8020_ykl-40.indd

An enzyme immunoassay for the
quantitation of YKL-40 in serum
Not for use in diagnostic procedures.
ENZYME IMMUNOASSAY KIT
For Research Use Only.
Not For Use in Diagnostic Procedures.
Catalog No. 8020
YKL-40 Enzyme Immunoassay Summary
Reagent Preparation
‰ Dilute 10X Wash Buffer 1:10 with DI Water‰ Prepare each vial of Enzyme Conjugate with 7 mL of Reconstitution Buffer (Use within 24 hours) Assay Procedure
and Specimens into assay wells (complete within 30 minutes) Pipette 100 μL of Capture Solution into assay wells NOTE: (use sufficient force to facilitate adequate mixing –
tap stripwell frame several times ) Incubate 60 ± 5 min at 18 – 28°C
Wash 4 times with Wash Buffer
Incubate 60 ± 5 min at 18 – 28°C
Prepare Substrate Solution 1 substrate tablet per bottle of Substrate Buffer (Allow 30 – 60 min to dissolve; shake vigorously) Wash 4 times with Wash Buffer
Incubate 60 ± 5 min at 18 – 28°C
Analyze the assay results using a linear curve fit SUMMARY AND EXPLANATION
YKL-40, also known as human cartilage glycoprotein 39 (HC gp-39), is a 40 kilodalton glycoprotein fi rst described in whey secretions of nonlactating cows.1 Subsequently, its production by chondrocytes, synovial cells, activated macrophages, neutrophils, and osteosarcoma cells (MG-63) has been reported.2-6 Though the role of YKL-40 is unknown at present, its pattern of expression and observed associations with various disease activities suggest it plays a role in tissue remodeling. It has both heparin and chitin binding domains and appears to be an autoantigen in rheumatoid arthritis.3,4,7The fi rst method for the measure of YKL-40 in serum (and synovial fl uid) – an RIA – was described by Johansen et al in 1993.8 They have used the RIA in a number of clinical investigations describing its possible utility as a marker of joint destruction activity in rheumatoid arthritis and osteoarthritis, liver fi brosis in alcoholic cirrhosis, and survival following recurring breast cancer or colorectal cancer.8-12 PRINCIPLE OF THE PROCEDURE
The YKL-40 assay is a sandwich enzyme immunoassay in a microtiter stripwell format. The Fab fragment of a monoclonal anti-YKL-40 antibody conjugated to biotin binds to streptavidin on the strip and captures YKL-40 in a standard, control, or sample. A polyclonal anti-YKL-40 antibody conjugated to alkaline phosphatase binds to the captured YKL-40. Bound enzyme activity is detected with p-nitrophenyl phosphate as substrate.
REAGENTS AND MATERIALS PROVIDED
96 Assays for YKL-40Metra YKL-40 EIA kit contains the following: A YKL-40 Standards:
Parts 4620-4625
B – F, 0.3 mL each
C (A = 0, B = 20, C = 50, D = 100, E = 200, F = 300 ng/mL)
D YKL-40 purifi ed from osteosarcoma MG-63 cells in a buff ered solution
E containing stabilizer and sodium azide (0.1%) as a preservative
F
L Low/High Controls
Parts 4626, 4627
H YKL-40 purifi ed from osteosarcoma MG-63 cells in a buff ered solution
containing stabilizer and sodium azide (0.1%) as a preservative q Coated Strips
Part 4634
Streptavidin adsorbed onto 12 eight-well breakaway strips in a resealable foil pouch w Stop Solution
Part 4702
e 10X Wash Buff er
Part 4703
Nonionic detergent in a buff ered solution containing sodium azide (0.05%) as a preservative r Reconstitution Buff er
Part 4628
Nonionic detergent in a buff ered solution containing food dye, stabilizers, and sodium azide (0.1%) as a preservative t Substrate Buff er
Part 4705
A diethanolamine and magnesium chloride solution containing sodium azide (0.05%) as a preservative y Substrate Tablets
Part 0012
u Enzyme Conjugate
Part 4633
Lyophilized rabbit polyclonal anti-YKL-40 antibody conjugated to alkaline phosphatase containing buff er salts and stabilizers i Capture Solution
Part 4629
Mouse monoclonal anti-YKL-40 Fab antibody conjugated to biotin in a buff ered solution containing food dye, stabilizers, and sodium azide (0.1%) as a preservative MATERIALS REQUIRED BUT NOT PROVIDED
• Micropipettes to deliver 25-300 μL and 500 μL
• Multichannel pipettes
• Items suitable for liquid measurement of 7-600 mL
• Container for wash buff er dilution
• Deionized or distilled water
• Plate reader capable of reading at 405 nm
• Linear curve fi tting software
WARNINGS AND PRECAUTIONS
1. For Research Use Only. Not for Use in Diagnostic Procedures.
2. Treat specimen samples as potentially biohazardous material. Follow Universal Precautions when handling contents of this kit and any patient samples.
