Free Testosterone AccuBind ELISA Test System

K181017 · Monobind, Inc. · CDZ · Jul 20, 2018 · Clinical Chemistry

Device Facts

• Record ID: K181017
• Device Name: Free Testosterone AccuBind ELISA Test System
• Applicant: Monobind, Inc.
• Product Code: CDZ · Clinical Chemistry
• Decision Date: Jul 20, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1680
• Device Class: Class 1

Indications for Use

The Free Testosterone AccuBind® ELISA Test System is an Enzyme Immunoassay (EIA) for the quantitative measurement of free testosterone in human serum. Measurement of free testosterone is used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males and in females; hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries and adrogenital syndromes.

Device Story

Competitive enzyme immunoassay (EIA) for quantitative measurement of free testosterone in human serum; utilizes immobilized testosterone antibody, enzyme-antigen conjugate (HRP-labeled), and native antigen from patient sample. Competitive reaction occurs between native free antigen and enzyme-antigen conjugate for limited binding sites on antibody-coated microplate. After equilibrium, unbound antigen is removed via decantation/aspiration. Substrate (TMB/hydrogen peroxide) added to produce blue color, followed by acid stop solution to yield yellow color. Microplate reader (450nm/620nm) measures absorbance; dose-response curve generated from serum calibrators determines unknown sample concentration. Used in clinical laboratories by trained personnel. Output assists clinicians in diagnosing/treating androgen-related disorders (hypogonadism, hirsutism, virilization).

Clinical Evidence

Bench testing only. Precision study (n=80 per sample) showed total CVs 7.8-12.4%. Linearity confirmed across 0.11-60 pg/mL (R^2=0.9888). LoB 0.0295 pg/mL, LoD/LoQ 0.0519 pg/mL. Interference and cross-reactivity studies showed no significant bias (>10%) for tested substances. Method comparison with predicate (n=137) yielded y=1.017x-0.244, r=0.997. Reference range established using 261 healthy subjects.

Technological Characteristics

Competitive ELISA; solid-phase microplate coated with testosterone antibody. Reagents: HRP-labeled testosterone analog conjugate, TMB substrate, stop solution. Detection: colorimetric microplate reader at 450nm. Standards: CLSI EP06-A (linearity), CLSI EP17-A (limits), CLSI EP07-A2 (interference/cross-reactivity), CLSI C28-A3 (reference ranges).

Indications for Use

Indicated for quantitative measurement of free testosterone in human serum for diagnosis and treatment of androgen-related disorders, including primary/secondary hypogonadism, impotence, hirsutism, and virilization in males and females.

Regulatory Classification

Identification

A testosterone test system is a device intended to measure testosterone (a male sex hormone) in serum, plasma, and urine. Measurement of testosterone are used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, delayed or precocious puberty, impotence in males and, in females hirsutism (excessive hair) and virilization (masculinization) due to tumors, polycystic ovaries, and adrenogenital syndromes.

Predicate Devices

• EiAsy Free Testosterone EIA (k030730)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k181017

B. Purpose for Submission:
New device

C. Measurand:
Testosterone (free)

D. Type of Test:
Quantitative, Enzyme Immunoassay (EIA)

E. Applicant:
Monobind, Inc.

F. Proprietary and Established Names:
Free Testosterone AccuBind ELISA Test System

G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| CDZ | Class I, reserved | 21 CFR 862.1680
Testosterone test system | Clinical Chemistry (75) |

H. Intended Use:

1. Intended use(s):
See Indication(s) for use below.

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2. Indication(s) for use:

The Free Testosterone AccuBind ELISA Test System is an Enzyme Immunoassay (EIA) for the quantitative measurement of free testosterone in human serum. Measurement of free testosterone is used in the diagnosis and treatment of disorders involving the male sex hormones (androgens), including primary and secondary hypogonadism, impotence in males and in females; hirsutism (excessive hair) and virilization (masculination) due to tumors, polycystic ovaries and adrogenital syndromes.

3. Special conditions for use statement(s):

For Prescription Use only.

4. Special instrument requirements:

Microplate Reader with 450nm and 620nm wavelength absorbance capability.

I. Device Description:

The Free Testosterone AccuBind ELISA Test System consists of the following:

- Seven vials of 1 mL serum reference calibrators for Free Testosterone containing testosterone in human serum with preservative at concentrations of 0, 0.2, 1.0, 2.5, 7.5, 20, and 60 pg/mL.
- Three vials of 1 mL controls (one low, one medium, and one high level) containing free testosterone in human serum with preservative.
- Free Testosterone Enzyme Reagent: One vial of 6 mL Testosterone (Analog)-horseradish peroxidase (HRP) conjugate in a protein stabilizing matrix.
- Substrate A: one vial of tetramethylbenzidine (TMB) in buffer.
- Substrate B: one vial of hydrogen peroxide in buffer.
- One 96-well microplate coated with testosterone antibody and packaged in an aluminum bag with a drying agent.
- One vial containing hydrogen peroxide in buffer.
- One vial of 20 mL concentrated wash solution.
- One vial of 8 mL stop reaction solution.

