O18 Novedoso inmunoensayo multiplex para la detección simultánea y discriminativa de los principales marcadores de autoinmunidad en diabetes mellitus

Adriana Victoria Sabljic; Silvia Sonia Bombicino; Juan Ignacio Marfía; Aldana Trabucchi; Liliana Trifone; Adriana Roussos; Rubén Francisco Iacono; Edgardo Poskus; Silvina Valdez

doi:10.47196/diab.v54i3Sup.379

Authors

Adriana Victoria Sabljic University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Silvia Sonia Bombicino University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Juan Ignacio Marfía University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Aldana Trabucchi University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Liliana Trifone Ricardo Gutiérrez Pediatric Hospital, Autonomous City of Buenos Aires, Argentina
Adriana Roussos Ricardo Gutiérrez Pediatric Hospital, Autonomous City of Buenos Aires, Argentina
Rubén Francisco Iacono University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Edgardo Poskus University of Buenos Aires, Autonomous City of Buenos Aires, Argentina
Silvina Valdez University of Buenos Aires, Autonomous City of Buenos Aires, Argentina

DOI:

https://doi.org/10.47196/diab.v54i3Sup.379

Keywords:

multiplex immunoassay, autoimmunity markers, diabetes

Abstract

Introduction: The main autoimmunity markers in Type 1 Diabetes Mellitus (DM1) are those that have specificity towards: glutamate decarboxylase (GADA), insulinomaassociated tyrosine phosphatase 2 (IA-2A) and isoform 8 of the Zinc transporter (ZnT8A). Its detection is essential to support the diagnosis of DM1 and to establish the presence of Latent Autoimmune Diabetes of the Adult (LADA) to guarantee adequate treatment in a timely manner. The reference method for its detection (Radioligand Binding Assay, RBA) is highly sensitive and specific, but expensive, polluting and its execution is limited to centers with infrastructure and personnel authorized by the regulatory entity. Therefore, it is essential to develop alternative methods that facilitate access to diagnosis to a bigger number of patients.

Aim: The aim of this work was to develop an immunoassay based on Flow Cytometry (FC) for the combined and discriminative determination of the main DM1 markers: GADA, IA-2A and ZnT8A.

Materials and methods: 10 sera from pediatric patients with a recent diagnosis of DM1 and 20 sera from normal human controls (NHC) were tested. A double paratope model was used incubating the samples with polystyrene microspheres of 4, 5 and 7 µm adsorbed with the recombinant antigens Trx-GAD, Trx-IA-2 and Trx-ZnT8, respectively, and the corresponding biotinylated proteins. After an overnight incubation at 4 ºC, the immune complexes formed were detected using streptavidin-Phycoerythrin and were acquired on a flow cytometer. All samples were evaluated in parallel by RBA.

Results: Of the 10 patients studied, 8 (80%) were positive for GADA, 10 (100%) for IA2A and 7 (70%) for ZnT8A. The sensitivities relative to RBA were: 100% for GADA and IA-2A and 75% for ZnT8A. The specificities, calculated as 100 minus the percentage of NHC detected as positive, were 100, 95 and 90% for GADA, IA-2A and ZnT8A, respectively.

Conclusions: The preliminary results obtained show the feasibility of using this novel multiplex immunoassay by FC for the simultaneous detection and in a single analytical act of the different autoimmunity markers in DM1, being necessary to increase the number of samples studied to establish the analytical performance of the test. This represents an operational advantage over making each determination individually,
reducing costs, time, and environmental impact.