DLD GAD65 Antibody - Elisa

ASSAY PRINCIPLE
In RSR’s GADAb ELISA, GADAb in patients’ sera, calibrators and controls are allowed to interact with GAD65 coated onto ELISA plate wells. After a 1 hour incubation, the samples are discarded leaving GADAb bound to the immobilised GAD65 on the plate. GAD65-Biotin is added in a 2nd incubation step where, through the ability of GADAb in the samples to act divalently, a bridge is formed between GAD65 immobilised on the plate and GAD65-Biotin. The amount of GAD65- Biotin bound is then determined in a 3 rd incubation step by addition of Streptavidin Peroxidase, which binds specifically to Biotin. Excess, unbound Streptavidin Peroxidase is then washed away and addition of 3,3’,5,5’ – tetramethylbenzidine (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 450 nm and 405 nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of GADAb the test sample. Reading at 405 nm allows quantitation of high absorbances (and should be used for concentrations of 120 u/mL or more).

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. Do not use plasma in the assay. When required, bring test sera to room temperature and mix gently to ensure homogeneity. Centrifuge serum 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.

RESULT ANALYSIS 
A calibration curve can be established by plotting calibrator concentration on the x-axis (log scale) against the absorbance of the calibrators on the yaxis (linear scale). The GADAb concentrations in patients’ sera can then be read off the calibration curve [plotted at RSR as a spline log/lin curve (smoothing factor = 0)]. Other data reduction systems can be used. The negative control can be assigned a value of 0.5 u/mL to assist in computer processing of assay results. Most test sera will have values below 250 u/mL and the 2000 u/mL calibrator need not always be included. Samples with high Ab concentrations can be diluted in GADAb negative serum or the kit negative control (D). For example, 20 L of sample plus 180 L of diluent to give a 10x dilution. Other dilutions (e.g. 100x) can be prepared from a 10x dilution or otherwise as appropriate. Some sera will not dilute in a linear way according to the kit calibrators (standardised against NIBSC 97/550).