3. Dispose of containers and unused contents in accordance with Federal, State and Local regulatory requirements.
4. Use the supplied reagents as an integral unit prior to the expiration date indicated on the package label.
5. Store assay reagents as indicated.
6. Do not use Coated Strips if pouch is punctured.
7. Test each sample in duplicate.
8. 0.5N NaOH is considered corrosive and can cause irritation. Do not ingest. Avoid contact with skin, eyes or clothing. If contact is made, wash with water. If ingested, call a physician.
9. Sodium azide is used as a preservative. Incidental contact with or ingestion of buff ers containing sodium azide can cause irritation to the skin, eyes, or mouth. Only use buff ers for intended purposes and avoid contact with acids. Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Upon disposal, fl ush with a large volume of water to prevent azide build-up.
10. The substrate buff er contains diethanolamine and may cause irritation to the eyes and/or skin with prolonged contact. Contacted areas should be immediately washed with soap and water.
11. Wear suitable protective clothing, gloves, and eye/face protection when handling contents of this kit. 12. Use of multichannel pipettes or repeat pipettors is recommended to ensure timely delivery of reagents.
13. For accurate measurement of samples, add samples and standards precisely. Pipette carefully using only calibrated equipment.
14. Dilute samples greater than 300 ng/mL in Standard A and retest. Be sure to include the dilution factor in the fi nal calculation.
15. Perform this assay with any validated washing method.
16. Perform a standard curve with each assay.
17. Adequate mixing of Standards, Controls and samples with Capture Solution is required for acceptable assay performance. Dispense Capture Solution forcefully into microtiter wells.
18. If room temperature cannot be maintained between 18-28°C, monitor the development of signal in the YKL-40 Standard F wells; stop the reaction when the optical density reaches at least 1.2; then read the strip(s).
19. If the plate reader is incapable of linear optical density readings between 2.0 and 3.0, monitor the development of substrate in the YKL-40 Standard F wells; stop the reaction when the optical density reaches at least 1.2 but less than 2.0; then read the strip(s).
REAGENT PREPARATION
All reagents should be equilibrated to 18-28°C prior to use.
Coated Strips
Remove Stripwell Frame and the required number of Coated Strips
from the pouch (see table in Assay Procedure section). Ensure that
the pouch containing any unused strips is completely resealed and
contains desiccant.
Wash Buff er
Prepare required amount of 1X Wash Buff er (see table) by diluting
10X Wash Buff er 1:10 with deionized water. Store at 18-28°C. Use
1X Wash Buff er within 4 weeks of preparation.
Enzyme Conjugate
Prepare Enzyme Conjugate within 24 hours of use. Reconstitute
each required vial of Enzyme Conjugate (see table) with 7 mL of
Reconstitution Buff er. Store reconstituted Enzyme Conjugate at
18-28°C until use. Discard remaining Enzyme Conjugate after use.
Working Substrate Solution
The Substrate Buff er must be brought to 18-28°C before beginning
the assay (two hours to overnight recommended). Prepare Working
Substrate Solution within 1 hour of use. Put one Substrate Tablet
into each required bottle of room temperature Substrate Buff er
(see table). Allow 30-60 minutes for tablet(s) to dissolve. Vigorously
shake bottle(s) to completely mix.
Store the kit at 2-8°C. Store unused reagents at 2-8°C. Store 1X Wash Buff er (10X diluted) at 18-28°C.
INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS
Cloudiness, discoloration, or off ensive odor may indicate instability or deterioration of kit reagents. If this occurs, discard the reagent.
SPECIMEN COLLECTION AND PREPARATION
Handle and dispose of all specimens using Universal Precautions.
