DLD Zinc Transporter 8 (ZnT8) Autoantibody Elisa Kit

ASSAY PRINCIPLE 
In RSR’s ZnT8 Ab ELISA, ZnT8 Ab in test patients’ sera, calibrators and controls are allowed to interact with ZnT8 coated onto ELISA plate wells. After a 16-20 hour incubation, the samples are discarded leaving ZnT8 Ab bound to the ZnT8 coated wells. ZnT8-Biotin is added in a 2nd incubation step where, through the ability of ZnT8 Ab in the samples to act bivalently (or polyvalently), a bridge is formed between ZnT8 bound to the wells and ZnT8-Biotin. Unbound ZnT8-Biotin is then removed in a wash step and the amount of bound ZnT8-Biotin determined (in a 3 rd incubation step) by addition of Streptavidin Peroxidase (SA-POD), which binds specifically to Biotin. Excess, unbound SA-POD is then washed away and addition of the peroxidase substrate 3,3’,5,5’ tetramethyl-benzidine (TMB) results in formation of a blue colour. This reaction is stopped by addition of stop solution causing the well contents to turn yellow. The absorbance of the yellow reaction mixture at 405 nm and 450 nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of ZnT8 Ab in the test sample. Reading at 405 nm allows quantitation of high absorbances and should be used when the OD at 450 nm is greater than 3.0. If it is possible to read at only one wavelength 405nm may be used. The measuring interval is 15 – 2000 u/mL (arbitrary RSR units).

Clinical Accuracy 
Sera containing rheumatoid factor (n=26) and sera containing autoantibodies to thyroglobulin (n=20), to thyroid peroxidase (n=24), to aquaporin-4 (n=3) and to the acetylcholine receptor (n=9) were negative for ZnT8 Ab. 4% (n=24) of sera positive for antibodies to the TSH receptor, and 9% (n=23) of sera positive for 21-hydroxylase Ab were positive for ZnT8 Ab using the RSR ZnT8 Ab ELISA kit.

STORAGE AND PREPARATION OF TEST SERUM SAMPLES 
Sera to be analysed should be assayed soon after separation or stored, preferably in aliquots, at or below –20oC. 50 L is sufficient for one assay (duplicate 25 L determinations). Repeated freeze thawing or increases in storage temperature should be avoided. Do not use lipaemic or haemolysed serum samples. Citrate and heparin plasma may be used in the assay. Studies in which EDTA plasma samples were spiked with ZnT8 Ab positive sera showed that lower signals were observed compared with spiked serum from the same donor. In particular OD450 values with spiked EDTA plasma were 33% - 65% of spiked serum or 37% - 64% in terms of u/mL (19 samples with spiked serum concentrations ranging from 11 u/mL – 326 u/mL). When required, bring test sera to room temperature and mix gently to ensure homogeneity. Centrifuge sera prior to assay (preferably for 5 min at 10- 15,000 rpm in a microfuge) to remove particulate matter. Please do not omit this centrifugation step if sera are cloudy or contain particulates.

CLINICAL EVALUATION
Clinical Specificity and Sensitivity 
In the IASP 2016 study the RSR ZnT8 Ab ELISA kit achieved 99% (n=90) specificity and 72% (n=50) sensitivity. 
Assay of 297 healthy blood donor sera gave a mean value of 1.9 ± 3.84 u/mL. 3 sera (1%) were above the assay cut off giving values of 45, 41 and 19 u/mL. 

Lower Detection Limit 
The negative control was assayed 20 times and the mean and standard deviation calculated. The lower detection limit at +2 standard deviations was 1.2 u/mL when the negative control was assigned a value of 1 u/mL.