J. Substantial Equivalence Information:

1. Predicate device name(s):

EiAsy Free Testosterone EIA

2. Predicate 510(k) number(s):

k030730

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3. Comparison with predicate

Similarities and Differences

| Items | Candidate Device
Free Testosterone AccuBind
ELISA Test System
(k181017) | Predicate Device
EiAsy Free Testosterone EIA
(k030730) |
| --- | --- | --- |
| Intended Use | The direct quantitative determination of free testosterone by enzyme immunoassay in human serum. | Same |
| Antibody | Utilizes a highly specific rabbit polyclonal antibody at a low binding capacity. | Same |
| Sample Type | Human serum | Same |
| Test Principle | Competitive Enzyme Immunoassay | Same |
| Detection Instrument | Microplate Colorimeter Reader | Same |
| Microplate coating | Antibody coated microwell plate | Same |
| Calibrators | Seven vials containing testosterone in human serum with preservative. | Six vials containing testosterone in human serum with preservative. |
| Controls | Three vials containing testosterone in human serum. | Two vials containing testosterone in human serum. |
| Measuring range | 0.11 - 60 pg/mL | 0.018 - 60 pg/mL |

K. Standard/Guidance Document Referenced (if applicable):

CLSI EP05-A3 Evaluation of Precision Performance of Quantitative Measurement Procedures (October 2014).

CLSI EP06-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach (April 2003).

CLSI EP17-A2 Evaluation of Detection Capacity for Clinical Laboratory Measurements Procedures (June 2012).

CLSI EP28-A3C Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory (October 2010).

CLSI EP07-A2 Interference Testing in Clinical Chemistry (November 2005).

CLSI EP25-A Evaluation of Stability of In-Vitro Diagnostic Reagents (September 2009).

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L. Test Principle:

The Free Testosterone AccuBind ELISA test system uses a competitive enzyme immunoassay technology. The essential reagent includes an immobilized antibody, enzyme-antigen conjugate and a native antigen. Upon mixing the immobilized antibody, enzyme-antigen conjugate and a serum containing the free native antigen, a competitive reaction results between the native free antigen and the enzyme-antigen conjugate for a limited number of insolubilized binding sites. After equilibrium is attained, the antibody-coated fraction is separated from unbound antigen by decantation or aspiration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown sample can be ascertained. The serum calibrators are prepared in human serum matrix. The enzyme-antigen conjugate is labelled with horseradish peroxidase (HRP) and the substrate reagent contains tetramethylbenzidine (TMB), a blue color is produced. The reaction is stopped with addition of an acid and a yellow color is developed. The plate is read in a microtiter plate reader at 450nm.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

A study was performed by testing three levels of human serum pools and three levels of control material using three reagent lots. The samples were tested in duplicate, two times a day for a period of 20 days for a total of 80 measurements per sample. Each of the three lots of reagents produced similar precision results. The precision results from one representative lot are summarized in the table below:

| | | Within-Run | | Total | |
| --- | --- | --- | --- | --- | --- |
| Sample | Mean (pg/mL) | SD | %CV | SD | %CV |
| Control 1 | 2.51 | 0.09 | 3.7% | 0.20 | 7.8% |
| Control 2 | 10.98 | 0.40 | 3.6% | 0.96 | 8.7% |
| Control 3 | 22.72 | 0.83 | 3.6% | 2.18 | 9.6% |
| Serum 1 | 0.98 | 0.06 | 5.9% | 0.12 | 12.4% |
| Serum 2 | 4.53 | 0.26 | 5.7% | 0.36 | 8.0% |
| Serum 3 | 53.62 | 4.24 | 7.9% | 4.32 | 8.1% |

b. Linearity/assay reportable range:

A study was performed to evaluate the linearity of the Free Testosterone AccuBind ELISA Test System. Test samples were prepared by performing serial dilutions of a high human serum free testosterone pool with a low human serum free testosterone pool. The ten free testosterone concentrations tested were as follows: 0.11, 6.58, 13.05, 19.52, 25.99, 32.46, 45.40, 51.87, 58.34, and 64.81 pg/mL.

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The samples were tested in replicates of 4. The following linearity regression equation was obtained:

$$
y = 1.0149x - 0.6028, R^2 = 0.9888
$$

The results of the linearity study support a measuring range of 0.11 - 60 pg/mL.

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

The test system is traceable to a certified reference material, Cerrlian testosterone, T-037.

d. Detection limit:

Detection limit studies were performed in accordance with CLSI EP17 guideline.

