DRG Diagnostics GmbH EIA-2124 96 Crosslaps Serum ELISA Instruction Manual

DRG Diagnostics GmbH EIA-2124 96 Crosslaps Serum ELISA

Product Information

Specifications

• Product Name: Crosslaps Serum ELISA EIA-2124
• Manufacturer: DRG International, Inc.
• Address: 841 Mountain Ave., Springfield, NJ 07081, USA
• Phone: 973-564-7555
• Fax: 973-564-7556
• Website: www.drg-international.com
• Email: [email protected]
• Expiration Date: 09.05.2023

Product Usage Instructions

Intended Use
The Crosslaps Serum ELISA is an enzyme immunological test designed for the quantification of degradation products of C-terminal telopeptides of Type I collagen in human serum and plasma. It is intended for in vitro diagnostic use to indicate human bone resorption.

The assay can be used as an aid in:

• Monitoring bone resorption changes of anti-resorptive therapies (such as hormone replacement therapies (HRT) with hormones or hormone-like drugs and bisphosphonate therapies) in postmenopausal women and individuals diagnosed with osteopenia.
• Predicting skeletal response (Bone Mineral Density) in postmenopausal women undergoing anti-resorptive therapies, such as HRT with hormones or hormone-like drugs and bisphosphonate therapies.

Precautions
Before using the Crosslaps Serum ELISA, please take note of the following precautions:

• This test is for in vitro diagnostic use only.
• Do not use expired reagents or materials.
• Follow the instructions carefully to ensure accurate results.
• Handle all specimens and reagents with care to avoid contamination.
• Dispose of all waste materials according to local regulations.

Materials Included
The Crosslaps Serum ELISA kit includes the following:

• Microplate coated with anti-human Crosslaps antibody
• Calibrator set
• Control set
• Wash solution concentrate
• Enzyme conjugate
• Substrate solution
• Stop solution
• Plate cover
• Instructions for use

Storage and Stability
Store the Crosslaps Serum ELISA kit at 2-8°C in a dark place. The kit is stable until the expiration date printed on the label. Do not freeze any components of the kit.

Assay Procedure
Follow these steps to perform the Crosslaps Serum ELISA assay:

1. Prepare all reagents and samples as instructed.
2. Add the prepared samples, calibrators, and controls to the appropriate wells of the microplate.
3. Incubate the plate at the recommended temperature and time.
4. Wash the plate with the provided wash solution.
5. Add the enzyme conjugate to each well and incubate.
6. Wash the plate again and add the substrate solution.
7. Incubate the plate in the dark until the desired color develops.
8. Add the stop solution to each well to terminate the reaction.
9. Measure the absorbance of each well using a microplate reader.

Interpretation of Results
Refer to the provided instructions for interpreting the results of the Crosslaps Serum ELISA assay. The absorbance values obtained from the samples, calibrators, and controls can be used to determine the concentration of degradation products of C-terminal telopeptides of Type I collagen in the tested specimens.

FAQ

1. Q: Can this kit be used for diagnosing bone-related conditions?
   A: The Crosslaps Serum ELISA kit is intended for in vitro diagnostic use as an indication of human bone resorption. It can help monitor bone resorption changes and predict skeletal response in certain therapeutic contexts. However, it is not intended for diagnosing specific bone-related conditions. Consult with a healthcare professional for a proper diagnosis.

2. Q: How should I handle and dispose of waste materials?
   A: All waste materials, including used reagent containers, should be disposed of according to local regulations for laboratory waste disposal. Follow your institution’s guidelines and
   regulations to ensure proper handling and disposal of waste materials.

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE
For In Vitro Diagnostic Use.
The Crosslaps Serum ELISA is an enzyme immunological test for the quantification of degradation products of C-terminal telopeptides of Type I collagen in human serum and plasma.

The Crosslaps Serum ELISA assay is intended for in vitro diagnostic use as an indication of human bone resorption and may be used as an aid in:

• Monitoring bone resorption changes of anti-resorptive therapies (such as hormone replacement therapies (HRT) with hormones or hormone like drugs and bisphosphonate therapies) in postmenopausal women and individuals diagnosed with osteopenia
• Predicting skeletal response (Bone Mineral Density) in postmenopausal women undergoing anti resorptive therapies, such as HRT with hormones or hormone like drugs and bisphosphonate therapies

SUMMARY AND EXPLANATION
Type I collagen accounts for more than 90% of the organic matrix of bone and is synthesized primarily in bone (1). During renewal of the skeleton, Type I collagen is degraded, and small peptide fragments are excreted into the bloodstream. These fragments can be measured by Crosslaps Serum ELISA. The measurements of the specific degradation products of Type I collagen in both urine (2) and serum (3) by a competitive CrossLaps have been reported. The sandwich assay has been reported as useful for follow up of anti resorptive treatment of patients with metabolic bone diseases (3-17).

