Acta Diabetol. 2014;51(3):429-34. doi: 10.1007/s00592-013-0532-4.

Novel enzyme-linked immunosorbent assay for bivalent ZnT8 autoantibodies.

 

Kawasaki E1, Tanaka M, Miwa M, Abiru N, Kawakami A.
  • 1Department of Metabolism/Diabetes and Clinical Nutrition, Nagasaki University Hospital, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan, eijikawa@nagasaki-u.ac.jp.

 

Abstract

Autoantibodies to zinc transporter 8 (ZnT8A) are 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 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 to or slightly higher than that of 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.

PMID: 24292540

 

Supplements:

The diagnosis of type 1A diabetes has been done by the combined detection of anti-islet autoantibodies, for example anti-GAD antibody, anti-IA-2 antibody and anti-insulin autoantibody. Anti-zinc transporter 8 (ZnT8) antibody (ZnT8A) is also one of the autoantibodies associated with type 1 diabetes [1]. The cation efflux ZnT8 has been first described as a novel target autoantigen in patients with type 1 diabetes in 2007. ZnT8 belonging to the SLC30 protein family is multipass transmembrane protein with a role in the transportation of zinc cations out of the cytoplasm or into the vesicles [2]. ZnT8 is specifically expressed in the pancreatic islet β cells and localized into insulin secretory granules [3]. Its reference gene encodes a 369 amino acid protein. In genome-wide association studies, an association between a non-synonymous variant in SLC30A8 (rs13266634; Arg325Trp) and susceptibility to type 2 diabetes was reported [4]. In our previous report, most ZnT8-reactive sera recognized the carboxy-terminal 102 amino acids of the molecule (amino acids 268-369) and an amino acid encoded by the polymorphic codon 325 was a key determinant of humoral autoreactivity to this protein [5].

The diagnostic kit with this autoantigen was developed as radioligand binding assay (RBA) and the evaluation of the ZnT8A was demonstrated by the Diabetes Autoantibody Standardization Program (DASP). In this society, the ZnT8A was acquired a high degree of sensitivity and specificity [6]. However, this detection system, fluid-phase ZnT8A RBA, has proved to be difficult for many laboratories to implement.

In this paper, we described the construction of ELISA system for detecting ZnT8A without radioactivity (ZnT8A-ELISA) and characterized this assay system. The new ELISA used dimeric carboxy-terminal domains of ZnT8A carrying either 325Trp or 325Arg as antigen. Moreover, this ELISA employed a modified ELISA format which was based on the ability of the autoantibodies to form a bridge between ZnT8 coated on the ELISA plate and ZnT8-biotin (Figure 1), and the detection of autoantibody-bound antigen rather than immunoglobulin itself. Furthermore, we also indicated the clinical significance of ZnT8A in patients with type 1 diabetes.

 

EK FIG1

Figure 1. Principles of ELISA for bivalent ZnT8A

 

To estimate our new assay system, we studied healthy subjects and patients with type 1 diabetes. We found high correlation between the assays with ELISA-ZnT8A and RBA, and the sensitivity of ELISA-ZnT8A is equivalent to or slightly higher than that of conventional RBA. A blinded IASP2012 workshop set of 90 control subjects and 50 new-onset patients with type 1 diabetes was also tested for ELISA-ZnT8A. In this test, their two different assays had good concordance.

In this study, the sera were also tested for the reactivity to the carboxy-terminal ZnT8 constructs bearing 325Arg or 325Trp. We found that the higher titer of ELISA-ZnT8A was associated with the humoral autoreactivity to ZnT8A-325Arg.

We concluded that this ELISA assay had high performance and was to facilitate large-scale autoantibody screening (Figure 2). Moreover, these results suggested that the humoral autoimmunity against ZnT8 was related to a high risk of faster development of type 1 diabetes and the ZnT8A levels are associated with the known amino acid 325 variants.

 

ek fig2

Figure 2. ZnT8Ab ELISA kit

 

References

  1. Wenxlau JM, Juhl K, Yu L, Moua O, Sarkar SA, Gottlieb P, Rewers M, Eisenbarth GS, Jensen J, Davidson HW, Hutton JC (2007) The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type I diabetes. Proc Natl Acad Sci USA 104(43):17040-17045
  2. Kambe T, Yamaguchi-Iwai Y, Sasaki R, Nagao M (2004) Overview of mammalian zinc transporters. Cell Mol Life Sci 61:49-68
  3. Chimienti F, Devergans S, Favier A, Seve M (2004) Identification and cloning of a bete-cell-specific zinc transporter, ZnT-8, localized into insulin secretory granules. Diabetes 53:2330-2337
  4. Sladak R, Rocheleau G, Rung J et al (2007) A genome-wide association study identifies novel risk loci for type 2 diabetes. Nature 445:881-885
  5. Kawasaki E, Uga M, Nakamura K, Kuriya G, Satoh T, Fujishima K, Ozaki M, Abiru N, Yamasaki H, Wenzlau JM, Davidson HW, Hutton JC, Eguchi K (2008) Association between anti-ZnT8 autoantibody specificity and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes. Diabetologia 51(12):2299-2302
  6. Lampasona V, Schlosser M, Mueller PW, Williams AJ, Wenzlau JM, Hutton JC, Achenbach P (2011) Diabetes antibody standardization program: first proficiency evaluation os assays for autoantibodies to zinc transporter 8. Clin Chem 57(12): 1693-1702

 

Contact:

Eiji Kawasaki, M.D., Ph.D.

Director, Diabetes Center,

Shin-Koga Hospital

120 Tenjin-cho, Kurume 830-8577, Japan

TEL 81-942-38-2222 Fax 81-942-38-2248

E-mail: e-kawasaki@tenjinkai.or.jp

 

 

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