Use of polyethylene glycol in separating bound from unbound ligand in radioimmunoassay of thyroxine.

In most rapid radioimmunoassay methods, absorbents are used that disturb the equilibrium of the ligand-antibody interaction; thus the separation process must be rigidly timed. We show here that polyethylene glycol (mol wt. 6000) abolishes this constant. We measured gamma-globulin precipitation by polyethylene glycol (150g/liter) in a thyroxine radioimmunoassay system involving use of a sheep anti-thyroxine antiserum, quantitatively and qualitatively, with either normal human serum or completely precipitated at pH 6-9 at 4 and 25 degrees C, but incompletely if salt concentration was high (1 mol/liter). Specificity of gamma-globulin precipitation increased with increasing pH and temperature. All gamma-globulin-bound thyroxine was precipitated, and also some not bound to gamma-globulin. This was taken into account by including "blank" tubes (no antibody, but with normal sheep serum) in all assays. Because this reaction is predominantly entropic and temperature independent, the entire procedure can be done at 37 degrees C and room temperature without rigid timing. The assay requires 10 mul of serum, its reproducibility is 4-8% (CV) in the euthyroid and hyperthyroid range, and its accuracy is close to 100%.