METHOD DESCRIPTION
The Crosslaps Serum ELISA is an enzyme immunological assay which is based on two highly specific monoclonal antibodies against the amino acid sequence of EKAHD-β-GGR, where the aspartic acid residue (D) is β-isomerised. To obtain a specific signal in the Crosslaps Serum ELISA, two chains of EKAHD-β-GGR must be cross linked. 50 μL of each standards, controls or unknown serum samples are pipetted into microtiter wells coated with streptavidin, followed by application of a mixture of a biotinylated antibody and an enzyme (horseradish peroxidase – HRP) conjugated antibody. Then, a complex between CrossLaps® antigens, biotinylated antibody and HRP conjugated antibody is generated; this complex binds to the streptavidin surface via the biotinylated antibody. Following the one step incubation at room temperature (18 °C – 22 °C), the wells are emptied and washed. The colour is developed using a chromogenic substrate (TMB). The colour reaction is stopped and the absorbance of the stopped reaction mixture is read in a microtiter plate reader. The colour intensity of the reaction mixture is proportional to the concentration of the cross-linked CTX-I in the original sample.

WARNINGS AND PRECAUTIONS

The Crosslaps Serum ELISA is for in vitro diagnostic use only and is not for internal use in humans or animals. This product must be used strictly in accordance with the instructions set out in these Instructions for Use (IFU). DRG will not be held responsible for any loss or damage (except as required by statute), howsoever caused, arising out of non-compliance with the instructions provided.
CAUTION: This kit contains material of animal origin. Handle kit reagents as if capable of transmitting an infectious agent. Appropriate precautions and good laboratory practice must be used in the storage, handling and disposal of the kit reagents. Disposal of kit reagents should be in accordance with local regulations.

Human materials
Human material used in the preparation of this product has been tested by FDA recommended assays for the presence of antibody to Human Immunodeficiency Virus (HIV I and II), Hepatitis B surface antigen, antibody to Hepatitis C, and found negative. As no test can offer complete assurance that infectious agents are absent, the reagents should be handled according to Biosafety Level 2.

Reagents containing Sodium Azide
Some reagents in this kit contain sodium azide (NaN3) <0.1 % (w/w) which may react with lead, copper or brass plumbing to form highly explosive metal azides. On disposal, flush with large volumes of water to prevent azide build up.

Classification according to Regulation (EC) CLP:
Skin sensitivity, Category 1

Warning
Contains ProClin 300

Hazard statements:
H317 – May cause an allergic skin reaction.

Precautionary statements:

• P261 – Avoid breathing dust/fume/gas/mist/vapour/spray.
• P280 – Wear protective gloves/protective clothing/eye protection/face protection/hearing protection.
• P321 – Specific treatment /see supplemental first aid instructions on this label).
• P333 + P313 – If skin irritation or rash occurs: Get medical advice/attention.
• P362 + P364 – Takeoff contaminated clothing and wash it before use.

SHELF LIFE AND STORAGE OF REAGENTS

This kit is stable until the expiry date printed on the box if stored as specified. Upon receipt, store all reagents at 2 °C – 8 °C. Do not use any kit component beyond their expiry date.

Indications of possible deterioration of kit reagents include:

• The presence of abnormal particulate matter in any of the reagents.
• A decrease in the maximum binding.
• A high non-specific binding.
•  A shift in the slope of the curve from its normal position.

SAMPLE COLLECTION AND STORAGE
For optimal results it is recommended to draw blood as fasting morning samples (18). For monitoring individual patients, it is recommended that follow up samples should be collected under same conditions as the baseline sample. Collect blood by venipuncture taking care to avoid haemolysis. Separate the serum from the cells within 3 hours after collection of blood. It is recommended to freeze (< -18 °C) samples immediately. The assay should be performed using serum or plasma (heparin or potassium EDTA) samples.
Note:

