Cortisol

• R1a — Cortisol-ALP conjugate and capture particles reagent: Cortisol-alkaline phosphatase (bovine) conjugate and paramagnetic particles coated with goat anti-rabbit IgG in TRIS buffered saline, with surfactant, BSA matrix, and <0.1% sodium azide
• R1b — Rabbit anti-cortisol antiserum reagent: Rabbit antiserum to cortisol in TRIS buffered saline, with surfactant, BSA matrix, and <0.1% sodium azide
• Lumi-Phos PRO (chemiluminescent substrate):
• S0 — Access Cortisol Calibrator S0 (zero calibrator / dilution diluent):
• UniCel DxI Wash Buffer II:

• Beads:
  • Base material: Paramagnetic particles
  • Coating: Goat anti-rabbit IgG
• Capture reagent:
  • Host: Rabbit
  • Clonality: polyclonal
  • Recombinant: No
• Competitor analog:
  • Host: Bovine (alkaline phosphatase source)
  • Molecule type: Non-Ab hapten-enzyme conjugate (cortisol chemically conjugated to bovine alkaline phosphatase)
  • Conjugate/label: Alkaline phosphatase (bovine)
• Reagent diluent/carrier (in R1a and R1b):
  • Composition: TRIS buffered saline with surfactant, BSA matrix, and <0.1% sodium azide
• Wash buffer:
  • Purpose: Removes unbound reagent/conjugate from the magnetically retained particles before substrate addition
• Zero calibrator / dilution diluent (S0):
  • Purpose: Serves as the assay zero matrix for manual sample dilution and as the reconstitution diluent for the dried ethyl-acetate urine extract prior to analysis

Reaction procedure:

1. (Urine samples only, optional) Sample pretreatment/extraction: 1.0 mL urine is mixed with 1.0 mL ethyl acetate, vortexed, and centrifuged; 200 μL of the ethyl acetate (organic) layer is transferred, evaporated to dryness, and reconstituted in 200 μL of Calibrator S0 before being loaded as the 'sample.' This removes water-soluble conjugated cortisol metabolites so the extracted result better approximates free/extractable cortisol, consistent with the insert's separate extracted vs. unextracted urine reference ranges.
2. 25 μL of sample (serum, plasma, or urine — direct or extracted) is combined in a single reaction vessel with R1b (rabbit polyclonal anti-cortisol antibody) and R1a (paramagnetic particles coated with goat anti-rabbit IgG, together with cortisol-alkaline phosphatase conjugate) in one co-incubation step. Endogenous cortisol and the cortisol-ALP conjugate in R1a compete simultaneously for a limited number of binding sites on the rabbit anti-cortisol antibody, while that antibody (bound or unbound) is simultaneously captured onto the particles via the goat anti-rabbit IgG bridge — a one-step design, since competition and solid-phase capture occur in the same incubation rather than being separated by an intervening wash.
3. Particles, with whatever antibody/antigen complexes are bound, are retained in a magnetic field while unbound material — including any cortisol-ALP conjugate that lost the competition for antibody sites — is washed away with Wash Buffer II.
4. Lumi-Phos PRO chemiluminescent substrate is added; ALP on the captured cortisol-ALP conjugate hydrolyzes the substrate, generating light measured by the luminometer.
5. Light output is inversely proportional to sample cortisol concentration (more endogenous cortisol displaces more cortisol-ALP conjugate from the limited antibody, leaving less ALP on the bead and less signal); concentration is read from the stored calibration curve. Results above the top calibrator can be rerun after a manual 1:1 dilution with Calibrator S0.

