DLD Voltage-Gated Potassium Channel (VGKC) Autoantibody RIA Elisa

ASSAY PRINCIPLE 
In RSR’s VGKC Ab radioimmunoassay (RIA), VGKC Ab in patient sera and controls are allowed to interact with detergent solubilised VGKCs extracted from rabbit brain tissue and complexed with 125Ilabelled -dendrotoxin (known to react with Kv1.1, 1.2 and 1.6 subtypes of the VGKC). After incubation at 2-8 oC overnight, the resulting antigen-antibody complexes are immunoprecipitated by the addition of anti-human IgG. After a second incubation of 1½ hours, assay buffer is added and the samples centrifuged. Unbound 125I-labelled -dendrotoxin-VGKC complex is removed from the tubes by aspiration of the supernatant. The level of radioactivity remaining in the tube is proportional to the antibody level in the test sample.

STORAGE AND PREPARATION OF SERUM SAMPLES 
Sera to be analysed should be assayed soon after separation or stored, preferably in aliquots, at 2- 8 oC for up to 2 weeks, or at –20oC or below for longer periods. 15 L is sufficient for one assay. Repeated freeze thawing or increases in storage temperature must be avoided. Do not use lipaemic or haemolysed serum samples. Citrate, EDTA and heparin plasma may be used in the assay. When required, thaw test sera at room temperature and mix gently to ensure homogeneity. Dilute 1:10 using assay buffer (e.g. 15 µL serum plus 135 µL assay buffer). Centrifuge diluted serum prior to assay (preferably for 5 min at about 10,000rpm i.e. about 10,000g in a microfuge) to remove any particulate matter.

CLINICAL EVALUATION 
Clinical Specificity 
Sera from 100 individual healthy blood donors were assayed in the VGKC Ab RIA. 98 (98%) were identified as being negative for VGKC Ab. 
Clinical Sensitivity Serum samples from 30 patients with suspected Voltage-Gated Potassium Channelopathies and related neurological disorders were assayed in the VGKC Ab RIA. 27 (90%) were positive for VGKC Ab. 
Lower Detection Limit The negative control was assayed 20 times and the mean and standard deviation calculated. The lower detection limit at 2 standard deviations was 4.5 pmol/L.