• Some commercially available sample collection tubes may affect the results of testing in particular cases.
• Follow the blood collection tube manufacturer’s recommendations for handling and processing the samples.
• Samples containing particulate matter must be centrifuged before performing the assay. Centrifuged samples with a lipid layer on the top must be transferred to a sample cup or secondary tube. Care must be taken to transfer only the clarified samples without the lipemic material
• Samples displaying microbial contamination, highly lipemic or grossly haemolysed should not be assayed with the kit.
• Before performing assays, make sure that samples, calibrators and controls are at room temperature (18 °C – 22 °C).
• Do not use heat-inactivated samples.
• Avoid repeat freeze/thaw cycles for samples.
• Each laboratory should follow the guidelines or requirements of local, state, and/or federal regulations or accrediting organizations to establish its own specimens handling and storage stability. For guidance on appropriate practices, please refer to the CLSI GP44-A4, “Procedures for the Handling and Processing of Blood Specimens for Common Laboratory Tests”.

MATERIALS

Materials Provided
The kit contains reagents sufficient for 96 determinations.

Streptavidin coated plate MICROPLAT
Microwell strips (12 × 8 wells) pre-coated with streptavidin, supplied in a plastic frame.

Biotinylated Antibody Ab BIOTIN
Concentrated solution of biotinylated monoclonal anti-CTX-I, in buffered solution with protein stabiliser and sodium azide as preservative (<0.1 %); 1 vial, 0.25 mL

Peroxidase Conjugated Antibody ENZYMCONJ
Concentrated peroxidase conjugated monoclonal antibody specific for CTX-I; provided in a buffered solution with protein stabiliser; 1 vial, 0.25 mL

Incubation Buffer BUF
Ready to use buffered solution containing protein stabiliser and detergents, with Proclin 300 as preservative (<0.1 %); 1 vial, 19.0 mL

Substrate Solution SUBS TMB
Ready to use tetramethylbenzidine (TMB) substrate in an acidic buffer; 1 vial, 12.0 mL Please note that the chromogenic substrate might appear slightly blueish.

Stopping Solution H2SO4
Ready to use solution of 0.18 mol/L Sulfuric acid; 1 vial, 12.0 mL

Washing Buffer WASHBUF 50x
Concentrated washing buffer with detergent; 1 vial, 20.0 mL

Standard CAL 0
Ready for use Phosphate buffered solution containing protein stabiliser and ProClin 300 as preservative (<0.1 %); 1 vial, 5.0 mL

Standards CAL 1 – 5
Ready for use Phosphate buffered solution containing CTX-I antigen, protein stabiliser ProClin 300 as preservative (<0.1 %); 1 each of 5 concentration levels, 0.4 mL per vial.
The exact value of each standard is printed on the QC report.

Controls CTRL 1 – 2
Ready for use Phosphate buffered solution containing desalted urinary antigen of human origin with protein stabilizers and ProClin 300 as preservative (<0.1 %); 1 each of 2 concentration levels, 0.4 mL pervial.
The established ranges for the controls are printed on the QC report.
Adhesive Plate Sealer 2 per kit.
Documentation Instructions for Use and QC report.

Materials Required But Not Provided

• Containers for preparing the Antibody Solution and the Washing Solution
• Precision pipetting devices to deliver 50 – 200 μL
• Distilled water
• Precision 8 or 12 channel multipipette to deliver 100 μL to 300 μL
• Vortex mixer
• Microwell mixing apparatus
• Automatic microplate washer (optional)
• Photometric microplate reader and data analysis equipment

PREPARATION OF REAGENTS
Allow all reagents to come to room temperature for a minimum of 60 minutes before use. Do not interchange kit components from different lots.

Antibody Solution
Prepare the Antibody Solution a maximum of 30 minutes before starting the assay. Mix the Biotinylated Antibody Ab BIOTIN, Peroxidase Conjugated Antibody ENZYMCONJ and Incubation Buffer  BUF  in  the volumetric ratio 1 + 1 + 100 in an empty container. Mix carefully and avoid formation of foam. Prepare a fresh solution before each run of the assay.

Wash buffer preparation
Prepare by adding 1-part Wash Concentrate WASHBUF 50x  to 50-parts distilled water. All other reagents are supplied ready for use.
N.B. To avoid potential microbial and / or chemical contamination, unused reagents should never be returned into the original vials.

