Novel enzyme-linked immunosorbent assay for bivalent ZnT8 autoantibodies

Abstract

Autoantibodies to zinc transporter 8 (ZnT8A) is a powerful diagnostic or predictive marker in type 1 diabetes. However, the widely used current ZnT8A radioligand binding assay (RBA) has proved to be difficult for many laboratories to implement. The aim of this study was the development and characterization of the performance of a novel fluid phase ZnT8A enzyme-linked immunosorbent assay (ELISA) in relation to standard radioligand binding assay (RBA) in type 1 diabetes. Sera from 114 patients with type 1 diabetes and 140 blinded Islet Autoantibody Standardization Program (IASP2012) samples were studied. The sensitivity of ELISA-ZnT8A is equivalent or slightly higher than conventional RBA with similar specificity. Furthermore, the median SD score using this ELISA was significantly higher than that obtained with RBA (P<0.0001). Multiple logistic regression analysis revealed that ELISA-ZnT8A positivity was associated with younger age of onset (≤ 20 years; OR 15.91, P=0.0002), acute-onset form of type 1 diabetes (OR 3.38, P=0.019), and the presence of IA-2 autoantibodies (OR 3.75, P=0.014). Furthermore, the levels of ELISA-ZnT8A were associated with the reactivity to ZnT8-325Arg, but not ZnT8-325Trp. We conclude that this nonradioactive bivalent ZnT8A assay has high performance and should facilitate large-scale autoantibody screening.Moreover, these results suggest that the humoral autoimmunity against ZnT8 is related to a high risk of faster development of type 1 diabetes and the ZnT8A levels are associated with the known aa325 variants.

Zinc transporter 8 autoantibodies complement glutamic acid decarboxylase and insulinoma‐associated antigen‐2 autoantibodies in the identification and characterization of Japanese type 1 diabetes

“…As the bridging‐type ELISA for ZnT8A used in the present study would be easy to implement and has proven to achieve a high degree of sensitivity and specificity in the recent proficiency evaluations by the Islet Autoantibody Standardization Program, this assay should be deemed a useful diagnostic method for determining type 1 diabetes. Although the provided cut‐off value for this kit (ElisaRSR™ ZnT8 Ab) by the manufacturer is <15 U/mL 6 , we have established a distinctive cut‐off value of <10 U/mL for Japanese populations based on the 99th percentile and 3SD above the mean value obtained through the 288 healthy Japanese control participants. Although the exact reasons for the cut‐off difference between Japanese people and white people are unknown, one possibility might be attributed to the effect of ethnicity.…”

“…ZnT8A, GADA and IA‐2A were determined using bivalent ELISA kits (RSR Ltd., Cardiff, UK) using biotinylated ZnT8, GAD65 and IA‐2, respectively, as described previously 6,11,12 . All of these determinations were based on the sandwich type principle with dimeric carboxy‐terminal domains of ZnT8 (aa 275–369) carrying either 325Trp or 325Arg, full‐length GAD65 and intracellular domain of IA‐2 (aa 604–979) as antigens.…”

“…In a recent report carried out by our team, we detailed the development and the characterization of bridging‐type enzyme‐linked immunosorbent assay (ELISA) with dimeric carboxy‐terminal ZnT8 domains carrying either 325Trp or 325Arg as antigens 6 . Although this assay achieved high sensitivity and specificity in the Islet Autoantibody Standardization Program or Diabetes Antibody Standardization Program 7 , its clinical significance in the Japanese populations remains ambiguous, as the serum samples used in these programs are derived from white people.…”

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

“…To ensure the diversity of the screened scFv, we chose ZnT8 N/C fusion protein for bio-panning. Since previous studies showed that principal epitopes targeted by ZnT8A were influenced by a single amino acid at position 325 encoded as arginine or tryptophan in T1D, the ZnT8 C terminal dimer with Arg325Trp mutation protein has been proved to possess both specificity and sensitivity to detect ZnT8A (Kawasaki et al, 2014;Kawasaki et al, 2008;Wenzlau et al, 2008). We identified the specificity of scFvs through ZnT8 C terminal dimer with Arg325Trp mutation.…”

Circulating anti‐glutamic acid decarboxylase‐65 antibody titers are positively associated with the capacity of insulin secretion in acute‐onset type 1 diabetes with short duration in a Japanese population

“…Glutamic decarboxylase‐65 autoantibodies titers were measured by the RIA and ELISA, with cut‐off values of 1.5 and 5.0 U/mL, respectively. Titers of IA‐2A and ZnT‐8A were measured by RIA, with a cut‐off value of 0.4 U/mL (range 0.4–50 U/mL), and by ELISA, with a cut‐off value of 15 U/mL (range 15–2000 U/mL), respectively. If the IA‐2A and ZnT‐8A titers fell below the lower limits of detection, a fill value of 0.01 was used.…”