Process and test kit for determining free active compounds in biological fluids

A method for determining the concentration of the free fraction of thyroxine present in a biological fluid containing natural binders, with said free fraction and the bound fraction of thyroxine being in equilibrium, comprising the steps of:

• (a) contacting a sample of said biological fluid with an unlabeled antibody to produce a reacted unlabeled antibody, with the quantity of said unlabeled antibody and the affinity of said unlabeled antibody for said free fraction of said thyroxine selected such that said unlabeled antibody does not substantially affect said equilibrium between said free fraction and said bound fraction of said thyroxine;
• (b) separating said sample from said reacted unlabeled antibody of step (a);
• (c) incubating said reacted unlabeled antibody of step (b) with a labelled tracer substance for cross-reaction with said unlabeled antibody, with the affinity of said tracer for said unlabeled antibody being substantially higher than or substantially lower than the affinity of said unlabeled antibody for said free fraction of said thyroxine, said tracer being selected from the group consisting of 3,3',5,5'-tetraiodothyroacetic acid, 3,5-diiodo-4-(3,5-diiodo-4-oxyphenyl)-benzenesulfonic acid, N-(methylphosphinoacetyl)-3,3',5,5'-tetraiodothyrosine, and N-(.epsilon.-aminocaprol)-3,3',5,5'-tetraiodothyrosine;
• and
• (d) measuring the amount of said tracer which is or is not bound to said unlabeled antibody and calculating from said tracer amount the concentration of said free fraction of said thyroxine.