Rapid, automated assay for progesterone on the Abbott AxSYM™ analyzer

Abstract

We describe an automated assay for progesterone (P4) in human serum and plasma with the Abbott AxSYMTM random-access immunoassay analyzer. In this one-step competitive assay, P4 immobilized onto latex microparticles competes with sample P4 for binding to a conjugate of alkaline phosphatase (AP) and anti-P4 antibody. Total CVs ranged from 3.4% to 8.2% in multiple precision studies conducted according to the 20-day NCCLS EP5-T protocol. The detection limit (zero calibrator + 2 SD) was 0.10 μg/L across 36 experiments. Values for diluted samples were 83–116% of expected. Recovery of P4 added to serum specimens was 92–115%. Cross-reactivities with 43 natural and synthetic steroids were 0–6.3%. No significant interference was detected from bilirubin, protein, erythrocytes, hemoglobin, triglycerides, or cholesterol. In a multisite correlation study, AxSYM P4 results compared well with results from a commercial RIA method (n = 1156; r = 0.976; slope = 1.03; y-intercept = 0.04). Assay throughput is >80 tests per hour in batch mode, 60 tests per hour with mixed load list configurations.

Liquid chromatography–mass spectrometry applications for quantification of endogenous sex hormones

“…Like estradiol and testosterone, immunoassays for progesterone quantification have been available since the 1970s but continue to suffer from limitations related to specificity and sensitivity (Kharma et al, 1972; O'Rorke, Kane, Gosling, Tallon, & Fottrell, 1994). For progesterone quantification, immunoassays can be affected by up to 10% cross‐reactivity from steroids like 17 α ‐hydroxyprogesterone and 11‐deoxycorticosterone (Wilson et al, 1998) as well as contraceptive steroids (Kharma et al, 1972). As such, NIST approached the standardization of progesterone by the development of an LC–MS/MS method using isotope dilution, which was validated and published in 2006 (Tai et al, 2006).…”

“…Zhang and colleagues developed an LC–MS/MS approach to quantify progesterone in plasma, in the presence of 17 α ‐hydroxyprogesterone, as this steroid is known to interfere with the immunoassay quantification of progesterone (Wilson et al, 1998; Zhang et al, 2008). Using a reversed‐phase gradient separation, the progesterone was chromatographically resolved from this interferant, and resolved using mass transitions.…”

Simultaneous quantitation of 17 endogenous adrenal corticosteroid hormones in human plasma by UHPLC-MS/MS and their application in congenital adrenal hyperplasia screening

“…As an approach, with simple and rapid operation, immunoassay-based methods have been routinely used for the measurement of plasma biomolecules in clinical settings, but nonspecific interaction remains a challenge for these methods. For instance, 17α-hydroxyprogesterone (17OHP), 11-deoxycorticosterone (DOC), and steroids were found to interfere with the measurement of progesterone in the immunoassay ( Wilson et al, 1998 ). Meanwhile, immunoassays cannot simultaneously measure multiple hormones in the same specimen.…”

Three‐dimensional structure, binding, and spectroscopic characteristics of the monoclonal antibody 43.1 directed to the carboxyphenyl moiety of fluorescein

“…The high quantum efficiencies, characteristic spectroscopic signatures and sensitivity to the microenvironment enabled implementation of fluorescein based probes for sensing intermolecular interactions in various laboratory and diagnostics applications. Subsequently, it prompted the use of anti‐fluorescein antibodies for the capture and detection of fluorescein labeled molecules . Typically, anti‐fluorescein antibodies have remarkable specificity to fluorescein and strongly quench its emission and therefore, may also present an interest as parts of molecular sensors and switches …”