ASSAY PROCEDURE

Prepare reagents as described in § 8. Preparation of Reagents. Mix all reagents and samples before use (avoid formation of foam).
NOTE: To ensure consistent results between runs, between operators, and to minimise any drift effect; strictly adhere to the following procedure:

• Bring all reagents to room temperature (18 °C – 22 °C) prior to use – this will take approximately 60 minutes.
• Set up the assay within 30 minutes of preparing the Antibody Solution.
• Seal the plate during incubations using the plate sealers which are supplied with the assay kit.
• Do not stack plates during incubation in order to ensure a consistent temperature for all plates.
• Do not under or over-fill the assay wells during the washing steps.
• Add reagents in the same sequence each time to reduce time deviation between reactions

Do not pipette directly from the vial containing TMB substrate. The required volume should first be transferred to a clean container. Solution remaining in the container should be discarded following use and NOT returned to the stock vial SUBS TMB . Determine the number of strips needed for the assay; it is recommended to test all samples in duplicate. In addition, for each run a total of 16 wells are needed for the standards and controls. Place the appropriate number of strips in the plastic frame. Store any unused strips in the tightly closed foil bag with desiccant capsules.

1. Pipette 50 μL of each standard CAL 0 – 5, control CTRL 1 – 2 or sample SPE to the appropriate wells on the Streptavidin coated plate MICROPLAT followed by 150 μL of the Antibody Solution AB SOLN.

2. Cover the plate with an adhesive plate seal.

3. Incubate at room temperature (18 °C – 22 °C) for 120 ±5 minutes on a microtiter plate mixer (300 rpm).

4. Wash all wells 5 times with diluted wash solution WASHBUF SOLN. Automatic plate wash Set plate washer to dispense 300 μL of wash solution per well. Fill and aspirate for 5 cycles. Manual wash Decant the contents of the wells by inverting sharply. Pipette 300 μL of wash solution into each well, decant and repeat 5 times. Remove excess wash buffer by tapping firmly on absorbent tissue before proceeding.

5. Pipette 100 μL of Substrate Solution SUBS TMB into each well.

6. Cover the plate with an adhesive plate seal.

7. Incubate at room temperature (18 – 22°C) for 15 minutes in the dark on a microtiter plate mixer (300 rpm).
   NOTE: do not pipette directly from the vial containing TMB substrate. The required volume should first be transferred to a clean container. Solution remaining in the container should be discarded following use and NOT returned to the stock vial SUBS TMB.

8. Pipette 100 μL of Stopping Solution H2SO4 into each well.

9. Measure absorbance at 450 nm with reference at 650 nm using a microplate reader within 2 hours of stopping the reaction.

N.B. Microplate readers measure vertically; when pipetting, do not touch the bottom of the wells

Automated Platforms
The Crosslaps Serum ELISA kit was designed and developed to be performed manually using the protocol described above. The protocol is not necessarily applicable to automated platforms. If automated platforms are used it is the responsibility of the user to ensure the kit has been appropriately tested. To improve the performance of the kit on automated platforms, it is recommended to increase the number of wash cycles at each wash step.

CALCULATION OF RESULTS
A variety of data reduction software packages are available, which may be employed to generate the mean calibration curve and to calculate the mean concentrations of unknown samples and controls. A quadratic curve fit, including Calibrator 0 is required. Alternatively, a calibration curve may be prepared on semi-log graph paper by plotting mean absorbance on the Y-axis against concentration of CTX-I on the X-axis. Calibrator 0 should be included in the calibration curve. Read the mean absorbance value of each unknown sample off the curve.
NOTE: If the absorbance of a sample exceeds that of Standard 5, the sample should be diluted in Standard 0 and re-analyzed.

QUALITY CONTROL
Good Laboratory Practice (GLP) requires the use of quality control specimens in each series of assays in order to check the performance of the assay. Controls should be treated as unknown samples, and the results analyzed with appropriate statistical methods. The two kit controls provided in the kit should be tested as unknowns and are intended to assist in assessing the validity of results obtained with each assay plate. DRG recommends the users to maintain graphic records of the control values generated with each assay run, including the running means, SDs and %CVs. This information will facilitate the controls trending analysis relating to the performance of current and historical control lots relative to the supplied Quality Control data. The trending will assist in the identification of assays which give control values significantly different from their average range. When interpreting control data, users should note that this product was designed and developed as a manual product. The range stated on the QC report should be appropriate for assays that are performed manually and with strict adherence to the Assay Procedure described above. It is recognized by Quality Control professionals, that as a result of differences in conditions and practices, there will always be variability in the mean values and precision of control measurements between different laboratories (27).

MEASUREMENT RANGE (REPORTABLE RANGE)
The reportable range of the assay is from 0.020 ng/mL. Any value that reads below 0.020 ng/mL should be reported as “< 0.02 ng/mL”. Samples with CTX-I concentrations above the absorbance of calibrator 5 should be diluted with calibrator 0. The results for diluted samples must be multiplied by the appropriate dilution factor.