• What's measured: Measures total immunoreactive cortisol (not specifically free/unbound cortisol) in serum, plasma, or urine; because the format is competitive with a polyclonal anti-cortisol antibody, structurally related steroids also cross-react to varying degrees (e.g., 11-deoxycortisol 17.8%, cortisone 8.06%, prednisolone 23.9% at the concentrations tested), so results reflect cortisol immunoreactivity broadly rather than one pure molecular species. In urine, unextracted results also include immunoreactive cortisol metabolites; solvent extraction removes water-soluble conjugated metabolites, giving a lower, more cortisol-specific result.
• Interference implications: Bilirubin, hemolysis, lipemia, and elevated total protein were all validated as having no effect at the tested levels. Heterophile antibodies (HAMA, human anti-goat antibodies) and other factors (rheumatoid factor, endogenous alkaline phosphatase, fibrin, ALP-binding proteins) are flagged only as a general, unquantified risk of 'erroneous results' — the insert does not state a direction and gives no assay-specific data, and does not mention biotin interference at all. Two directional effects are called out specifically: prednisolone/prednisone use causes falsely elevated cortisol via cross-reactivity, while pregnancy or estrogen-containing hormone therapy can cause falsely decreased serum cortisol (confirm with urinary free cortisol if inconsistent with the clinical picture).
• Non-linear response: The insert does not report a specific hook-effect/high-dose study for this assay. Structurally, because this is a one-step competitive format with a small-molecule, monovalent hapten analyte (cortisol has no second, spatially distinct site for a separate detection antibody the way a sandwich-format protein analyte would), there is no two-antibody 'sandwich' to disrupt at high analyte — very high endogenous cortisol simply out-competes more of the cortisol-ALP conjugate for the limited antibody, driving signal down monotonically toward the assay floor rather than producing the false-low 'hook' seen when a sandwich assay's capture and detection antibodies are each saturated separately by excess antigen.
• Intended use: Reported as a quantitative concentration (μg/dL or nmol/L) used alongside the clinical picture and dynamic testing (e.g., ACTH stimulation, dexamethasone suppression) to evaluate suspected Cushing's syndrome or Addison's disease; there is no qualitative cutoff/S-CO interpretation for this assay. If a serum result is inconsistent with the clinical picture (e.g., pregnancy or estrogen therapy, which can lower serum cortisol), the insert recommends a urinary free cortisol to confirm.
• Known bias: Access 2 vs. DxI 9000 comparison (CLSI EP09c, Passing-Bablok/Pearson): N=116, range 1.6-59 μg/dL, slope 1.01 (95% CI 0.99-1.03), intercept -0.20 (95% CI -0.41 to 0.056), r=1.00 — indicating essentially negligible bias between the two platforms.

• M — Streptavidin-coated microparticles: Streptavidin-coated microparticles 0.72 mg/mL; preservative
• R1 — Anti-cortisol-Ab~biotin: Biotinylated monoclonal anti-cortisol antibody (ovine) 20 ng/mL; danazol 20 μg/mL; MES buffer 100 mmol/L, pH 6.0; preservative
• R2 — Cortisol-peptide~Ru(bpy): Cortisol derivative (synthetic), labeled with ruthenium complex 20 ng/mL; danazol 20 μg/mL; MES buffer 100 mmol/L, pH 6.0; preservative

• Beads:
  • Coating: Streptavidin
• Capture reagent:
  • Host: ovine
  • Clonality: monoclonal
  • Conjugate/label: biotin
• Competitor analog:
  • Molecule type: Non-Ab, synthetic cortisol derivative (small-molecule hapten conjugate), not an antibody
  • Conjugate/label: ruthenium complex (Ru(bpy))
• Reagent diluent / analyte-liberating agent (co-formulated within R1 and R2):
  • Composition: Danazol 20 μg/mL in MES buffer 100 mmol/L, pH 6.0
  • Purpose: Danazol displaces/liberates endogenous cortisol from its plasma-binding proteins (corticosteroid binding globulin, albumin) so total cortisol -- not only the 2-5% free fraction -- is available to react in the assay.
• Sample dilution diluent:
  • Purpose: Used to dilute serum/plasma samples above the measuring range (recommended 1:10) so the diluted result falls back within the reportable range.

