Anti-ZnT8 (Zinc Transporter 8) ELISA

$1,290.00

The Eagle Biosciences Anti-ZNT8 (Zinc Transporter 8) ELISA Assay kit is intended for the quantitative determination of Anti-ZNT8 (Zinc Transporter 8) in serum or plasma by enzyme linked immunoassay (ELISA). The Eagle Biosciences Anti-ZNT8 ELISA Assay kit is for research use only and not to be used in diagnostic procedures.

SKU: ZT831-K01 Categories: , ,

Anti-ZnT8 (Zinc Transporter 8) ELISA

For Research Use Only

Size: 1×96 wells
Sensitivity: 10 U/ml
Dynamic Range: 10 – 500 U/ml
Incubation Time: overnight
Sample Type: Serum, Plasma
Sample Size: 25 µL

Additional Information

Assay Background

Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM), results from a chronic autoimmune destruction of the insulin-secreting pancreatic beta cells, probably initiated by exposure of genetically susceptible host to environmental agents. Autoimmune destruction of beta cells is thought to be completely asymptomatic until 80 – 90% of the cells are lost. This process may take years to complete and may occur at any time in all ages.

During the preclinical phase, this autoimmune process is marked by circulating autoantibodies to beta cell antigens. These autoantibodies, such as anti-insulin (IAA), anti-glutamic acid decarboxylase (GAD), anti-tyrosine phosphatase ICA 512 (IA2) and zinc transporter 8 (ZnT8), are present years before the onset of type 1 diabetes and prior to clinical symptoms.

ZnT8 autoantibodies are directed principally to the C terminal domain of ZnT8 (residues 268 – 369). Human population gene polymorphism at the codon for the 325th amino acid results in the expression of three protein variants: Arginine (R) 325, Tryptophan (W) 325 and very rarely Glutamine (Q) 325. ZnT8 autoantibodies may be specific to the R 325 or W 325 variant, or may be residue 325 non-specific. Sera that react with the Q allele only are extremely rare.   The Eagle Biosciences anti-ZnT8 ELISA Assay kit is capable of detecting, and quantifying, autoantibodies specific to R 325 or to W 325, or to residue 325 non-specific variants.

Assay Principle

The Anti-ZnT8 ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of autoantibodies to zinc transporter 8 (ZnT8 Ab) in human serum or plasma.  The assay utilizes the ability of ZnT8 Abs to act divalently, and to form a bridge between immobilized ZnT8 and ZnT8-Biotin in the fluid phase.

In the first step, ZnT8 Ab present in samples binds with ZnT8 immobilized onto the microtiter plate. In the second step, ZnT8-Biotin binds to this complex. The amount of ZnT8-Biotin bound correlates with the level of antibodies present in patient samples. Unbound ZnT8-Biotin is then removed by washing. Bound ZnT8-Biotin can then be quantified by addition of streptavidin peroxidase (SA-POD) and a colorigenic substrate (TMB), and reading the optical density at 450nm.

Assay Procedure

  1. Pipette into the corresponding wells according to assay scheme:  25 µl negative control (C I) and calibrators (1 – 4), control sera (C II, C III) and patient samples.
  2. Cover the plate, shake for approximately 5 seconds on a plate shaker at >500 rpm and incubate overnight, for 16 – 20 hours, at 2 – 8°C.
  3. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any residual droplets. Wash 3 times with 300 µl washing solution (diluted from B) with 5 seconds soaking time each.
  4. Add 100 µl of cold reconstituted ZnT8-Biotin solution (prepared from H and J) to each well.
  5. Cover the plate and incubate for 60 min at 2 – 8 °C, without shaking.
  6. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any residual droplets. Wash 3 times with 300 µl washing solution (diluted from B) with 5 seconds soaking time each.
  7. Add 100 µl reconstituted SA-POD (prepared from D and G) to each well.
  8. Cover the plate and incubate for 20 min at room temperature (18 – 25 °C) while shaking >500 rpm.
  9. Aspirate or “flick out” by striking the wells sharply onto absorbent paper to remove any residual droplets. Wash 3 times with 300 µl washing solution (diluted from B) with 5 seconds soaking time each.
  10. Add 100 µl substrate solution (E) to each well.
  11. Incubate for 20 min in the dark at room temperature, without shaking.
  12. Add 100 µl stop solution (F) after exactly 20 min to each well. Shake the plates for 5 seconds at >200 rpm.
  13. Read the optical density at 450 nm against 620 or 690 nm with a micro plate reader, within 5 minutes after adding the stop solution.

Typical Standard Curve

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The Role of Anti-Zinc Transporter 8 in Diabetes

The appearance of autoantibodies is one of the first measurable signs of development of beta cell autoimmunity. Several studies have shown that there are four disease-related autoantibodies associated linked to clinical T1D (Type 1 Diabetes) manifestation.   These antibodies have been identified as ICA (classical islet cell autoantibody), IAA (insulin autoantibody), GADA (autoantibody to glutamic acid decarboxylase, GAD65), and IA2A (autoantibody to protein tyrosine phosphatase-related IA-2 molecule). It has been discovered that when screening at risk or pre-diabetic subjects, that the most cost effective approach is screening for IA-2A and ZnT8 alone.5,7

