DLD Insulin Antibody - RIA Elisa

ASSAY PRINCIPLE 
In RSR’s insulin antibody (IAb) assay test serum samples are incubated first with 125I-(A14)- monoiodinated insulin. This is followed by addition of anti-human IgG to precipitate any labelled insulin-anti insulin complexes which have formed. After centrifugation the precipitates are counted for 125I and the amount of radioactivity in the precipitate is proportional to the concentration of IAb in the test sample. The measuring range is 0.4 – 50 u/mL (arbitrary RSR units).

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. 40 L is sufficient for one assay (duplicate 20 L determinations). When required, thaw test samples at room temperature and mix gently to ensure homogeneity. Lipaemic or haemolysed sera should not be used. Remove any particulate matter by centrifugation prior to assay (preferably for 5 min at about 10,000rpm i.e. about 10,000g in a microfuge).

CLINICAL EVALUATION 
Clinical Specificity and Sensitivity 
100% (n=100) of healthy blood donor sera samples gave values of less than 0.4 units per mL. Out of 62 type 1 DM patients who had never received insulin treatment, 21 (34%) were positive for IAb. However, in patients who have received insulin treatment, IAb prevalence is much higher suggesting that insulin antibodies are being induced by insulin treatment in many patients. 
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 0.03 u/mL.

RESULT ANALYSIS 
A calibration curve can be established by plotting calibrator concentration on the x-axis (log scale) against the % 125I-insulin bound for the calibrators on the y-axis (linear scale). IAb concentrations in test sera can then be read off the calibration curve [plotted at RSR as a spline log/lin curve (smoothing factor = 0)]. Other data reduction methods can be used. The negative control can be assigned a value of 0.04 u/mL to assist in computer processing of assay results. Samples with high IAb concentrations can be diluted in kit negative control (E). For example, 10 L of sample plus 90 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.