Reaction procedure:

1. First incubation (co-incubation): 6 μL sample is mixed simultaneously with R1 (biotinylated anti-cortisol monoclonal antibody) and R2 (ruthenium-labeled cortisol derivative). Danazol in R1/R2 liberates endogenous cortisol from its serum binding proteins so total, not free, cortisol is available to react. Endogenous (unlabeled) cortisol and the labeled R2 derivative then compete for the limited biotinylated-antibody binding sites on R1 -- the classic single-antibody competitive design (not a two-binder sandwich, so the analyte does not need to bridge two reagents).
2. Second incubation: M (streptavidin-coated microparticles) is added; whatever R1-antibody complex formed (whether it captured endogenous cortisol or the labeled R2 derivative) becomes bound to the solid phase through the biotin-streptavidin interaction.
3. The reaction mixture is aspirated into the measuring cell, the microparticles are magnetically captured onto the electrode, and unbound material (including unbound R2 label) is washed away with ProCell II M.
4. A voltage is applied to the electrode, inducing chemiluminescent emission from bound Ru-label, read by a photomultiplier. Because R2 and endogenous cortisol compete for the same antibody sites, signal is inversely proportional to sample cortisol concentration.
5. Concentration is read from an instrument-specific calibration curve (2-point calibration plus a leading calibration curve supplied via cobas link). Total assay duration is 18 minutes.

• What's measured: The assay measures total serum/plasma cortisol (danazol in R1/R2 liberates cortisol from CBG and albumin, so the normally-bound ~95-98% of circulating cortisol is included, not just the 2-5% free fraction) or, for saliva, the largely unbound cortisol present there. Because it is a single-antibody competitive format, results reflect the antibody's overall specificity for the cortisol structure -- cross-reacting steroids (6-α-methylprednisolone 14.7%, prednisolone 7.32%, cortisone 4.68%, 11-deoxycortisol 3.62%, prednisone 2.33%, 21-deoxycortisol 0.515%) will read as cortisol-equivalent signal, which matters for patients on glucocorticoid therapy or undergoing metyrapone testing.
• Interference implications: Bilirubin, hemoglobin, lipemia, biotin (≤287 nmol/L), rheumatoid factor, IgG/IgA/IgM, and 16 commonly used drugs showed no significant effect at the concentrations tested. Because the assay uses biotin-streptavidin capture, patients on high-dose biotin (>5 mg/day) still need an ≥8-hour washout before sampling, despite the validated-none finding at the tested concentration. Rare, very-high-titer antibodies against the assay's own anti-cortisol antibody, streptavidin, or ruthenium label can interfere (a heterophile-type risk), though the design is said to minimize this. Separately, several true cross-reactivity effects (not matrix interference) push results falsely high: glucocorticoid therapy (prednisone/prednisolone/6-α-methylprednisolone), 21-hydroxylase deficiency, and metyrapone testing.
• Non-linear response: The insert does not report a specific high-dose hook/prozone check for this assay. Mechanistically this is a single-antibody competitive format, not a two-binder sandwich -- signal comes from how much labeled cortisol derivative (R2) remains bound to the one antibody (R1) after competition, so there is no second (detection) antibody for excess antigen to saturate independently. That removes the classic sandwich-assay hook mechanism (which requires both capture and detection antibodies to be separately saturated by monovalent binding at very high antigen); a competitive assay instead simply approaches a floor signal as cortisol rises, without a reversal/false-low high-dose hook.
• Intended use: Quantitative result reported in nmol/L, μg/dL, or μg/L. Used to diagnose Cushing's syndrome (cortisol overproduction) or Addison's disease (cortisol deficiency) and to monitor therapy (e.g. dexamethasone suppression testing, hormone replacement). Because of the pronounced circadian rhythm, results must be read against a time-of-day-matched reference interval (morning vs. afternoon vs. midnight values differ several-fold), and interpreted alongside clinical history rather than as a stand-alone cutoff.
• Known bias: Versus ID-GC/MS reference method (IRMM/IFCC-451 panel, n=34): Passing/Bablok y=1.00x+4.96, r=0.998, essentially unbiased. Cross-platform comparisons (cobas e601 vs e801, n=145; e402 vs e801, n=198) also showed high correlation (r=0.998-0.999) with slopes of 0.93-1.03, indicating close agreement across cobas e platforms.

