Labeling of Steroids on Solid Phase

Up to four tetra-tert-butyl-1-[4-aminoacetamido)benzyl]diethylenetriaminetetrakis(acetato) derivatives of Fmoc glutamic acid (1) were attached to two steroids (17(alpha)-hydroxyprogesterone-3-O-carboxymethyloxime 2 and 1,3,5(10)-estratriene3,16(alpha),17(beta)-triol-6-one-6-O-carboxymethyloxime, 3)) on solid phase using an oligopeptide synthesizer. Upon deprotection and conversion to the corresponding europium(III) chelates, these steroid conjugates were used in DELFIA-based competitive fluoroimmunoassays. The more chelates conjugated to 17-(alpha)-hydroxyprogesterone, the more diluted antiserum could be used in an immunoassay for 17-(alpha)-hydroxyprogesterone, without any alteration of the measurement range. Hence, 17-(alpha)-hydroxyprogesterone tracers with several chelates are useful when a high serum dilution factor is desired i.e., when only a limited quantity of antiserum is available. The result demonstrates the suitability and usefulness of lanthanide(III) chelates as multilabels in bioaffinity assays.