TRACEABILITY
The Crosslaps Serum ELISA has been standardised against in-house reference standards (CTX-I in analyte-free buffered protein matrix).

LIMITATIONS OF USE

• As in the case of any diagnostic procedure, results must be interpreted in conjunction with the patient’s clinical presentation and other information available to the physician.
• The performance characteristics of this assay have not been established in a paediatric population.
• Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro immunoassays (28). Patients routinely exposed to animals or to animal serum products can be prone to this interference and anomalous values may be observed.
• The recommended daily allowance of Biotin (defined as 0.03 mg) does not typically cause interference with immunoassays that use biotin-streptavidin technology. Higher biotin levels may cause interference and affect corresponding test results, false highs or false lows (29,30). Biotin has a very rapid elimination half-life of about 2 hours; theoretically, most of it should clear from the body within 4–5 hours (31). Concentrations of biotin up to 1200 ng/mL may be present in specimens collected from patients taking up to 300 mg per day (30).
• For results that are not consistent with the patient’s clinical presentation, biotin interference may be considered. Physicians and other practitioners should inquire and advise patients to abstain from biotin intake before blood draw31. The lowest Biotin level that does not significantly raise the results (<10% bias) within the Crosslaps Serum ELISA kit is 10 ng/mL.
• The following substances do not interfere in the Crosslaps Serum ELISA when the concentrations presented in the following table are below the stated threshold.

EXPECTED VALUES
The following ranges were determined using the Crosslaps Serum ELISA and are provided for information only. For further reading, please refer to the reference list. All samples were morning fasting samples from healthy individuals.

Populations| Number of subjects| *Mean Values (ng/mL)| 95% Confidence Interval (ng/mL)**
---|---|---|---
Post-menopausal women| 193| 0.439| 0.142 – 1.351
Pre-menopausal women| 226| 0.287| 0.112 – 0.738
Males| 125| 0.294| 0.115 – 0.748

The above ranges should be considered as guidelines only; it is recommended that each laboratory establish its own expected range based upon its own patient population.

CLINICAL STUDIES

The Crosslaps Serum ELISA has been used to monitor treatment in several clinical studies and the CrossLaps values have been compared to Bone Mineral Density (BMD-spine) measurements. All the clinical studies presented below were performed according to the European Standard for good clinical practice (GCP and GLP). Most of the clinical studies presented here were conducted on white Danish women. However, several studies have been published showing that other demographic groups display similar CrossLaps decrease in response to anti resorptive therapies (9-13). For all the data presented below fasting morning samples have been used. The Bone mineral density was measured at the Lumbar spine (L1, L4). The change in the bone mineral density is presented below α-BMD. α-BMD is defined as the slope of the linear regression line for BMD-spine versus time (years) for the period of treatment. In most cases the calculation of α BMD involves a minimum of 5 BMD-spine measurements. The α-BMD thus represents the % change in BMD-spine per year. Because there to this date is no universal agreement as to what constitutes positive BMD response we have calculated the sensitivity and specificity using two different cut off values for α-BMD; α-BMD > 0 and α-BMD > 1. The sensitivity is defined as the percent of the study population with a positive BMD response and who have a % change from baseline of Crosslaps Serum ELISA which is 40% or greater. The specificity is defined as the percent of the study population without a positive BMD response and who have a % change from baseline of Crosslaps Serum ELISA that is less than 40%.

Bisphosphonate studies
Below is shown the Crosslaps Serum ELISA data from two different bisphosphonate studies.

Alendronate

• Women between age 40 and 59 years, 6 months to 3 years since menopause
• 12 participants on placebo (500 mg calcium)
• 42 participants on active treatment (5 mg (n=16), 10 mg (n=14), 20 mg (n=12)) Alendronate and 500 mg calcium)
• Treatment period: 2 – 3 years

SEM
Baseline| 0.676 (0.543 – 0.809)| 0.235| 0.068| 0.603 (0.540 – 0.666)| 0.208| 0.032
After 6 months treatment| 0.623 (0.498 – 0.748)| 0.221| 0.064| 0.191 (0.144 – 0.238)| 0.153| 0.024

Ibandronate

• Women less than 75 years, more than ten years after menopause and have a BMD forearm 1.5 SD or more below the standard for healthy pre-menopausal women
• 17 participants on placebo (1000 mg calcium)
• 36 participants on active treatment: (2.5 mg (n=20), 5 mg (n=16)) ibandronate and 1000 mg calcium
• Treatment period: 1 year

SEM
Baseline| 0.590 (0.502 – 0.678)| 0.185| 0.045| 0.614 (0.536 – 0.692)| 0.240| 0.040
After 6 months treatment| 0.325 (0.241 – 0.409)| 0.178| 0.043| 0.136 (0.093 – 0.179)| 0.131| 0.022

Crosslaps Serum ELISA versus α-BMD for patients treated with bisphosphonates

Using a cut-off for Crosslaps Serum ELISA of 40% change from baseline the following sensitivities, specificities and 95% confidence intervals are obtained.

