NOVEL FLOW CYTOMETRY IMMUNOASSAY TO STUDY THE PREVALENCE OF ANTI-PROINSULIN AUTOANTIBODIES IN ARGENTINE CHILD-ADOLESCENT PATIENTS WITH TYPE 1 DIABETES MELLITUS

SABLJIC, ADRIANA V.; BOMBICINO, SILVINA S.; MARFÍA, JUAN IGNACIO; TRABUCCHI, ALDANA; TRIFONE, L.; ROUSSOS A; IACONO, RUBÉN F.; POSKUS, EDGARDO; VALDEZ, SILVINA N.

Buenos Aires

Congreso; Reunión Anual de Sociedades de Biociencia; 2020

Sociedad Argentina de Inmunología

Anti-proinsulin autoantibodies (PAA) are often the first markers that appear in patients with type 1 Diabetes Mellitus (T1DM) and its prevalence varies from 10-60% in child-adolescent patients. The gold standard method for PAA detection is the Radioligand Binding Assay (RBA), a highly specific and sensitive technique, but expensive and polluting; thus, it is imperative to develop an alternative method. The aim of this work was studying the prevalence of PAA in Argentine pediatric patients with T1DM using a novel flow cytometric microsphere-based immunoassay (FloCMIA). Materials and methods: Human proinsulin (PI) was expressed as Thioredoxin fusion protein (TrxPI) in E. coli and a fraction was biotinylated. Sera from 100 normal human controls and 51 T1DM patients -all PAA positive by RBA- were used to optimize FloCMIA. A double paratope model was used in which samples were incubated with TrxPI-biotin and microspheres adsorbed with TrxPI. The immune complexes were revealed using streptavidin-Phycoerythrin. The geometric mean of the signals was analyzed, and the results were expressed in Standard Deviation scores. With the optimized FloCMIA, the prevalence of PAA was evaluated in 60 samples of patients with T1DM (age range 0.1-18 years).Results: The study of ROC curves allowed choosing a cut-off value of 3.7 SDs and the AUC was 0.884, indicating that the method has good ability to distinguish between samples from each group. The specificity of FloCMIA was 97% and the analytical sensitivity 69%, calculated as the percentage of patients RBA positive that were also positive by FloCMIA. There was a substantial agreement between methods (kappa statistic=0.700). A prevalence of 30% for PAA was obtained in the population of T1DM patients studied.Conclusions: An alternative method to RBA was developed with good performance and less operational complexity and environmental impact. The novel assay was implemented in Argentine patients with T1DM to study the prevalence of PAA.