DLD IA - 2 Antibody - Elisa

ASSAY PRINCIPLE 
In RSR’s IA-2 Ab ELISA Version 2, IA-2 Ab in patients’ sera, calibrators and controls are allowed to interact with IA-2 coated onto ELISA plate wells. After a 16 – 20 hour incubation, the samples are discarded leaving IA-2 Ab bound to the IA-2 coated wells. IA-2-Biotin is added in a 2nd incubation step where, through the ability of IA-2 Ab to act divalently, a bridge is formed between the IA-2 immobilised on the plate and IA-2-Biotin. The amount of IA-2-Biotin is then determined in a third incubation step by the 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’ – tetramethylbenzidine (TMB) results in formation of a blue colour.

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. 100 L is sufficient for one assay (duplicate 50 L determinations). Repeated freeze thawing or increases in storage temperature must be avoided. Do not use lipaemic or haemolysed serum samples. Studies in which EDTA, citrate and heparin plasma samples were spiked with IA-2 Ab positive sera showed minor changes in signal compared with spiked serum from the same donor. In particular the absorbance values at 450 nm with spiked EDTA, citrate and heparin plasmas were 84% - 112% of spiked serum (8 samples with serum concentrations ranging from 2.3 u/mL – 505 u/mL) or 81% - 114% in terms of u/mL.

CLINICAL EVALUATION 
Clinical Specificity and Sensitivity 
In the IASP 2016 study the RSR IA-2 Ab ELISA Version 2 kit showed 98% (n=90) specificity and 76% (n=50) sensitivity. 
Sera from 533 individual healthy blood donors were tested in the RSR IA-2 Ab ELISA Version 2 kit. 523 (98.1%) sera were identified as being negative (less than the cut off of 7.5 u/mL) for IA2 Ab. 

Lower Detection Limit 
The kit negative control was assayed 20 times, and the mean and standard deviation calculated. The lower detection limit at +2 standard deviations was 0.95 u/mL.

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 IA-2 Ab 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 (B) can be assigned a value of 0.75 u/mL to assist in computer processing of assay results. Many test sera will have values below 350 u/mL and the 4000 u/mL calibrator need not always be included. Samples with high IA-2 Ab concentrations can be diluted in kit negative control (B). For example, 15 L of sample plus 135 L of negative control 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.