The corresponding data is reported below:

| Ibandronate| Alendronate
---|---|---
| Sensitivity| Specificity| Sensitivity| Specificity
α-BMD > 0| 93% (81 – 99) 40/43| 30% (7 – 65) 3/10| 89% (73 – 97) 31/35| 92% (64 – 100) 12/13
α-BMD > 1| 94% (81 – 99) 33/35| 22% (6 – 48) 4/18| 90% (73-98) 26/29| 68% (43 – 87) 13/19

Below are shown distribution plots for the combined bisphosphonate studies. The number over each bar indicates the number of participants in each class.

HRT Studies

Below is shown the Crosslaps Serum ELISA data from three different HRT studies.

Tibolone

• Women less than 75 years and more than ten years after meno pause
• 13 participants on placebo (400 mg calcium/day)
• 49 participants on active treatment (1.25 mg (n=25) or 2.5 mg (n=24) Tibolone and 400 mg calcium/day
• Treatment period 2 years

SEM
Baseline| 0.264 (0.217 – 0.311)| 0.085| 0.024| 0.339 (0.302 – 0.376)| 0.130| 0.019
After 6 months treatment| 0.287 (0.232 – 0.342)| 0.099| 0.028| 0.192 (0.161 – 0.223)| 0.113| 0.016

HRT I

• Women more than 45 years, 1 to 6 years since menopause
• 42 participants on placebo (400 mg calcium/day)
• 120 participants on active treatment:
  • Days 1 – 16 Days 17 – 28
  • E 1 mg E 1 mg + G 25 μg
  • E 2 mg E 2 mg + G 25 μg
  • E 2 mg E 2 mg + G 50 μg
  • E 1 mg + G 25 μg  E 1 mg + G 25 μg continuously
  • E = estradiol 17β, G = gestodene, active treatment also receive 400 mg calcium/day
• Treatment period: 2 years.

SEM
Baseline| 0.389 (0.348 – 0.430)| 0.136| 0.021| 0.411 (0.386 – 0.437)| 0.140| 0.013
After 6 months treatment| 0.396 (0.349 – 0.443)| 0.153| 0.024| 0.182 (0.164 – 0.200)| 0.098| 0.009

HRT Il

• Women between 65 and 70 years and BMC forearm below 1 SD of healthy pre-menopausal women
• 17 participants on placebo (1000 mg calcium/day)
• 15 participants on active treatment: 50 μg estradiol, 1 mg norethisterone and 1000 mg calcium/day

SEM
Baseline| 0.350 (0.301 – 0.399)| 0.099| 0.025| 0.371 (0.306 – 0.436)| 0.135| 0.033
After 6 months treatment| 0.293 (0.240 – 0.346)| 0.106| 0.027| 0.159 (0.108 – 0.210)| 0.106| 0.026

Crosslaps Serum ELISA versus α-BMD for patients treated with HRT

Using a cut off for Crosslaps Serum ELISA of 40% change from baseline the following sensitivities, specificities and 95% confidence intervals are obtained.

The corresponding data is reported below:

| Tibolone| HRT I| HRT II
---|---|---|---
| Sensitivity| Specificity| Sensitivity| Specificity| Sensitivity| Specificity
α-BMD > 0| 58%| 90%| 80%| 90%| 60%| 100%
| (44 – 72)| (56 – 100)| (72 – 87)| (77 – 97)| (39 – 79)| (59 – 100)
| 31/53| 9/10| 16/20| 37/41| 15/25| 7/7
α-BMD > l| 65%| 80%| 83%| 78%| 62%| 91%
| (49 – 79)| (56 – 94)| (74 – 94)| (64 – 88)| (38 – 82)| (59 – 100)
| 28/43| 16/20| 90/109| 42/54| 13/21| 10/11

Below is shown distribution plots for the combined HRT studies. The number over each bar indicates the number of participants in each class

PERFORMANCE DATA

Representative performance data are shown. Results obtained at individual laboratories may vary.