A limit of blank (LoB) study was performed using three different blank samples that were measured using 3 reagent lots over 6 days to yield 144 measurements. The LoB was determined nonparametrically using the following equation:

$[\mathrm{N_B(p / 100)} + 0.5] =$ result at position $[0.95*\mathrm{N_B} = 0.5] = \mathrm{P}(1 - \alpha)$. The LoB was determined to be largest result of the 3 reagent lots, $0.0295~\mathrm{pg / mL}$.

The limit of detection (LoD) study was performed by obtaining 10 measurements of 10 low-level samples across 3 different reagent lots and 2 analyzers. LoD was calculated using the following equation: $\mathrm{LoD = LoB + Cv \times SDs}$. The LoD was determined to be $0.0519~\mathrm{pg / mL}$.

Limit of Quantitation (LoQ): The test results from the LoD study were used to calculate total error. The goal for the total error was set as $0.05~\mathrm{pg / mL}$. The TE was lower than $0.05~\mathrm{pg / mL}$, therefore the sponsor claims that the LoQ is equal to the LoD, $0.0519~\mathrm{pg / mL}$.

| Limit of Blank | Limit of Detection | Limit of Quantitation |
| --- | --- | --- |
| 0.0295 pg/mL | 0.0519 pg/mL | 0.0519 pg/mL |

The sponsor's claimed measuring range is 0.11 - 60 pg/mL.

e. Analytical specificity:

Interference:

An interference study was performed following CLSI EP07-A2 guideline. Aliquots from pools of human serum with a free testosterone concentration of $7.916~\mathrm{pg / mL}$ and $38.5~\mathrm{pg / mL}$ were spiked with potentially interfering substances at one or more concentrations. The sponsor defines significant interference as $>10\%$ bias.

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The results are summarized in the following table:

| Substance | Highest concentration at which no significant interference was observed |
| --- | --- |
| Acetaminophen | 20 mg/dL |
| Acetylcysteine | 150 mg/dL |
| Ascorbic Acid | 6 mg/dL |
| Bilirubin, conjugated | 15 mg/dL |
| Bilirubin, unconjugated | 20 mg/dL |
| Biotin | 100 ng/mL |
| Caffeine | 6 mg/dL |
| Cholesterol | 503 mg/dL |
| Creatine | 30 mg/dL |
| Dextran | 5000 mg/dL |
| Digoxin | 6.1 ng/ mL |
| Doxycycline | 50 mg/dL |
| Erythromycin | 6 mg/dL |
| Gentamicin | 1 mg/dL |
| Human mouse antibodies | 440 ng/mL |
| Hemoglobin | 500 mg/dL |
| Heparin | 3 U/mL |
| Human serum albumin | 2.5 g/dL |
| Ibuprofen | 50 mg/dL |
| Immunoglobulin G | 4 g/dL |
| Levodopa | 20 mg/L |
| Lidocaine | 1.2 mg/dL |
| Lipemia (glycerides) | 1000 mg/dL |
| Methyldopa | 20 mg/dL |
| Nicotine | 0.1 mg/dL |
| Phenobarbital | 15 mg/dL |
| Protein, total | 10.5 g/dL |
| Rheumatoid factor | 1110 IU/mL |
| Salicylic Acid | 60 mg/dL |
| Sex hormone binding globulin | 200 μg/mL |
| Triglycerides | 900 mg/dL |
| Urea | 500 mg/dL |

Cross-Reactivity:
A cross-reactivity study was performed in accordance with CLSI EP07-A2 to evaluate whether various analytes or substances cross-react with the quantitation of free testosterone using the device. Aliquots from a pool of human serum with a free testosterone concentration of 7.408 pg/mL were spiked with substances at the concentrations listed in the table below. Cross-reactivity was determined using the following equation: (observed value - unspiked value)/ concentration of cross-reactant x 100%. The sponsor defines significant cross-reactivity as >10% difference.