• M — Streptavidin-coated microparticles: Streptavidin-coated microparticles 0.72 mg/mL; preservative
• R1 — Anti-cortisol-Ab~biotin (biotinylated anti-cortisol monoclonal antibody): Biotinylated monoclonal anti-cortisol antibody (mouse) 18 ng/mL; danazol 20 μg/mL; MES buffer 100 mmol/L pH 6.0; preservative
• R2 — Cortisol-peptide~Ru(bpy) (ruthenium-labeled cortisol derivative): Cortisol derivative (synthetic), labeled with ruthenium complex, 5 ng/mL; danazol 20 μg/mL; MES buffer 100 mmol/L pH 6.0; preservative

• Beads:
  • Base material: Magnetic microparticle
  • Coating: Streptavidin
• Capture reagent:
  • Host: mouse
  • Clonality: monoclonal
  • Conjugate/label: biotin
• Competitor analog:
  • Molecule type: Non-Ab synthetic hapten-label conjugate (cortisol derivative, not a binding protein)
  • Conjugate/label: ruthenium(bipy)3 complex (Ru(bpy))
• R1/R2 assay diluent (MES buffer with danazol), 100 mmol/L pH 6.0:
  • Composition: MES (2-morpholino-ethane sulfonic acid) buffer 100 mmol/L, pH 6.0; danazol 20 μg/mL; preservative
  • Purpose: Danazol liberates endogenous cortisol from its binding proteins (corticosteroid-binding globulin, albumin) so it is free to compete for antibody binding sites; MES buffer at pH 6.0 maintains conditions for antibody-hapten binding
• System wash/measuring-cell solution (ProCell II M):
  • Purpose: Removes unbound substances (unbound R2, unbound endogenous cortisol, other sample matrix components) from the magnetically captured microparticles before electrochemiluminescent measurement

Reaction procedure:

1. Sample (6 μL) is co-incubated with R1 (biotinylated anti-cortisol monoclonal antibody) -- 1st incubation. Endogenous urinary cortisol (present as free/unconjugated UFC, with danazol in R1 additionally liberating any residual protein-bound cortisol) binds free antibody sites, forming immunocomplex in an amount proportional to sample cortisol concentration.
2. M (streptavidin-coated microparticles) and R2 (Ru-labeled cortisol derivative) are added together -- 2nd incubation. R2 competes for whatever biotinylated-antibody sites remain unoccupied after step 1, and all biotinylated-antibody-containing species become bound to the microparticles via biotin-streptavidin interaction. R1, sample, and R2 are combined without an intervening wash before bead capture, but this is expected/benign competitive-assay logic (not sandwich hook risk) since R1 is a fixed limiting reagent and only one antibody site per molecule is being partitioned between endogenous cortisol and R2.
3. The reaction mixture is aspirated into the measuring cell; microparticles are magnetically captured onto the electrode surface, and unbound substances (including unbound R2 and unbound endogenous cortisol) are washed away with ProCell II M.
4. A voltage applied to the electrode induces electrochemiluminescent emission from bead-bound Ru(bpy) label; a photomultiplier measures the light. Because R2 and endogenous cortisol compete for the same limited antibody sites, signal is inversely proportional to sample cortisol concentration.
5. Concentration is read from an instrument-specific calibration curve (2-point calibration adapted to a master curve provided via cobas link).

