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

A method for determining the concentration of the free fraction of an active compound, present in a biological fluid, in the presence of natural binders, the free and bound fractions of the active compound being in mutual equilibrium, by a) contacting a sample of the fluid with an unlabeled antibody, b) separating the sample from the unlabeled antibody, c) incubating the unlabeled antibody with a labeled substance (tracer) for cross-reaction with the antibody and d) measuring the amount of the tracer which is or is not bound to the antibody and calculating from this the concentration of the free fraction of the active compound, wherein the quantity of the unlabeled antibody and/or its affinity for the active compound are so small that they do not substantially effect the equilibrium between the free and bound fractions of the active compound, and the affinity of the tracer for the antibody is substantially higher or substantially lower than that of the active compound itself, and a test kit suitable for this method.