DLD LEMS Assay - RIA Elisa

ASSAY PRINCIPLE 
In RSR’s VGCC Ab RIA, VGCC Ab in patient sera and controls are allowed to interact with detergent solubilised P-type VGCCs extracted from rabbit brain tissue and complexed with 125I-labelled wconotoxin MVIIC. After a 1 hour incubation, the resulting antigen-antibody complexes are immunoprecipitated by the addition of anti-human IgG. After a second incubation of 1 hour, assay buffer is added and the samples centrifuged. Unbound 125I-labelled w-conotoxin MVIIC 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. Non-specific binding in the assay is determined using a preparation of VGCCs supplied in the kit which have been labelled with 125I-conotoxin MVIIC in the presence of an excess of unlabelled conotoxin.

STORAGE AND PREPARATION OF TEST 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. Do not use plasma. When required, bring test sera to room temperature and mix gently to ensure homogeneity. Dilute 1:10 using dilution buffer (e.g. 15 µL serum plus 135 µL dilution buffer). Centrifuge diluted serum prior to assay (preferably for 5 min at about 10,000 rpm i.e. about 10,000g in a microfuge) to remove any particulate matter.

CLINICAL EVALUATION 
Clinical Specificity 
Samples from 160 individual healthy blood donors were assayed in the VGCC Ab RIA. 160 (100%) were identified as being negative for VGCC Ab. 
Clinical Sensitivity 
Samples from 50 patients diagnosed with LEMS were assayed in the VGCC Ab RIA. 50 (100%) were identified as being positive for VGCC 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 2.86 pmol/L.

RESULT ANALYSIS 
After subtracting the background count for each tube, the radioactivity in the pellet for the T tracer (T cpm) represents the amount of 125I VGCC bound by the VGCC Ab plus non-specific binding, and the radioactivity in the pellet for the NSB tracer (NSB cpm) represents the amount of 125I VGCC that is bound non-specifically (expressed as fmoles of labelled toxin bound). These values can then be converted to pmoles of labelled toxin bound per litre of test serum (pmol/L) by calculating the specific binding for each pellet and multiplying by 400 to adjust for the sample volume and dilution, and the change of unit (fmoles to pmoles). The following information that is required can be found on the QC record sheet.