DLD Aquaporin-4 (AQP4) Antibody - Elisa

ASSAY PRINCIPLE 
In RSR’s AQP4 Ab ELISA Version 2 kit, AQP4 Ab in patients’ sera, calibrators and controls are allowed to interact with AQP4 coated onto ELISA plate wells and liquid phase biotinylated AQP4 (AQP4-Biotin). After incubation at room temperature for 2 hours with shaking, the well contents are discarded. AQP4 Ab bound to the AQP4 coated on the well will also interact with AQP4-Biotin through the ability of AQP4 Ab in the samples to act divalently leaving AQP4-Biotin bound to the well via an AQP4 Ab bridge. The amount of AQP4-Biotin bound is then determined in a second incubation step involving addition of streptavidin peroxidase (SA-POD), which binds specifically to biotin.

STORAGE AND PREPARATION OF 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 should be avoided. Do not use lipaemic or haemolysed samples. Studies in which EDTA, citrate and heparin plasma samples were spiked with AQP4 Ab positive sera showed minor changes in signal compared with spiked serum from the same donor. In particular OD450 values with spiked EDTA, citrate and heparin plasmas were 79% - 128% of spiked serum (15 samples with serum concentrations ranging from 2.6 u/mL – 30 u/mL) or 87% - 130% in terms of 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 AQP4 Ab concentrations in patient 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 0.15 u/mL to assist in computer processing of assay results. Samples with AQP4 Ab concentrations above 80 u/mL can be diluted (e.g. 10 x and/or 100 x) in AQP4 Ab negative serum. Some sera will not dilute in a linear way.