Radioimmunoassay of steroids in biological fluids

Abstract

The principle of radioimmunoassay has been applied to many organic compounds of biological interest. One of the requirements for the application of radioimmunoassay to the measurement of a compound is that this compound must be antigenic and must elicit high affinity antibodies. Steroids when covalently linked to protein carriers behave like haptenic groups, and specific antibodies may be evoked against them by active immunization. Strong covalent bonds between the steroid and the protein are required to prevent in vivo lysis of these bonds during immunization. Peptide bonds are biologically stable. To prepare such bonds, a free carboxyl group is attached to the steroid molecule using the hydroxyl and ketone groups of the steroid molecule as anchoring points. Peptide bonds are formed between these carboxyl groups and the ϵ-amino groups of lysine residue of albumin. Some of these steroid-protein conjugates were found to be effective in producing specific antibodies against the steroid residues, and the antisera obtained have been used by many investigators, including us, as specific binding reagents in the radioimmunoassay of these steroids in biological fluids. Because of the multiplicity of closely related steroids in biological fluids, some form of Chromatographic purification is usually required to obtain specificity. For some steroids with unique configuration and for synthetic steroids not normally present in biological fluids, it may be possible to perform the assay directly, without extraction and chromatography. Also, if a particular steroid exists in a particular biological fluid in relatively large concentration compared to related interfering steroids, it may be measured directly. Because of the ease of performance, radioimmunoassay lends itself to automation, and such an adaptation for the measurement of steroids ought to be forthcoming.