The reasons for these discrepancies are related to the origins of steroid immunoassays, which were developed in 1969 (
29) but delayed for a decade after the invention of peptide immunoassay because additional steps were required to adapt immunoassay methodology to valid measurement of nonimmunogenic small molecules such as steroids. Developing steroid-specific antibodies required conjugating steroids as haptens to larger immunogenic carrier proteins via small multivalent reactive bridge compounds. However, this made steroid antibodies that were epitope “blind” to the conjugation site, allowing for undesirable cross-reactivity with structurally related steroids (e.g., structurally related steroid precursors, metabolites, and conjugates produced by phase II metabolism). The original in-house steroid immunoassays developed in the 1970s used solvent extraction, chromatography, and structurally authentic (H3, C14) tracers, a triplet of validity criteria for steroid immunoassay that removed structurally related steroids as well as nonspecific matrix interference in the immunoassay reactions. In the ensuing decades, E2 immunoassays were commercialized primarily to monitor ovarian responses to gonadotropin stimulation, where excessive serum E2 response was a risk indicator for dangerous overdosage effects such as high-order multiple pregnancies and/or life-threatening ovarian hyperstimulation syndrome. However, making a quantal distinction between dangerously high E2 concentrations (>2000 pmol/L) and typical premenopausal concentrations (200–800 pmol/L) did not require quantitative accuracy at physiological E2 concentrations. Subsequently, ultrasound monitoring of follicular growth has reduced the importance of this application of serum E2 immunoassays. The subsequent growing demand for steroid immunoassays in clinical practice and research resulted in assay simplification to adapt steroids into semi-automated multiplex platforms and 1-plate/tube kit formats, preferred by routine pathology and research labs, respectively.
This simplification eliminated preassay purification steps (extraction, chromatography) as well as β-scintillation counting, with the latter forcing the replacement of structurally authentic steroid tracers with bulky conjugated steroids, which allowed for more convenient nonradioactive assay readouts. However, eliminating the triplet of validity criteria (preassay extraction and chromatography, authentic tracers) sacrificed the specificity of E2 immunoassays and revealed the vulnerabilities of direct steroid immunoassay to artifacts from steroid cross-reactivity and matrix interference. Although the original validated steroid immunoassays are now confined to a few long-established laboratories, there is evidence that some but not all the limitations of direct E2 immunoassays can be overcome by preassay solvent extraction (
12,
28). However, since our study did not include any classical (indirect) estradiol immunoassays, it is not clear whether meeting the original triplet validity criteria is sufficient to overcome all limitations of direct E2 immunoassays, and it remains possible that suboptimal antibody specificity could also contribute to the nonspecificity of steroid immunoassays