Sensitivity
The sensitivity, defined as the concentration corresponding to the mean minus 3 standard deviations of 21 replicates of the zero calibrator, is 0.020 ng/mL.

Precision
The imprecision of the Crosslaps Serum ELISA was evaluated for three serum samples.

The results are summarised in the table below:

| Inter Assay Variation (n=10)| Intra Assay Variation (n=10)
---|---|---
Mean (ng/mL)| SD
(ng/mL)| CV (%)| SD
(ng/mL)| CV (%)
0.121
0.444
1.967| 0.013
0.043
0.050| 10.9
9.7
2.5| 0.004
0.007
0.035| 3.0
1.7
1.8

Intra-individual variation was assessed by evaluating patient sera, collected as morning fasted sample from 11 healthy postmenopausal women on 5 occasions within a 2-week period:

| Visit Number| Mean| SD| CV
---|---|---|---|---
ID| 1| 2| 3| 4| 5| (ng/mL)| (ng/mL)
1| 0.423| 0.428| 0.396| 0.46| 0.445| 0.430| 0.024| 6%
2| 0.461| 0.523| 0.500| 0.535| 0.539| 0.512| 0.032| 6%
3| 0.850| 0.731| 0.761| 0.782| 0.764| 0.778| 0.044| 6%
4| 0.377| 0.468| 0.455| 0.499| 0.440| 0.448| 0.045| 10%
5| 0.918| 0.834| 0.791| 0.781| 0.714| 0.808| 0.075| 9%
6| 0.268| 0.249| 0.246| 0.257| 0.258| 0.256| 0.009| 3%
7| 0.431| 0.457| 0.468| 0.494| 0.506| 0.471| 0.030| 6%
8| 0.666| 0.587| 0.670| 0.595| 0.728| 0.655| 0.063| 10%
9| 0.323| 0.357| 0.341| 0.409| 0.345| 0.355| 0.033| 9%
10| 0.419| 0.520| 0.541| 0.491| 0.470| 0.488| 0.047| 10%
11| 0.353| 0.472| 0.429| 0.464| 0.400| 0.424| 0.049| 12%

Dilution/Linearity
The Crosslaps Serum ELISA is linear in the range 0.020 ng/mL to 3.380 ng/mL of CTX-I. Serum samples with the concentration of 0.460 – 0.668 ng/mL CTX-I were diluted with standard 0 and the concentration of CTX-I were determined with Crosslaps Serum ELISA. The serum neat sample is set to 100%.

The data below is calculated from 3 different runs:

Dilution Procedure

Serum [%]| Standard 0 [%]| Recovery
[% of expected value]
100| 0| 100
90| 10| 103
80| 20| 103
70| 30| 101
60| 40| 102
50| 50| 105
40| 60| 109
30| 70| 107
20| 80| 97
10| 90| 100
Mean| | 103

BIBLIOGRAPHY

1. Bjarnason NH. & Christiansen,C. Early Response in Biochemical Markers Predicts Long-term
   Response in Bone Mass During Hormone Replacement Therapy in Early Postmenopausal Women.
   Elsevier 26; 561-569 (2000).

2. Bonde M, Garnero P, Fledelius C, Qvist P, Delmas PD, Christiansen C. Measurement of bone degradation products in serum using antibodies reactive with an isomerized from of an 8 aminoacid sequence of the C-telopeptide of Type I collagen.
   J Bone Miner Res 12:1028-1034 (1997).

3. Bonde M, Qvist P, Fledelius C, Riis BJ, Christiansen C. Applications of an enzyme immunoassay for a new marker of bone resorption (CrossLaps®) – follow up on hormone replacement therapy and osteoporosis risk assessment.
   J Clin Endocrinol & Metab 80:864-8 (1995).

4. Buclin T, Rochat MC,Burckhardt P, Azria M, Attinger M. Bioavailability and biological efficacy of a new oral formulation of salmon calcitonin in healthy volunteers.
   J Bone Miner Res 17;8-1478 (2002).

5. Burgeson RE. New collagens, new concepts.
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6. Chailurkit LO, Aunphongpuwanart S, Ongphiphadhanakul B, Jongjaroenprasert W, Sae-Tung S, and Rajatanavin R. Efficacy of intermittent low dose alendronate in Thai postmenopausal osteoporosis.
   Endocr Res 30:29-36 (2004).

