Glutamic Acid Decarboxylase (GAD) Autoantibody …

1 ASSAY PRINCIPLE. ElisaRSRTM GADAb In RSR's GADAb ELISA, GADAb in patients' sera, Glutamic acid Decarboxylase (GAD) calibrators and controls are allowed to interact with Autoantibody ELISA kit from RSR GAD65 coated onto ELISA plate wells. After a 1. Instructions for use hour incubation, the samples are discarded leaving GADAb bound to the immobilised GAD65 on the RSR Limited plate. GAD65-Biotin is added in a 2nd incubation Avenue Park Pentwyn Cardiff step where, through the ability of GADAb in the CF23 8HE United Kingdom samples to act divalently, a bridge is formed Tel.: +44 29 2073 2076 Fax: +44 29 2073 2704 between GAD65 immobilised on the plate and Email: Website: GAD65-Biotin.

2 The amount of GAD 65-Biotin bound INTENDED USE is then determined in a 3rd incubation step by The RSR GAD65 Autoantibody (GADAb) ELISA kit is addition of Streptavidin Peroxidase, which binds intended for use by professional persons only, for specifically to Biotin. Excess, unbound Streptavidin the quantitative determination of GADAb in human Peroxidase is then washed away and addition of serum. Autoantibodies to pancreatic beta cell 3,3',5,5' tetramethylbenzidine (TMB) results in antigens are important serological markers of type formation of a blue colour. This reaction is stopped 1 diabetes mellitus (type 1 DM).

3 The antigens by addition of stop solution causing the well recognised by these antibodies include insulin, contents to turn yellow. The absorbance of the Glutamic acid Decarboxylase (GAD65 kDa isoform), yellow reaction mixture at 450 nm and 405 nm is the islet cell antigen IA-2 or ICA-512 and zinc then read using an ELISA plate reader. A higher transporter 8 (ZnT8). absorbance indicates the presence of GADAb the test sample. Reading at 405 nm allows REFERENCES quantitation of high absorbances (and should be H. Brooking et al used for concentrations of 200 u/mL or more). A Sensitive non-isotopic assay for GAD65 Low values (less than 10 u/mL) should be read off autoantibodies the 450 nm calibration curve.

4 If it is possible to Clinica Chimica Acta 2003 331:55-59 read at only one wavelength 405nm may be used. The measuring interval is 5 2000 u/mL (units are S. Chen et al NIBSC 97/550). Sensitive non-isotopic assays for autoantibodies to IA2 and to a combination of both IA2 and STORAGE AND PREPARATION OF TEST. GAD65. SERUM SAMPLES. Clinica Chimica Acta 2005 357:74-83 Sera to be analysed should be assayed soon after separation or stored, preferably in aliquots, at or E. Nilson et al below 20oC. 50 L is sufficient for one assay Calcium addition to EDTA plasma eliminates (duplicate 25 L determinations).

5 Repeated freeze falsely positive results in the RSR GADAb ELISA. thawing or increases in storage temperature should Clinica Chimica Acta 388 (2008) 130-134 be avoided. Do not use lipaemic or haemolysed K. Rahmati et al serum samples. Do not use plasma in the assay. When required, bring test sera to room temperature A Comparison of Serum and EDTA Plasma in the and mix gently to ensure homogeneity. Centrifuge Measurement of Glutamic acid Decarboxylase serum prior to assay (preferably for 5 min at 10- Autoantibodies (GADA) and Autoantibodies to 15,000 rpm in a microfuge) to remove particulate Islet Antigen-2 (IA-2A) Using the RSR.

6 Matter. Please do not omit this centrifugation step Radioimmunoassay (RIA) and Enzyme Linked if sera are cloudy or contain particulates. Immunosorbent Assay (ELISA) Kits. Clin. Lab. 2008 54:227-235 SYMBOLS. C. T rn et al Symbol Meaning Diabetes Antibody Standardization Program: evaluation of assays for autoantibodies to EC Declaration of Conformity Glutamic acid Decarboxylase and islet antigen-2. Diabetologia 2008 51:846-852 IVD In Vitro Diagnostic Device PATENTS REF Catalogue Number The following patents apply: RSR patents: European patent EP 1448 993 B1, LOT Lot Number Chinese patent ZL , Indian patent 226484, Japanese patent 5711449 and US Consult Instructions patents 8,129,132 B1 and 9,435,797 B2.

7 Manufactured By Patents licensed to RSR: US patents 6,682,906 B1. and 6,277,586 B1. 7th August 2017 Page 1 of 4 RSR/24 Rev 34. Streptavidin Peroxidase (SA-POD). Sufficient for 1 x mL. Concentrated Expiry Date G Dilute 1 in 20 with diluent for diluting SA- POD (H). For example, mL (G) + Store mL (H). Store at 2 8oC for up to 16. weeks after dilution. Negative Control Diluent for SA-POD. H 15 mL. Positive Control Ready to use Peroxidase Substrate (TMB). MATERIALS REQUIRED AND NOT SUPPLIED I 15 mL. Pipettes capable of dispensing 25 L and 100 L. Ready to use Means of measuring out various volumes to Concentrated Wash Solution reconstitute or dilute reagents.

8 125 mL. Pure water. J Concentrated ELISA Plate reader suitable for 96 well formats and Dilute 10 X with pure water before use. capable of measuring at 450nm and 405nm. Store at 2 8oC up to kit expiry date. ELISA Plate shaker, capable of 500 shakes/min (not Stop Solution an orbital shaker). K 12 mL. ELISA Plate cover. Ready to use PREPARATION OF REAGENTS SUPPLIED ASSAY PROCEDURE. Store unopened kit and components at 2 - 8oC Allow all reagents to stand at room temperature GAD65 Coated Wells (20-25 oC) for at least 30 minutes before use. A. 12 breakapart strips of 8 wells (96 in total) repeating Eppendorf type pipette is recommended in a frame and sealed in a foil bag.

9 Allow for steps 4, 7, 10 and 11. to stand at room temperature (20-25 oC) for 1. Pipette 25 L of patient sera, calibrators at least 30 minutes before opening. (B1-6) and controls (C and D) into A Ensure wells are firmly fitted into frame respective wells, in duplicate, leaving one provided. After opening return any unused well empty for blank (see step 12). wells to the original foil bag with desiccant 2. Cover the frame and shake the wells for 1. provided and seal with adhesive tape. hour at room temperature on an ELISA. Place foil bag in the self-seal plastic bag plate shaker (500 shakes per min.)

10 And store at 2-8oC for up to 16 weeks. 3. Use an ELISA plate washer to aspirate and Calibrators wash the wells three times with diluted 5, 18, 35, 120, 250, 2000 u/mL wash solution (J). If a plate washer is not B1-6 (units are NIBSC 97/550) available, discard the well contents by 6 x mL briskly inverting the frame of wells over a Ready to use suitable receptacle, wash three times Positive Control manually and finally tap the inverted wells (see label for concentration range) gently on a clean dry absorbent surface. C. mL 4. Pipette 100 L of reconstituted GAD65- Ready to use Biotin (E) into each well (except blank).