Myoglobin elisa package insert

DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
Enzyme Immunoassay for the Quantitative Determination of Myoglobin Concentration in Human Serum
FOR IN VITRO DIAGNOSTIC USE ONLY
PROPRIETARY AND COMMON NAMES

INTENDED USE
The Myoglobin ELISA is intended for the quantitative determination of myoglobin in human serum.
INTRODUCTION
Myoglobin, a heme protein with a molecular weight of approximately 17,500 Daltons is found in both cardiac and
skeletal muscle. Damage to either type of muscle following conditions such as trauma, ischemia, and diseases that
cause myopathy, is associated with the release of myoglobin into serum.1,2 Specifically, following cardiac necrosis
associated with myocardial infarction (MI), myoglobin is one of the first markers to rise above normal levels.
Myoglobin levels increase measurably above baseline within 2-4 hours post-infarct, peaking at 9-12 hours, and
returning to baseline within 24-36 hours.1,3,4,5
In the absence of skeletal muscle trauma or other factors associated with a non-cardiac related increase in circulating
myoglobin, its levels have been used as an early marker for myocardial infarct.4,6,7 A number of reports suggest
using the measurement of myoglobin as a diagnostic aid in ruling out myocardial infarction5,8 with negative
predictive values of up to 100% reported at certain time periods after the onset of symptoms.9-15 Unlike the other
cardiac enzymes such as creeatine kinase and the MB isoform (i.e., CK and CK/MB) which do not reach serum
levels until several hours post-infarction (approx. 19 hours), myoglobin levels can be expected to peak within 6 to 9
hours.16
The Myoglobin Enzyme Immunoassay provides a rapid, sensitive, and reliable assay for the quantitative
measurement of myoglobin in serum. The antibodies developed for the test will determine a minimal concentration
of 5.0 ng/ml, and there is no cross-reactivity with related cardiac or skeletal enzymes.
PRINCIPLE OF THE TEST
The Myoglobin ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay.17,18 The
assay system utilizes a unique monoclonal antibody directed against a distinct antigenic determinant on the
myoglobin molecule. Mouse monoclonal anti-myoglobin antibody is used for solid phase immobilization (on the
microtiter wells). A goat anti-myoglobin antibody is in the antibody-enzyme (horseradish peroxidase) conjugate
solution. The test sample is allowed to react simultaneously with the two antibodies, resulting in the myoglobin
molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 45 minute incubation at
room temperature, the wells are washed with water to remove unbound labeled antibodies. A TMB (Tetramethyl-
benzidine) Reagent is added and incubated for 20 minutes, resulting in the development of a blue color. The color
development is stopped with the addition of Stop Solution changing the color to yellow. The concentration of
myoglobin is directly proportional to the color intensity of the test sample. Absorbance is measured
spectrophotometrically at 450 nm.
REAGENTS

Materials provided with the kit:
1. Antibody-Coated Wells (1 plate, 96 wells)
Microtiter wells coated with murine monoclonal anti-myoglobin. 2. Reference Standard Set (1.0 ml/vial, 1 set/kit) DRG International Inc., USA
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
Contains 0, 25, 100, 250, 500, and 1000 ng/ml myoglobin, liquid, ready-to-use. These standards have been pre-
diluted 10-fold. Please do not dilute them again.
Contains bovine serum and 1.0% (w/v) Pro-Clin as preservative. 4. Enzyme Conjugate Reagent (22 ml/vial) Contains anti-myoglobin conjugated to horseradish peroxidase in Tris Buffer-BSA solution with preservatives. 6. Stop Solution (1 bottle, 11 ml/bottle) Contains diluted hydrochloric acid (1N HCl).
Materials required but not provided:
• Precision pipettes: 20 μl , 50 μl, 200 μl, and 1.0 ml
• Disposable pipette tips
• Distilled water
• Vortex mixer or equivalent.
• Absorbent paper or paper towels
• Graph paper
• Microtiter plate reader
WARNINGS AND PRECAUTIONS FOR USERS
1. CAUTION: This kit contains human material. The source material used for manufacture of this component
tested negative for HBsAg, HIV 1/2 and HCV by FDA-approved methods. However, no method can completely assure absence of these agents. Therefore, all human blood products, including serum samples, should be considered potentially infectious. It is recommended that the reagents and patient samples be handled according to the OSHA Standard on Bloodborne Pathogens19 or other appropriate national biohazard safety guidelines or regulations.20-21 2. Avoid contact with 1N HCl. It may cause skin irritation and burns. If contact occurs, wash with copious amounts of water and seek medical attention if irritation persists. 3. Do not use reagents after expiration date and do not mix or use components from kits with different lot numbers.
4. Replace caps on reagents immediately. Do not switch caps.
5. Do not pipette reagents by mouth.
6. For in vitro diagnostic use.
STORAGE CONDITIONS
1. Store the unopened kit at 2-8°C upon receipt and when it is not in use, until the expiration shown on the kit
label. Refer to the package label for the expiration date. 2. Keep microtiter plate in a sealed bag with desiccant to minimize exposure to damp air.