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| Substance | Concentration. of substance (ng/mL) | % Cross Reactivity |
| --- | --- | --- |
| 11-Deoxycortisol | 1000 | 0.000% |
| 11-KetoTestosterone | 10 | 0.647% |
| 11β-Hydroxytestosterone | 100 | 0.065% |
| 17α-ethynyl estradiol | 1000 | 0.000% |
| 17α-Estradiol | 1000 | 0.000% |
| 17β-Estradiol | 100 | 0.000% |
| 17-Hydroxypregnenolone | 1000 | 0.000% |
| 17-Hydroxprogesterone | 10 | 0.000% |
| 3-EstriolGluc | 1000 | 0.000% |
| 3-EstriolSul | 1000 | 0.000% |
| 3β-Androstanediol | 500 | 0.000% |
| 5α-Dihydrotestosterone | 100 | 0.054% |
| Aldosterone | 8000 | 0.000% |
| Amitriptyl HCI | 1000 | 0.000% |
| Androsterone | 1000 | 0.000% |
| Andronstenedione | 1000 | 0.004% |
| Clomiphene Citrate | 1000 | 0.000% |
| Corticosterone | 1000 | 0.000% |
| Cortisone | 1000 | 0.000% |
| Cortisol | 1000 | 0.000% |
| Cyproterone acetate | 1000 | 0.000% |
| D-5-Androstene-3β,17β-diol | 1000 | 0.000% |
| Danazol | 1000 | 0.000% |
| Dehydroepiandrosterone | 100000 | 0.000% |
| Dehydroepiandrosterone Sulfate | 1000 | 0.000% |
| Desogestrel | 100 | 0.000% |
| Dexamethasone | 1000 | 0.000% |
| Epitestosterone | 1000 | 0.001% |
| Estriol | 1000 | 0.000% |
| Estrone | 1000 | 0.000% |
| Ethisterone | 1000 | 0.000% |
| Ethynediol | 1000 | 0.000% |
| Ethynediol diacetate | 50 | 0.000% |
| Flunisolide | 1000 | 0.000% |
| Fluoxymesterone | 1000 | 0.000% |
| Lynestrol | 1000 | 0.000% |
| Medroxyprogesterone acetate | 1000 | 0.000% |
| Methyl Testosterone | 100 | 0.000% |
| Mestranol | 1000 | 0.000% |
| Norethindrone | 50 | 0.000% |
| Norethindrone acetate | 50 | 0.000% |
| Norgestimate | 1000 | 0.000% |

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| Substance | Concentration of substance (ng/mL) | % Cross Reactivity |
| --- | --- | --- |
| Norgestrel (Levonorgestrel) | 50 | 0.000% |
| Norethynodrel | 50 | 0.000% |
| Oxymetholone | 100 | 0.000% |
| Prednisolone | 1000 | 0.000% |
| Prednisone | 800 | 0.000% |
| Progesterone | 1000 | 0.000% |
| Salbutamol | 1000 | 0.000% |
| Spironolactone | 1000 | 0.000% |
| Stanozolol | 1000 | 0.000% |
| Testosterone enanthate | 10 | 0.000% |
| Testosterone SO4 | 1000 | 0.004% |
| Testosterone Propionate | 1000 | 0.000% |
| Triamcinolone | 50 | 0.000% |

An additional study was performed to evaluate the cross-reactivity effects of testosterone cypionate and testosterone undecanoate. Aliquots from pool of human serum with a free testosterone concentration of 38.4 pg/mL were spiked with 12 ng/mL of testosterone cypionate and testosterone undecanoate. Cross-reactivity was determined using the following equation: observed value - unspiked value/concentration of cross-reactant x100%. The sponsor defines significant cross-reactivity as >10% difference. The results are summarized in the chart below:

| Substance | Concentration of Substance (ng/mL) | % Cross Reactivity |
| --- | --- | --- |
| Testosterone cypionate | 12 | 0.000% |
| Testosterone undecanoate | 12 | 0.000% |

f. Assay cut-off:

Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

A method comparison study was performed by testing 137 (133 native and 4 spiked) human serum samples with the candidate and predicate devices. The test results on the candidate device ranged from 0.11-59.63 pg/mL. The following regression equation was obtained using Passing-Bablok analysis: y= 1.017x - 0.244, r= 0.997

b. Matrix comparison:

Not applicable. Serum is the only claimed sample type.

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3. Clinical studies:

a. Clinical Sensitivity:
Not applicable.

b. Clinical specificity:
Not applicable.

c. Other clinical supportive data (when a. and b. are not applicable):
None.

4. Clinical cut-off:
Not applicable.

5. Expected values/Reference range:

A reference range study was performed by testing a total of 261 serum samples obtained from 130 females (aged 22-93 years), and 131 males (aged 22-89 years) on the candidate device. The subjects were not pregnant, not on hormone therapy, not taking contraceptives, glucocorticoids or corticosteroids; and had no history of thyroid, autoimmune, Cushing's, or Addison's disease. The resulting reference interval is summarized in the following table:

| Cohort | N | 95% Confidence Range (pg/ml) |
| --- | --- | --- |
| Male, 20-39 yrs. | 45 | 9.2-34.6 |
| Male, 40-59 yrs. | 43 | 6.1-30.3 |
| Male, ≥60 yrs. | 43 | 6.1-27.9 |
| Female, 20-39 yrs. | 44 | 0.2-6.1 |
| Female, 40-59 yrs. | 42 | 0.3-4.4 |
| Female, ≥60 yrs. | 44 | 0.5-3.4 |

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.