme-Linked ImmunoMagnetic Electrochemical) assay for a sensitive detection of specific IgE (sIgE) towards the aeroallergens G5 and D2 in serum samples. The system, with a detection limit of 0.1KIU/L, uses magnetic beads as support of the immunological chain and a strip of eight-magnetized screen-printed electrodes, connected to a portable instrument, for a multiplexed detection. The analysis of 46 serum samples performed using both the ELIME and the Immulite 2000 assays, suggests that our method is able to measure very low concentration of sIgE and to discriminate all positive samples from negative ones. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Adornetto G.,University of Rome Tor Vergata | Volpe G.,University of Rome Tor Vergata | De Stefano A.,University of Rome Tor Vergata | Martini S.,Radim SpA | And 4 more authors.
Analytical and Bioanalytical Chemistry

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphataseconjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to "blank" sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r 2 of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory. © Springer-Verlag 2012. Source

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