INSTRUMENTATION
A microtiter well reader with a bandwidth of 10 nm or less and an optical density range of 0 to 3 OD or greater at
450 nm wavelength is acceptable for absorbance measurement.
REAGENT PREPARATION
1. All reagents should be brought to room temperature (18-25°C) before use.
2. Patient serum and control serum should be diluted 10 fold before use. Prepare a series of small tubes (such
as 1.5 ml microcentrifuge tubes) and mix 20 μl serum with 180 μl (0.18 ml) Sample Diluent. PLEASE DO
NOT DILUTE THE STANDARDS - THEY HAVE ALREADY BEEN PRE-DILUTED 10-FOLD.
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
3. Samples with expected myoglobin concentrations over 1000 ng/ml may be quantitated by further dilution 10-
SPECIMEN COLLECTION AND PREPARATION
1. The use of SERUM samples is required for this test.
2. Specimens should be collected using standard venipuncture techniques. Remove serum from the coagulated or
packed cells within 60 minutes after collection. 3. Specimens which cannot be assayed within 24 hours of collection should be frozen at -20°C or lower, and will 4. Specimens should not be repeatedly frozen and thawed prior to testing. DO NOT store in "frost free" freezers, 5. Specimens which have been frozen, and those which are turbid and/or contain particulate matter, must be PROCEDURAL NOTES
1. Pipetting Recommendations (single and multi-channel): Pipetting of all standards, samples, and controls should
2. All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are 3. It is recommended that the wells be read within 15 minutes following addition of Stop Solution.
ASSAY PROCEDURE
1. Patient serum and control serum should be diluted 10 fold before use. Prepare a series of small tubes (such
as 1.5 ml microcentrifuge tubes) and mix 20 μl serum or plasma with 180 μl (0.18 ml) Sample Diluent.
PLEASE DO NOT DILUTE THE STANDARDS - THEY HAVE ALREADY BEEN PRE-DILUTED 10-
FOLD.
2. Secure the desired number of coated wells in the holder.
3. Dispense 20 μl of myoglobin standards, diluted specimens and diluted controls into the appropriate wells.
4. Dispense 200 μl of Enzyme Conjugate Reagent into each well.
5. Thoroughly mix for 30 seconds. It is very important to mix completely.
6. Incubate at room temperature (18-25°C) for 45 minutes.
7. Remove the incubation mixture by flicking plate contents into a waste container.
8. Rinse and flick the microwells 5 times with distilled or deionized water. (Please do not use tap water.)
9. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water drops.
10. Dispense 100 μl of TMB Reagent solution into each well. Gently mix for 5 seconds.
11. Incubate at room temperature for 20 minutes.
12. Stop the reaction by adding 100 μl of Stop Solution to each well.
13. Gently mix 30 seconds. It is important to make sure that all the blue color changes to yellow color completely.
14. Read absorbance at 450 nm with a microtiter well reader within 15 minutes.