• What's measured: Measures urinary free cortisol (UFC) -- cortisol excreted into urine essentially unaltered, used directly without extraction (unlike serum, where the majority of cortisol circulates protein-bound, urine cortisol is largely free, though danazol in R1/R2 is included to liberate any residual protein-bound fraction). Notable cross-reactivity with structurally related steroids: 11-deoxycortisol (24.3%) and allotetrahydrocortisol (11.3%) are the largest cross-reactants, so conditions or tests that raise these metabolites (metyrapone testing, 21-hydroxylase deficiency raising 21-deoxycortisol) can falsely elevate the reported cortisol result.
• Interference implications: Endogenous substances (bilirubin, hemoglobin, lipemia, rheumatoid factor, immunoglobulins, albumin, creatinine, glucose, NaCl, urea) and most tested drugs, including many corticosteroid-class drugs, show no interference at tested concentrations. Biotin is clean up to 50,000 ng/mL but causes progressively larger falsely elevated results above that -- relevant for patients on high-dose biotin supplementation, since urinary biotin from supplement users has been reported as high as ~31,700 ng/mL in the literature. Heterophile/anti-reagent antibodies (against the analyte-specific antibody, streptavidin, or ruthenium) can rarely interfere but are described as minimized, not eliminated, by test design. Prednisolone and hydrocortisone at therapeutic doses cause falsely elevated cortisol (expected cross-reactivity with closely related steroids), while 6-methylprednisolone does not interfere up to 0.157 mg/dL. Pregnancy, oral contraceptives, and estrogen therapy raise cortisol physiologically, independent of assay interference.
• Non-linear response: This is a competitive, not sandwich, format, so the classic bivalent-saturation hook mechanism doesn't directly apply -- there is no two-epitope sandwich to disrupt at high antigen. Instead, R1 (the biotinylated antibody) is a fixed, limiting reagent whose sites are partitioned between endogenous cortisol and the Ru-labeled R2 analog; as endogenous cortisol rises it progressively displaces R2, driving signal down monotonically toward the assay floor rather than producing a paradoxical high-dose hook. The insert does not report a discrete hook-effect verification study, consistent with competitive designs generally being far less hook-prone than two-site sandwich immunoassays.
• Intended use: Reported quantitatively (nmol/L, μg/dL, or μg/L); for 24-hour urine collections, multiply the analyzer result by the 24-hour urine volume (L/24h) to obtain total UFC excretion, which is the value used clinically. Reference interval from this insert: 24.8-238 nmol/24h (8.98-86.2 μg/24h). UFC is one of several first-line tests recommended for suspected Cushing's syndrome (alongside late-night salivary cortisol and dexamethasone suppression testing) and should be interpreted together with the patient's medical history and clinical exam, not as a standalone diagnostic.
• Known bias: Vs. a commercially available method (native 24-h urine, n=125): Passing/Bablok y=1.051x-0.810 (τ=0.933), linear regression y=1.092x-1.00 (r=0.991), in μg/dL. Cobas e402 vs. e801 (same assay, n=111): Passing/Bablok y=1.000x-0.0362 (τ=0.982), linear regression y=1.023x-0.128 (r=0.999), in μg/dL, showing good inter-analyzer agreement.

• Lite Reagent — Acridinium-ester-labeled cortisol (competitor): Cortisol (~5 ng/mL) labeled with acridinium ester in buffered saline; sodium salicylate (~50 mg/mL); sodium azide (0.1%); preservatives
• Solid Phase — Rabbit anti-cortisol antibody bound to mouse anti-rabbit-coated paramagnetic particles: Rabbit anti-cortisol antibody (~1.1 µg/mL) bound to mouse monoclonal anti-rabbit IgG antibody (~56 µg/mL) covalently coupled to paramagnetic particles in buffered saline; sodium azide (0.1%); preservatives
• Atellica IM Acid — Chemiluminescence trigger reagent (acid):
• Atellica IM Base — Chemiluminescence trigger reagent (base):
• Multi-Diluent 3 — Atellica IM Multi-Diluent 3 (ancillary reagent for automated dilutions): Human plasma; sodium azide (0.1%)
• Atellica IM Cortisol Urine Reconstitution Buffer (optional urine extraction reagent):

• Beads:
  • Base material: paramagnetic particles
  • Coating: Mouse monoclonal anti-rabbit IgG antibody (~56 µg/mL), covalently coupled
• Capture reagent:
  • Host: rabbit
  • Clonality: polyclonal
• Conjugate/bridge reagent:
  • Host: mouse
  • Clonality: monoclonal
• Competitor analog:
  • Molecule type: Cortisol-acridinium ester conjugate (small-molecule hapten analog, not a binder protein)
  • Conjugate/label: acridinium ester
• Wash buffer:
  • Purpose: Special reagent water used post-competitive-incubation to wash away unbound acridinium-ester-labeled cortisol, so only solid-phase-retained label contributes to the RLU signal.
• Assay/dilution diluent:
  • Composition: Human plasma; sodium azide (0.1%)
  • Purpose: Matrix-matched diluent used for automated onboard dilution of serum/plasma samples exceeding the upper measuring interval; the insert does not describe a complex-dissociating or complex-stabilizing function beyond general dilution.
• Sample pretreatment (urine, optional):
  • Purpose: Used to reconstitute extracted urine samples prior to analysis; the insert references a separate extraction procedure for this reagent but does not detail its mechanism in this document.

