Radioimmunoassay of Human Adenosine Deaminase

This chapter describes the radioimmunoassay of human adenosine deaminase. Human adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine to produce inosine and ammonia. ADA is purified from human erythrocytes, using antibody affinity chromatography. The highly purified enzyme is used for antiserum production, preparation of iodinated enzyme tracer, and served as a standard for the radioimmunoassay. Immunoreactive ADA protein in hemolysate samples is determined by a competitive radioimmunoassay. Levels of ADA, immunologically cross-reactive material, are measured, using serial dilutions of each hemolysate sample (in triplicate) over a fourfold displacement range. In the radioimmunoassay procedure, the percentage of ¹²⁵I-labeled ADA bound to antibody in the presence of unlabeled ADA is corrected for the background and nonspecific ¹²⁵I binding. The addition of a 100-fold excess of unlabeled purified ADA, over that required for 50% displacement of tracer from antibody, prevented greater than 99% of tracer from binding. It is found that, while the iodination conditions required to produce such a high incorporation of ¹²⁵I into the ADA molecule resulted in extensive damage to the protein, further purification of the tracer by adenosine sepharose column chromatography proved to be a key step in making the iodination technique practical.