DLD RSR 3 Screen ICA - Elisa

ASSAY PRINCIPLE 
In RSR’s 3 Screen ELISA, GAD, IA-2 and ZnT8 Ab in patients’ sera, reference preparation or calibrators (optional) and controls are allowed to interact with GAD65, IA-2 and ZnT8 coated onto ELISA plate wells. After a 16 - 20 hour incubation, the samples are discarded leaving any GAD, IA-2 and/or ZnT8 Ab in the patient sera, reference preparation or calibrators (optional) and controls bound to the GAD65, IA-2 and ZnT8 coated wells. A mixture of GAD65-Biotin, IA-2-Biotin and ZnT8- Biotin is then added and during a 2nd incubation step where, through the ability of GAD, IA-2 and ZnT8 Ab to act divalently, a bridge is formed between the GAD65, IA-2 or ZnT8 immobilised on the plate and GAD65-Biotin, IA-2-Biotin and ZnT8- Biotin respectively.

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 (RSR recommends duplicate 25 L determinations). Repeated freeze thawing or increases in storage temperature should be avoided. Do not use lipaemic or haemolysed serum samples. Do not use plasma in the assay. When required, bring test sera to room temperature and mix gently to ensure homogeneity. Centrifuge sera prior to assay (preferably for 5 min at about 10,000 rpm i.e. about 10,000 g in a microfuge) to remove particulate matter. Please do not omit this centrifugation step if sera are cloudy or contain particulates.

MATERIALS REQUIRED AND NOT SUPPLIED 
Pipettes capable of dispensing 25µL and 100µL. Means of measuring out various volumes to reconstitute or dilute reagents. Pure water. ELISA Plate reader suitable for 96 well formats and capable of measuring at 450nm and 405nm. ELISA Plate shaker, capable of 500 shakes/min (not an orbital shaker). ELISA Plate cover.

RESULT ANALYSIS 
Calculation of results without calibrators 
Index Calculation 
The index values are calculated as follows: 
The index value can also be calculated using absorbance data at 405nm 
97% of 1200 healthy adult male blood donor sera gave index values of less than 30 suggesting that index values of 30 or more could be considered positive in this group (see page 4)