Collect serum using standard venipuncture technique. Specimens
should be collected without anticoagulants and processed to
avoid hemolysis. Allow the blood to clot and separate the serum
by centrifugation. When left at room temperature, blood must be
processed into serum in less than 3 hours or into plasma within
8 hours. If this is not possible, the blood samples must be stored
at 4°C until processed. Serum can be stored for 7 days at 2-8°C, or
freeze the sample at ≤ -20°C for longer storage. Do not subject the
samples to more than 5 freeze/thaw cycles.
ASSAY PROCEDURE
Read entire product insert before beginning the assay.
See WARNINGS AND PRECAUTIONS and REAGENT PREPARATION.
Determine amount of each reagent required for the number of
strips to be used.
*When more than one bottle or vial is to be used, combine the contents and mix prior to use.
†Automated plate washers may require larger volumes.
Sample / Capture Solution Incubation
1. Allow pouch of Coated Strips to equilibrate to 18-28°C before
opening. Remove Stripwell Frame and the required number of Coated Strips from the pouch (see table). Ensure that the pouch containing unused strips is completely resealed and contains desiccant.
2. Place desired number of Coated Strips in Stripwell Frame just prior to use. Label strips to prevent mix-up in case of accidental removal from Stripwell Frame.
3. Add 20 μL Standard, Control, or sample to each well of the Coated Strips. This step should be completed within 30 minutes.
4. Add 100 μL of Capture Solution to each well. Dispense Capture cient force to ensure adequate mixing. Tap 5. Incubate 60 ± 5 minutes at 18-28°C.
Enzyme Conjugate Incubation
6. Prepare required amount of 1X Wash Buff er (see table) by
diluting 10X Wash Buff er concentrate 1:10 with deionized water. Store at 18-28°C. Use 1X Wash Buff er within 4 weeks.
7. Manually invert/empty strips. Add at least 250 μL of 1X Wash Buff er to each well and manually invert/empty strips. Repeat three more times for a total of four washes. Vigorously blot the strips dry on paper towels after the last wash.
8. Prepare Enzyme Conjugate within 24 hours of use. Reconstitute each required vial of Enzyme Conjugate (see table) with 7 mL of Reconstitution Buff er. Store reconstituted Enzyme Conjugate at 18-28°C until use.
9. Add 100 μL of reconstituted Enzyme Conjugate to each well. Discard remaining reconstituted Enzyme Conjugate after use.
10. Incubate 60 ± 5 minutes at 18-28°C.
11. Prepare Working Substrate Solution within 1 hour of use. Put one Substrate Tablet into each required bottle of 18-28°C Substrate Buff er (see table). Allow 30-60 minutes for tablet(s) to dissolve. Vigorously shake bottle(s) to completely mix.
Substrate Incubation
12. Manually invert/empty strips. Add at least 250 μL of 1X Wash
Buff er to each well and manually invert/empty strips. Repeat three more times for a total of four washes. Vigorously blot the strips dry on paper towels after the last wash. While the strips are inverted, carefully wipe the bottom of strips with a lint-free paper towel to ensure that the bottom of the strips are clean.
13. Add 100 μL of Working Substrate Solution to each well.
14. Incubate for 60 ± 5 minutes at 18-28°C.
Stop/Read
15. Add 100 μL of Stop Solution to each well. Add Stop Solution in
the same pattern and time intervals as the Working Substrate Solution addition.
16. Read the optical density at 405 nm. Assure that no large bubbles are present in wells and that the bottom of the strips
are clean. Strips should be read within 15 minutes of Stop
Solution addition.
17. Use a linear calibration curve to analyze Metra YKL-40 assay 18. Determine concentration of samples and Controls from the 19. Control values should be within the range specifi ed in the Certifi cate of Analysis supplied with the kit.
QUALITY CONTROL
The Certifi cate of Analysis included in this kit is lot specifi c and is to be used to verify that the results obtained by your laboratory are similar to those obtained at Quidel Corporation. The optical density values are provided and are to be used as a guideline only. The results obtained by your laboratory may diff er.
Quality control ranges are provided. The control values are intended to verify the validity of the curve and sample results. Each laboratory should establish its own parameters for acceptable assay limits. If the control values are NOT within your laboratory’s acceptance limits, the assay results should be considered questionable and the samples should be repeated.
If the optical density of the YKL-40 Standard A is greater than 0.4, the results should be considered questionable and the samples should be repeated.
