DLD Acetylcholine Receptor Autoantibody (ACHRAB) Assay Elisa

ASSAY PRINCIPLE 
RSR’s AChRAb ELISA depends on the ability of AChRAb in human serum to bind to similar sites on the AChR as various monoclonal antibodies such as MAb1 (coated on ELISA plate wells) and/or MAb2 and/or MAb3 (which are labelled with Biotin). In the absence of AChRAb a complex is formed between MAb1 coated on the plate wells, the AChR and MAb2- and MAb3-Biotin. MAb2- and MAb3-Biotin bound are then detected by 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. 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 is then read using an ELISA plate reader. In the presence of AChRAb the formation of the MAb1-AChR-MAb2-/MAb3-Biotin complex is inhibited, resulting in less SA-POD being bound and a reduction in final absorbance at 450 nm. The higher the concentration of AChRAb in the test serum, the greater the inhibition of MAb-Biotin binding. When using the kit calibrators, the measuring interval is 0.45 – 20 nmol/L toxin bound.

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 AChRAb 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 83% - 122% of spiked serum (20 samples with serum concentrations ranging from 0.28 nmol/L – 18 nmol/L) or 69% - 165% in terms of nmol/L.

RESULT ANALYSIS 
A calibration curve can be established by plotting calibrator concentration (including a value of 0.2 nmol/L for the negative control) on the x-axis (linear scale) against the absorbance of the calibrators on the y-axis (linear scale). The AChRAb concentrations in patients’ sera can then be read off the calibration curve. The data in these instructions are based on a 4 parameter curve fit. Samples with high AChRAb concentrations can be diluted in negative control (N). For example 10 L of sample plus 90 L of negative control (N) 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 and we suggest that the dilution giving a value closest to 50% inhibition is used for calculation of AChRAb concentration.