Abstract: The invention relates to a method for the selective separation of a given protein substance having an affinity for a ligand from other substances some of them having the same affinity for the ligand but also having a high molecular weight, higher than that of said protein substance, said substances being in solution.The method consists of filtering said substances on a gel which excludes said high molecular weight substances, said gel being coupled with a ligand for which said protein substance has affinity.The method may be applied to enzymatic and especially to immunoenzymatic assays.

Abstract: The invention relates to a novel enzymatic labelling agent for immunoenzymatic assays. The labelling agent is .DELTA..sub.5, 3-keto-steroid isomerase. The enzymatic activity bound to the antibody can be revealed advantageously by: (1) direct reading on the supernatent liquor or on the cake obtained by precipitation by means of a solvent, such as polyethylene glycol, with the use of soluble antibodies; (2) reading in solution after liberation of the enzyme from the antigen-antibody complex by a reducing agent, such as dithiotreitol; (3) immediate reading after filtration of a substrate solution on the insolubilized antibody-antigen-enzyme complex. The method can be used for immunoenzymatic assays automatically on substances present in very low concentrations, such as hypophysial hormones and plasmatic steroids.