Various studies have shown that ZnT8 is highly cell specific unlike GAD and IA-2. In fact, ZnT8 autoantibodies are detected in a significant proportion of patients withT2D (Type 2 Diabetes) and has been proven to be a useful marker to distinguish between different disease states and clinical phenotypes. 4 Other research has revealed that the presence of two or more autoantibodies reflects a progressive process and the presence of only one of these autoantibodies characterizes benign, non-progressive beta-cell autoimmunity. Thus, the detection of circulating autoantibodies provides valuable biomarkers for monitoring and identifying the development of autoimmunity preceding any clinical manifestation.1, 4, 7

Anti-Zinc Transporter 8, also known as ZnT8 is a product of the SLC30A8 gene and it is located on the membrane of the insulin secretory granules. The ZnT8 structure contains six transmembrane domains and comprises of a histidine-rich loop (presumed to be the zinc binding domain) between transmembrane domains 4 and 5. When ZnT8 is bound to insulin is known as the zinc dependent hexameric complex.   ZnT8’s main role is facilitating the uptake of the zinc required for efficient storage of insulin. Alterations in this marker’s activities have been associated with impaired glucose induced insulin response, thereby influencing the progression from glucose intolerance to full blown diabetes. 3,4,6

Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM), results from a chronic autoimmune destruction of the insulin-secreting pancreatic beta cells, probably initiated by exposure of genetically susceptible host to environmental agents. Autoimmune destruction of beta cells is thought to be completely asymptomatic until 80-90% of the cells are lost. This process may take years to complete and may occur at any time in all ages.

ZnT8 autoantibodies are directed principally to the C terminal domain of ZnT8 (residues 268–369). Human population gene polymorphism at the codon for the 325th amino acid results in the expression of three protein variants: Arginine (R) 325, Tryptophan (W) 325 and very rarely Glutamine (Q) 325. ZnT8 autoantibodies may be specific to the R 325 or W 325 variant, or maybe residue 325 non-specific. Sera that react with the Q allele only are extremely rare. The Eagle Biosciences Anti-ZnT8 ELISA Assay kit is capable of detecting, and quantifying, autoantibodies specific to R 325 or to W 325, or to residue 325 non-specific variants.

Zinc plays a vital role in all the insulin processes including synthesis, storage, secretion, and islet cell communication and signaling. Research has demonstrated that ZnT8 may be the chief factor and/or fuel for insulin maturation and other functions in insulin-secreting pancreatic cells. As a result, there is an intricate relationship and correlation between zinc and Type 1 and Type 2 diabetes with regards to disease states.2

 

Link: following to appropriate assays on Eagle website:

  1. ICA ELISA (classical islet cell autoantibody)
  2. IAA ELISA (insulin autoantibody)
  3. GAD ELISA (autoantibody to glutamic acid decarboxylase, GAD65)
  4. IA2 ELISA (autoantibody to protein tyrosine phosphatase-related IA-2 molecule)
  5. anti-ZnT8 ELISA Assay kit

 

References:

  1. Achenbach, P. et al. “Autoantibodies to Zinc Transporter 8 and SLC30A8 genotype stratify type 1 diabetes risk.” Diabetologia Clinical and Experimental Diabetes and Metabolism. Springer-Verlag 200910.1007/s00125-009-1438-0. http://link.springer.com/article/10.1007%2Fs00125-009-1438-0/fulltext.html
  2. Chimienti, Fabrice et al. “Identification and Cloning of a-Cell–Specific ZincTransporter, ZnT-8, Localized Into Insulin Secretory Granules.” Diabetes December 2013, 62 (12). http://diabetes.diabetesjournals.org/content/53/9/2330.full.pdf+html
  3. Chistiakov DA, et al. “Zn(2+)-transporter-8: a dual role in diabetes.” Biofactors 2009 Jul-Aug;35(4):356-63. doi: 10.1002/biof.49.   http://www.ncbi.nlm.nih.gov/pubmed/19655390
  4. Lampasona, Vito et al. “Zinc Transporter 8 Antibodies Complement GAD and IA-2 Antibodies in the Identification and Characterization of Adult-Onset Autoimmune Diabetes Non Insulin Requiring Autoimmune Diabetes (NIRAD) 4”,   Diabetes Care, December 2013, 36 (12) http://care.diabetesjournals.org/content/33/1/104.full.pdf+html
  5. Gorus, FK et al. “Screening for insulinoma antigen 2 and zinc transporter 8 autoantibodies: a cost-effective and age-independent strategy to identify rapid progressors to clinical onset among relatives of type 1 diabetic patients.” Clin Exp Immunol. 2013 Jan;171(1):82-90. doi: 10.1111/j.1365-2249.2012.04675.x. http://www.ncbi.nlm.nih.gov/pubmed/23199327
  6. Wenzlau, Janet M. “The Cation Efflux Transporter ZnT8 (Slc30A8) is a Major Autoantigen in Human Type 1 Diabetes.” PNAS October 23, 2007 vol. 104 no. 43 17040-17045. http://www.pnas.org/content/104/43/17040.full.pdf+html    
  7. Wu, Juli. “ZnT8 (Slc30A8) is a New Antigenic Target in Type 1 diabetes.” Beta Cell Biology Consortium. Article summary 2013. http://www.betacell.org/content/articleview/article_id/234/

 Recent Citations:

Bahendeka Silver, et al. (2018). Autoantibodies and HLA Class II DR-DQ Genotypes in Ugandan Children and Adolescents with Type I Diabetes Mellitus. International Journal of Diabetes in Developing Countries. 1-8.

Arroyo-Jousse et. al. “La metilación global del ADN y los niveles de homocisteína en plasma se encuentran disminuidos en pacientes con diabetes mellitus tipo 1.” Revista Medica Chile. 2015:14(5).

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