A fluid-phase routine method for the detection of insulin-anti-insulin complexes

A fast routine method has been devised to measure circulating insulin-anti-insulin complexes. The principle lies in the calculation of the difference between the insulin binding capacity of the free antibody and that of the total amount of insulin antibody. The pH of 1 aliquot of serum was lowered to 3 by adding glycine-HCl buffer. Free insulin was removed by charcoal precipitation and the pH was again neutralized by the simple addition of NaOH; the final dilution of serum was 1/5. Radiolabelled insulin was added to this and to a second aliquot of serum, also diluted 1/5. Free and bound insulin were separated using either dextran charcoal or PEG 6000 at a final dilution of 14.3%. The first technique of separation was preferred. This method has been used in normal controls and in insulin-treated diabetic patients and the results have been compared to those obtained using other methods to detect insulin-anti-insulin complexes and insulin antibodies. Insulin-anti-insulin complexes tended to be more frequently observed in patients with high insulin antibody values. The technique described is much less laborious than other methods for detecting insulin complexes since it requires only a few hours to complete. It is reproducible and sensitive enough for clinical research. This method is of value when both free and bound insulin antibodies have to be evaluated.