INTERPRETATION OF RESULTS
YKL-40 assay results are expressed in ng/mL and do not need to be corrected for dilution (unless sample was diluted prior to testing).
Representative Standard Curve
Standard YKL-40 levels: 0, 20, 50, 100, 200, 300 ng/mL
0
(4
ity
s
n
e
l D
a
YKL-40 (ng/mL)
EXAMPLE VALUES
In our testing of 329 adults ≤ 60 years of age, values obtained from the Metra YKL-40 kit had a range of 24-125 ng/mL. Each laboratory should establish its own reference range.
PERFORMANCE OF THE TEST
Limits of Detection
The minimum detection limit of the YKL-40 assay is 20 ng/mL.
Recovery - Spike Recovery
Spike recovery was determined by adding a known quantity
of purifi ed YKL-40 to serum samples with diff erent levels of
endogenous YKL-40. Typical results are provided below.
Recovery - Linearity
Linearity was determined by diluting samples in Standard A and
comparing observed values with expected values. Typical results
are provided below.
Precision
Within-run and between-run precision were determined by
assaying up to 22 serum samples in 6 diff erent runs.
Interfering Substances
The following substances were tested at the specifi ed
concentrations, and were found not to interfere with the assay.
Drug Interferences
Various concentrations of drugs were added to serum specimens.
The following drugs were found not to interfere at the highest
concentrations shown:
ASSISTANCE
To place an order or for technical assistance, please contact a Quidel
Representative at 800-524-6318 or 408-616-4301, Monday through
Friday, between 8:00 a.m. and 5:00 p.m., Pacifi c Time. Orders may
also be placed by fax at 408-616-4310.
For services outside the U.S., please contact your local distributor.
Protected by U.S. Patent No. 7,230,086; other patents pending.
REFERENCES
1. Rejman JJ, Hurley WL. Isolation and characterization of a novel 39 kilodalton whey protein from bovine mammary secretions collected during the non-lactating period. Biochem.Biophys.Res. 2. Hakala BE, White C, Recklies AD. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J.Biol.Chem. 3. Nyirkos P, Golds EE. Human synovial cells secrete a 39 kDa protein similar to a bovine mammary protein expressed during the non-lactating period. Biochem.J. 1990;268:265-8.
4. Renkema GH, Boot RG, Au FL, et al. Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosyl hydrolases secreted by human macrophages. Eur.J.Biochem. 1998;251:504-9.
5. Volck B, Price PA, Johansen JS, et al. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specifi c granules in human neutrophils. Proc.Assoc.Am.Physicians 6. Johansen JS, Williamson MK, Rice JS, Price PA. Identifi cation of proteins secreted by human osteoblastic cells in culture. J.Bone 7. Verheijden GF, Rijnders AW, Bos E, et al. Human cartilage glycoprotein-39 as a candidate autoantigen in rheumatoid arthritis. Arthritis Rheum. 1997;40:1115-25.
8. Johansen JS, Jensen HS, Price PA. A new biochemical marker for joint injury. Analysis of YKL-40 in serum and synovial fl uid. Br.J.Rheumatol. 1993;32:949-55.
9. Johansen JS, Hvolris J, Hansen M, et al. Serum YKL-40 levels in healthy children and adults. Comparison with serum and synovial fl uid levels of YKL-40 in patients with osteoarthritis or trauma of the knee joint. Br.J.Rheumatol. 1996;35:553-9.
10. Johansen JS, Møller S, Price PA, et al. Plasma YKL-40: a new potential marker of fi brosis in patients with alcoholic cirrhosis? Scand.J.Gastroenterol. 1997;32:582-90.
11. Johansen JS, Cintin C, Jorgensen M, et al. Serum YKL-40: a new potential marker of prognosis and location of metastases of patients with recurrent breast cancer. Eur.J.Cancer 12. Cintin C, Johansen JS, Christensen IJ, et al. Serum YKL-40 and colorectal cancer. Br.J.Cancer 1999;79:1494-9.
13. Centers for Disease Control. Recommendations for prevention of HIV transmission in health-care settings. MMWR 1987;36 REF 8020 – Metra® YKL-40 Enzyme Immunoassay Kit
Quidel Corporation
Worldwide Headquarters
10165 McKellar Court
San Diego, CA 92121 USA
www.quidel.com
0645G (2007/10)

Source: http://www.biolead.com.cn/datasheet%5Cquidel%5C8020_metra_YKL-40.pdf