Reaction procedure:

1. Dispense 20 µL of sample into a cuvette and incubate alone for 3 minutes at 37°C – equilibrates the sample before reagent addition; no competitor or antibody is present yet.
2. Dispense 50 µL of Lite Reagent (acridinium-ester-labeled cortisol) and 250 µL of Solid Phase (rabbit anti-cortisol antibody bound, via a mouse anti-rabbit IgG bridge, to paramagnetic particles) together and co-incubate for 5 minutes at 37°C – this single simultaneous co-incubation is the competitive step: endogenous patient cortisol and the fixed amount of labeled cortisol in Lite Reagent compete for the limited rabbit anti-cortisol antibody binding sites on the bead.
3. Magnetically separate, aspirate, and wash the cuvette with special reagent water – removes unbound (excess) labeled cortisol that lost the competition, leaving only bead-retained acridinium-ester label.
4. Dispense 300 µL each of Atellica IM Acid and Atellica IM Base – triggers the chemiluminescent reaction of the acridinium ester label remaining on the solid phase.
5. Report results – relative light units (RLUs) are inversely proportional to the amount of cortisol in the patient sample and converted to concentration via the stored master curve.

• What's measured: The assay measures immunoreactive cortisol in serum, EDTA/lithium-heparin plasma, or urine as detected by the rabbit polyclonal anti-cortisol antibody; no sample pretreatment step to isolate free vs. protein-bound cortisol is described, so the result reflects total immunoreactive cortisol in the sample. The polyclonal capture antibody shows substantial cross-reactivity with several structurally related endogenous steroids and, notably, with synthetic corticosteroids – prednisolone cross-reacts at up to 120% and prednisone at up to 35% – so results in patients on these therapies (or with metyrapone-induced 11-deoxycortisol accumulation, ≤25% cross-reactivity) should be interpreted with caution as they may not reflect endogenous cortisol alone.
• Interference implications: Common anticoagulants (dipotassium EDTA, heparin) and hemolysis/icterus/lipemia at the stated thresholds show no clinically significant interference (bias generally within a few percent). Urine matrix components (protein, salts, urea, creatinine, glucose, boric acid) also show no significant interference at tested levels. Biotin interference is not discussed in this insert. Heterophilic antibodies are flagged as a general immunoassay risk that could cause falsely elevated or falsely depressed results, though the assay is designed to minimize this; no insert-specific validation data is given, so this is a suspected rather than a validated-none risk. The clinically important interferences here are cross-reactivity-driven: prednisolone/prednisone therapy and metyrapone therapy both cause falsely elevated results due to structurally related steroids reacting with the anti-cortisol antibody.
• Non-linear response: This is a competitive, monovalent-binding format (a single rabbit anti-cortisol antibody site per bead, not a two-antibody sandwich), so the classic bivalent-sandwich high-dose hook mechanism does not apply here – there is no second detection antibody arm that excess antigen can saturate independently to falsely suppress signal at very high concentrations. The insert does not report a dedicated hook-effect verification study for this assay, consistent with hook effect being a sandwich-assay-specific concern; instead, serial dilution recovery (86.2%–114.0%) and spiking recovery (84.8%–113.0%) studies demonstrate that signal continues to respond proportionally (monotonically decreasing RLU with increasing cortisol) across the tested concentration ranges, with samples above the 75.00 µg/dL upper limit simply requiring dilution rather than being at risk of a falsely low hooked result.
• Intended use: This is a quantitative assay reported in µg/dL or nmol/L. Because circulating cortisol follows a diurnal pattern, a single random serum cortisol has limited diagnostic value alone and is typically interpreted alongside dynamic function testing – the ACTH stimulation test to evaluate Addison's disease, or the dexamethasone suppression test to evaluate Cushing's syndrome. A 24-hour urinary cortisol measurement (using the insert's conversion formulas) is described as the preferred initial screen for Cushing's syndrome since it is not subject to diurnal variation. Results should always be interpreted together with the patient's medical history and clinical presentation, and with awareness that prednisolone/prednisone or metyrapone therapy can cause falsely elevated results via cross-reactivity.
• Known bias: Designed to correlate with the ADVIA Centaur Cortisol assay at r ≥ 0.95 and slope 1.0 ± 0.1. Observed: serum y = 1.02x + 0.14 µg/dL (r = 1.00); direct urine y = 1.10x + 0.59 µg/dL (r = 0.98); extracted urine y = 1.07x + 0.20 µg/dL (r = 1.00).