7. Christgau S, Bitsch-Jensen O, Bjarnason NH, Gamwell Henriksen E, Qvist P, Alexandersen P, Bang Henriksen D. Serum CrossLaps for Monitoring the Response in Individuals Undergoing Antiresorptive Therapy.
   Elsevier 26; 505-511 (2000).

8. Christgau S, Rosenquist C, Alexandersen P, Bjarnason NH, Ravn P, Fledelius C, Herling C, Qvist P, Christiansen C. Clinical evaluation of the Serum CrossLaps® ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen Ctelopeptides.
   Clin Chem 44:11;2290-2300 (1998).

9. Christiansen C, Tanko LB, Warming L, Moelgaard A, Christgau S, Qvist P, Baumann M, Wieczorek L, and Hoyle N. Dose dependent effects on bone resorption and formation of intermittently administered intravenous Ibandronate. Osteoporos Int (2003).

10.  Delmas PD. Markers of bone turnover for monitoring treatment of osteoporosis with antiresorptive drugs. Osteoporos Int 11;S66-S76 (2000).

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    Osteoporos Int 11;295-303 (2000).

13. Garnero P, Gineyts E, Riou JP, Delmas PD. Assessment of bone resorption with a new marker of collagen degradation in patients with metabolic bone disease.
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14. Pedersen BJ, Ravn P, Bonde M. Type I Collagen C-telopeptide Degradation products as Bone Resorption Markers. J Clin Ligand Assay 21;2:118-127 (1998).

15. Qvist P, Munk M, Hoyle N, Christiansen C. Serum and plasma fragments of C-telopeptides of type I collagen (CTX) are stable during storage at low temperatures for 3 years.
    Clin Chim Acta 350:167-173 (2004).

16. Ravn P, Neugebauer G, Christiansen C. Association between pharmacokinetics of oral ibandronate and clinical response in bone mass and bone turnover in women with postmenopausal osteoporosis.
    Bone 30;320-324 (2002).

17. Ravn P. & Clemmesen B. Biochemical Markers Can Predict the Response in Bone Mass During Alendronate Treatment in Early Postmenopausal Women.
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19.  Reginster JY, Henrotin Y, Christiansen C, Gamwell-Henriksen E, Bruyere Collette J, Christgau S. Bone resorption in post-menopausal women with normal and low BMD assessed with biochemical markers specific for telopeptide derived degradation products of collagen type I.
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22.  Rosenquist C, Fledelius C, Christgau S, Pedersen BJ, Bonde M, Qvist P, Christiansen C. Serum CrossLaps® ELISA. First application of monoclonal antibodies for measurement in serum of bonerelated degradation products from
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23. Tanko LB, Bagger YZ, Alexandersen P, Devogelaer JP, Reginster JY, Chick R, Olson M, Benmammar H, Mindeholm L, Azria M, Christiansen C. Safety and Efficacy of a Novel Salmon Calcitonin (sCT) Technology-Based Oral Formulation in Healthy Postmenopausal Women: Acute and 3-Month Effects on Biomarkers of Bone Turnover.
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24. Warming L, Ravn P, Spielman D, Delmas P, Christiansen C. Trimegestone in a low-dose, continuous-combined hormone therapy regimen prevents bone loss in osteopenic postmenopausal women.
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26. Warming L, Ravn P, Christiansen C. Levonorgestrel and 17beta-estradiol given transdermally for the prevention of postmenopausal osteoporosis.
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27. Basic QC Practices On-line Course; http://www.Westgard.com.

28. Boscato, LM. and Stuart, MC., ‘Heterophilic antibodies: a problem for all immunoassays’.
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29. United States Food and Drug Administration. Biotin (Vitamin B7): Safety Communication – May interfere with lab tests. Published on Nov 28, 2017.
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30. Piketty, M. L.; Polak, M.; Flechtner, I.; Gonzales-Briceno, L.; Souberbielle, J. C. False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences. Clin Chem Lab Med 2017, 55 (6), 780.

31. Chun KY. Biotin Interference in Diagnostic Tests.
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SYMBOLS USED

DRG Instruments GmbH, Germany Frauenbergstraße 18, 35039 Marburg

• Phone: +49 (0)6421-1700 0
• Fax: +49 (0)6421-1700 50
• Website: www.drg-diagnostics.de
• E-mail: [email protected]

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081

• Phone: 973-564-7555
• Fax: 973-564-7556
• Website: www.drg-international.com
• E-mail: [email protected]

References

• Analyticon® Biotechnologies GmbH
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