QUALITY CONTROL
1. Good laboratory practice requires that quality control specimens (controls) be run with each calibration curve to
2. To ensure proper performance, control material should be assayed repeatedly to establish mean values and
CALCULATION OF RESULTS
1. Calculate the mean absorbance value (A450) for each set of reference standards, controls and samples.
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in ng/ml on graph paper, with absorbance values on the vertical or Y axis, and concentrations on the horizontal or X axis. 3. Use the mean absorbance values for each specimen to determine the corresponding concentration of myoglobin 4. Since the reference standards have already been pre-diluted 10-fold, there is no need for the patient samples or control sera observed values to be multiplied by the dilution factor of 10. However, if the patient samples are diluted to 100-fold, the observed values should be multiplied by 10.
EXAMPLE OF STANDARD CURVE
Results of a typical standard run with optical density readings at 450 nm shown in the Y axis against myoglobin
concentrations shown in the X axis. This standard curve is for illustrative purpose only, and should not be used to
calculate unknowns. Each laboratory should obtain its own data and standard curve.
Myoglobin
Absorbance
Absorbance (450 nm)
M yoglobin Concentration (ng/ml)

LIMITATIONS OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete
understanding of the package insert instructions and with adherence to good laboratory practice. 2. Diagnostic results obtained from the Myoglobin ELISA should be used in conjuction with other diagnostic procedures and information available to the physician.; e.g., additional clinical testing, ECG, symptoms, and clinical observations. 3. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance 4. Patient samples may contain human anti-mouse antibodies (HAMA) which are capable of giving falsely elevated results with assays that utilize mouse monoclonal antibodies. The Myoglobin ELISA assay has been designed DRG International Inc., USA
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
to minimize interference from HAMA-containing specimens; nevertheless complete elimination of this interference from all patient specimens cannot be guaranteed. 5. Test results that are inconsistent with the clinical picture and patient history should be interpreted with caution.
EXPECTED NORMAL VALUES
1. Normal serum myoglobin levels range from 12 to 100 ng/ml. Values increase slightly with age.22
2. Using the Myoglobin ELISA, an evaluation of the clinical data was conducted to determine the normal expected
value of the kit. THe study yielded normal range values in agreement with industry standards. Eighty-three (83) apparently healthy adults were assayed using the test to establish the normal expected value. The range was found to be between 8.1 and 54.5 ng/ml myoglobin. 3. Each facility should establish its own reference intervals for myoglobin as performed on ELISA test. Other factors should also be considered in the diagnosis of myocardial infarction, as any condition resulting in skeletal
or cardiac muscle damage may potentially increase myoglobinlevels above the expected normal range.
NOTE: Serial sampling may be required to detect elevated levels.

PERFORMANCE CHARACTERISTICS
1. Clinical Performance
A clinical investigation was conducted to determine the accuracy of the Myoglobin ELISA as compared to the Abbott AxSym Myoglobin MEIA. The data is presented below. A statistical study using 150 clinical patient serum samples, ranging in myoglobin concentration from 3.7 ng/ml to 919.8 ng/ml as analyzed using the Myoglobin ELISA (13.0 ng/ml to 1011.0 ng/ml Abbott Myoglobin MEIA), demonstrated equivalent correlation with the AxSym Myoglobin kit as shown below.
2. Sensitivity
The lowest detectable level of myoglobin by this assay is estimated to be 5 ng/ml. 3. Hook Effect
No high-dose hook effect is observed in this test with patient sample concentrations up to 10,000 ng/ml.
4. Precision
Within-run precision was determined by replicate determinations of five different serum samples in one assay. Within-assay variability is shown below: DRG International Inc., USA
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
Between-run precision was determined by replicate measurements of five different serum samples over a series of individually calibrated assays. Between-assay variability is shown below:
5. Recovery
Various patient serum samples of known myoglobin levels were combined and assayed in duplicate. The mean recovery was 102.8%. EXPECTED
OBSERVED [Myoglobin]
[Myoglobin]
RECOVERY

6. LINEARITY
Three patient samples were serially diluted to determine linearity. The mean recovery was 105.8%. Dilution
Expected Conc. (ng/ml)
Observed Conc. (ng/ml)
Expected
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007

7. Specificity
The following materials were tested for cross-reactivity at concentrations up to the levels indicated below. No
cross-reactivity was observed for any of the components.
MATERIAL TESTED
TEST CONCENTRATION
Interfering Substances
Cardiac Tnl
Endogenous Substances
Billirubin
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
Therapeutic Substances
Aspirin 0.3