• Coated wells (solid phase): Streptavidin (bacterial) coated on the microwell; binds ≥3 ng biotin/well
• Conjugate reagent (HRP-cortisol): HRP-cortisol conjugate ≥100 ng/mL in buffer with human plasma, bovine serum albumin, bovine gamma globulin and antimicrobial agent
• Biotinylated antibody reagent: Biotin-sheep polyclonal anti-cortisol antibody, binds ≥0.719 nmol cortisol/mL, in buffer with bovine serum albumin and antimicrobial agent
• Signal Reagent: Luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent (a substituted acetanilide)
• Universal Wash Reagent:
• High Sample Diluent B:

• Beads:
  • Coating: Streptavidin (bacterial-derived); binds ≥3 ng biotin/well
• Capture reagent:
  • Host: sheep
  • Clonality: polyclonal
  • Conjugate/label: biotin
• Competitor analog:
  • Molecule type: Non-Ab reagent -- hapten-enzyme conjugate (cortisol conjugated to horseradish peroxidase)
  • Conjugate/label: horseradish peroxidase (HRP)
• Urine sample pretreatment (extraction):
  • Composition: Dichloromethane (organic solvent)
  • Purpose: Liquid-liquid extraction of lipophilic cortisol (and structurally related, cross-reacting steroid metabolites) from urine into an organic phase, away from the polar urine matrix; the extract is evaporated and reconstituted in High Sample Diluent B before testing. This is a physical partition step, not an immune-complex dissociation.
• Sample resuspension / over-range dilution diluent (VITROS High Sample Diluent B):
  • Purpose: Reconstitutes the dried, dichloromethane-extracted urine cortisol residue for testing, and is separately used by the system for automated up-to-2-fold dilution of serum/plasma samples exceeding the measuring range; not stated to alter protein/complex structure.
• Wash buffer (VITROS Universal Wash Reagent):
  • Purpose: Removes unbound conjugate, unbound antibody, and uncomplexed cortisol from the well after the competitive binding incubation, prior to signal development.

Reaction procedure:

1. Sample (25 µL serum/plasma, or dichloromethane-extracted and reconstituted urine) is added to a streptavidin-coated well together with the Biotinylated Antibody Reagent (biotin-sheep anti-cortisol) and the Conjugate Reagent (HRP-cortisol), all combined in a single 30-minute, 37°C incubation.
2. During this co-incubation, endogenous sample cortisol and the HRP-cortisol conjugate compete for a limited number of cortisol-binding sites on the biotinylated antibody; simultaneously the biotinylated antibody (whichever species it has bound) is captured onto the well via the biotin-streptavidin interaction. Because capture antibody, labeled competitor, and sample analyte are all present together in one incubation (a one-step design), the amount of HRP-conjugate ultimately bound is inversely related to sample cortisol concentration.
3. Unbound conjugate, unbound antibody, and free (uncomplexed) cortisol are removed by washing with Universal Wash Reagent.
4. Signal Reagent (luminol derivative + peracid salt + acetanilide electron-transfer agent) is added; bound HRP catalyzes oxidation of the luminol derivative to generate a chemiluminescent signal, which the electron-transfer agent amplifies and prolongs.
5. The system reads the light signal, which is inversely proportional to sample cortisol concentration, and interpolates the result from the stored (rescaled) four-parameter logistic calibration curve.

• What's measured: Measures immunoreactive cortisol via a polyclonal sheep antibody. In serum/plasma this is intended to reflect total circulating cortisol; the insert does not describe an explicit protein-binding-disruption step, but notes cortisol circulates largely bound to corticosteroid-binding globulin. Because the capture antibody is polyclonal, it cross-reacts appreciably with structurally related corticosteroids and cortisol metabolites -- notably prednisolone (26.8% at 50% displacement), 6-α-methylprednisolone (5.94%), fludrocortisone (3.85%), and 11-β-hydroxyprogesterone (2.52%), with smaller cross-reactivity from tetrahydrocortisol, corticosterone, 11-deoxycortisol and cortisone -- so results in patients on exogenous corticosteroids (especially prednisolone) should be interpreted cautiously. For urine, the lack of a chromatographic purification step (only a dichloromethane liquid-liquid extraction) means these same cross-reacting steroids/metabolites can co-extract and contribute to the reported 'free cortisol' value.
• Interference implications: Hemolyzed and turbid specimens are not recommended for testing; hemoglobin produces a dose-dependent negative (falsely decreased) bias. Biotin up to 40 ng/mL causes ≤10% change, but above that threshold it falsely elevates results, with very large relative bias at high biotin levels, particularly at low baseline cortisol -- relevant given that high-dose biotin supplements can push serum biotin into that range. Notably, biotin does NOT interfere in the urine test even at very high concentrations, because the dichloromethane extraction step removes biotin before testing. Azide at a low spiked concentration (20 mg/dL) showed no interference, but the insert separately flags that control/proficiency materials with azide ≥0.05% have been shown to interfere -- a QC-matrix-specific caution rather than a patient-sample one. Heterophile/anti-animal antibodies are a general, unquantified risk in patients with animal exposure or prior animal-serum-product treatment; discordant results should prompt confirmatory testing. Bilirubin, triolein (a lipemia surrogate), dipyrone, and Intralipid showed no significant interference at the concentrations tested.
• Non-linear response: The insert does not report a dedicated high-dose hook study for this assay. By design, this is a one-step competitive immunoassay for a small steroid hapten: cortisol cannot bridge two antibody molecules the way a larger, multi-epitope antigen does in a sandwich assay, so the bivalent two-site saturation mechanism behind a classic sandwich hook effect does not apply here. Instead, at very high sample cortisol, essentially all antibody sites are expected to be occupied by unlabeled cortisol, driving the bound HRP-conjugate signal toward its lowest value -- i.e., extremely high concentrations should read toward the top of the measuring range or beyond it, not falsely mimic a normal/low result, though the insert presents no data confirming behavior above its stated 1700 nmol/L upper measuring limit.
• Intended use: Result is a quantitative cortisol concentration (nmol/L or µg/dL) to be interpreted against a time-of-day-specific serum reference interval (higher before 10:00 am, lower after 5:00 pm) or a 24-hour urine reference interval, reflecting cortisol's circadian secretion pattern. The insert cautions that a single measurement gives limited information about HPA-axis status and should be interpreted alongside other tests of HPA function (e.g., dynamic stimulation/suppression testing) rather than as a stand-alone diagnostic result.
• Known bias: Lot-to-lot agreement (reagent lots below 1000 vs. 1000 and above) was strong for serum (Deming slope 0.986, r=0.987) but weaker for urine (slope 0.798, r=0.976, intercept -22.0 nmol/L), suggesting some lot-dependent difference in urine recovery. Cross-system agreement (Passing-Bablok) was good: serum 3600 vs. ECi/ECiQ slope 0.941 (intercept 8.14 nmol/L), 5600 vs. ECi/ECiQ slope 0.966 (intercept 1.45 nmol/L); urine 3600 vs. ECi/ECiQ slope 0.991, 5600 vs. ECi/ECiQ slope 0.970. Calibration is traceable to in-house reference calibrators value-assigned against GC-MS.