STANDARDIZATION
Human myoglobin Complex was obtained from a qualified vendor, and myoglobin concentration was determined.
The material was further diluted with the Myoglobin Sample Diluent and served as "Standard Stock Solution" for
preparing myoglobin reference Standard Sets. The target value of the "Standard Stock Solution" was confirmed by
the Abbott AxSym Myoglobin immunoassay.
REFERENCES
1. Kagen, L.J.: Myoglobin: Methods and Diagnostic Uses, CRC Crit. Rev. Clin. Lab. Sci., 2:273; (1978).
2. Juronen, E.L., Viikmaa, M.H., and Mikelsarr, A.V.N.: Rapid, simple and sensitive antigen capture ELISA for
the quantitation of myoglobin using monoclonal antibodies, J. Imm. Meth.: 111, 109; (1988). 3. Chapelle JP. et al.: Serum myoglobin determinations in the assessment of acute myocardial infarction. Eur. 4. Cairns, J.P., et.al.: Usefulness of serial determinations of myoglobin and creatine kinase in serum compared for assessment of acute myocardial infarction, Clin. Chem. News, 29: 469, (1983). 5. Silva, D.P., et.al.: Development and application of antibodies to human cardiac myoglobin in rapid fluolrescence immunoassay, Clin. Chem., 37: 1356, (1991). 6. Ellis AK.: Patters of myoglobin release after reperfusion of injured myocardium. Clin. Chem., 72:639, (1985). 7. Mair J. et al.: Rapid diagnosis of myocardial infarction by immunoturbidimetric myoglobin measurement (letter). Lancet, ;337:1343, (1991). 8. Chapelle, J.P.: Myoglobin. Clin. Chem. News, 17:22, (1991). 9. Hamfelt, A., et. al.: Use of biochemical tests for myocardial infarction in the county of Vasternorrland, a clinical chemistry routine for the diagnosis of myocardial infarction. Scand. J. Clin. Lab. Invest. Suppl., 200:20, (1990). 10. Tucker, J.F., et.al.: Early diagnostic efficiency of cardiac troponin I and Troponin T for acute myocardial infarction, Academic Emergency Medicine: 4(1): 13-21; (1997). 11. de Winter, R.J., et.al.: Value of myoglobin, troponin T and CK-Mbmass in ruling out an acute myocardial infarction in the emergency room, Circulation: 92(12): 3401-7; (1995). 12. Montague, C., Kircher, T.: Myoglobin in early evaluation of acute chest pain, Amer. J. Clin. Path.: 104(4): 13. Tucker, J.F., et.al., Value of serial myoglobin levels in the early diagnosis of patients admitted for acute myocardial infarction, Annals of Emergency Medicine: 24(4): 704-8; (1994). 14. Roxin, L.E., et.al.: The value of serum myoglobin determinations in the early diagnosis of acute myocardial infarction, Acta Medica Scand.: 215(5): 417-25; (1984). 15. Sylven, C., Bendz, R.: Myoglobin, creatine kinase and its isoenzyme MB in serum after acute myocardial infarction, Eur. J. Cardiol.: 8(4-5): 515-21; (1978). 16. Norregaard-Hansen, K., et. al.: Early observations of S-myoglobin in the diagnosis of acute myocardial infarction. The influence of discrimination limit, analytical quality, patient's sex, and prevalence of disease. Scand. J. Clin. Lab. Invest., 46:561-569, (1986). 17. Engvall, E., "Methods in Enzymology", Volume 70, VanVunakis H. and Langone, J.J. (eds.), Academic Press, 18. Uotila, M., Ruouslahti, E. And Engvall, E., J. Immunol. Methods, 42, 11-15, (1981). DRG International Inc., USA
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DRG® Myoglobin (EIA-3955)
Revised 2 November, 2007
19. U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR Part 1910.1030. Occupational Exposure ot Bloodborne Pathogens; Final Rule. Federal Register; 56(235):64175, (1991). 20. USA Center for Disease Control/National Institute of Health Manual, "Biosafety in Microbiological and 21. National Committee for Clinical Laboratory Standards. Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue: Approved Guideline. NCCLS Document M29-A, (1997). 22. Clinical Guide to Laboratory Tests. N.W. Tietz, Ed., 3rd Edition, W.B. Saunders, Co., p. 482, (1995). VERS. 110207 ~sd/pm. DRG International Inc., USA
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