Myoglobin RIA
For the quantitative determination of myoglobin in human serum or urine
For Research Use Only. Not For Use In Diagnostic Procedures.
Catalog Number:
Size:
Version:
24-MYOHU-R100
100 determinations
ALPCO 1/13/2010
I.
INTENDED USE
The Myoglobin RIA test is used to measure myoglobin in human serum or urine. This kit is for
research use only.
II.
INTRODUCTION
Myoglobin is a single polypeptide chain of 153 amino acids and molecular mass of
approximately 17,500 Daltons. It contains one heme prosthetic group per molecule and is
found only in striated, skeletal, and cardiac muscle (1).
Serum myoglobin levels determined by radioimmunoassay are elevated in more than 90% of
patients with clinical and laboratory evidence of acute mvocardial infarction (2-5). In patients
with chest pain or congestive heart failure but without evidence of acute myocardial
infarction, serum myoglobin levels remain normal (2,3). Several clinical studies have
documented that hypermyoglobinemia often precedes elevation of serum creatine kinase in
patients with acute myocardial infarction (2,3). Because of the increasing use of thrombolytic
therapy (6) and acute percutaneous coronary angioplasty (7) for treating patients with acute
myocardial infarction a demand for a quick and reliable diagnosis is expected (8). Serum
myoglobin has received attention as a potential marker for the early diagnosis of acute
myocardial infarction. In dog model, it has been shown that the serum myoglobin values rise
within two hours and peak within six hours following coronary artery occlusion (9).
Elevated levels of serum myoglobin are also seen in conditions affecting striated muscle,
such as trauma to the muscles or degenerative or inflammatory muscle diseases (1,10).
Hypermyoglobinemia has been detected in patients and in genetic carriers of progressive
muscular dystrophy and thus may constitute a useful adjunct in carrier detection of this sexlinked disorder (11,12). Elevated serum myoglobin levels also have been observed during
the first 24-hour after open-heart surgery and in patients in shock or with severe renal failure
(4,9,13). Moderate to exhaustive bicycle ergometer exercise will not cause any elevated
levels of myoglobin (3,13), nor do uncomplicated intramuscular injections (2,4). Other
important causes of elevated myoglobin levels are heavy alcohol abuse (14) or reduced
glomerular filtration rate (15). Most of these causes are, however, easily sorted out by serial
blood sampling since the levels are generally unaltered in these conditions (16).
Therefore, for the research of early prediction of acute myocardial infarction, the serum
myoglobin assay is a useful tool, especially since the assay time is short. Using the Myoglobin
RIA test kit, values can be obtained in less than 1 hr.
III.
PRINCIPLE
The double antibody myoglobin procedure is a competitive RIA in which 125-I labeled
myoglobin competes with myoglobin in samples for limited antibody binding sites. After
incubation for a fixed time, separation of bound from free is achieved by the PEG
accelerated double antibody precipitation method. The radioactivity of the precipitated
antibody-bound couplex is counted in a gamma spectrometer. The concentration of
myoglobin in samples is determined from a calibration curve.
IV.
WARING AND PRECAUTIONS
Some of the reagents in this kit contain sodium azide. Sodium azide may react with lead and
copper plumbing to form highly explosive metal azides. On disposal, flush with a large volume
2/9
of water to prevent azide accumulation. Sodium azide is also toxic. Care should be taken to
avoid ingestion.
Handle all components and all samples as if capable of transmitting hepatitis and the
acquired immunedeficiency syndrome. Source materials derived from human body fluids and
used in the preparation of this kit were tested and found negative for hepatitis B surface
antigen and HIV antibody. However, no known test can guarantee that such material does
not contain the causative agent of viral hepatitis or HIV antigen.
Caution. Radioactive material not for use in human or animals. To minimize exposure to
radiation, the user should adhere to guidelines set forth in the National Bureau of Standards
publication on the Safe Handling of Radioactive Materials (handbook No. 92, issued March 9,
1964) and in subsequent publications issued by State and Federal authorities. Radioactive
materials should be confined to specifically designated, regularly monitored areas in the
laboratory, away from traffic and restricted to authorized personnel, with food, drink, smoking
and the application of cosmetics all expressly prohibited. Use disposable lab ware and
disposable absorbent bench covers. Always wear film badges, lab coats and disposable
gloves. Never pipet radioactive materials by mouth. Wipe up spills promptly, washing the
affected surface with a decontaminant and monitoring with a radiation detector. Place
contaminated tissues, tribes, bench eaters, gloves, etc., in a specially marked container for
disposal as solid radioactive waste. Wash thoroughly after work. Maintain complete records
of the receipt, use and disposal of all radioactive materials. Discard liquid, dispersible and
solid radioactive waste only as permitted by Federal, State and local ordinances.
V.
REAGENTS AND MATERIALS
Materials supplied in this test kit are sufficient for 100 determinations.
•
•
•
•
•
VI.
Myoglobin calibrators, catalog Nos. 11-11, 11-12, 11-13, 11-14, and 11-15
respectively. Buffered reagent containing five different concentrations of myoglobin.
Concentration (ng/ml) is indicated on the labels (approximately 25-500 ng/ml). 0.01%
sodium azide added as preservative. Zero calibrator 4 ml, other calibrators 1.0 ml per
vial.
125-I Myoglobin reagent, catalog No. 11-30. Buffered reagent containing 125-I
myoglobin tracer 0.01% sodium azide added as preservative. < 185 kBq (5µ.Ci). 10
ml. Red color.
Myoglobin antiserum reagent, catalog No. 11-40. Buffered reagent containing rabbit
anti human myoglobin antiserum. 0.01% sodium azide added as preservative. 20 ml.
Green color.
Myoglobin controls, catalog No. 11-21 and 11-22 respectively. Human serum
containing two different concentrations of myoglobin values are listed on the product
inserts. 0.01% sodium azide is added as preservative. 1.0 ml per vial.
Myoglobin precipitating reagent, catalog No. 11-50. Buffered reagent containing goat
anti rabbit gamma globulin, and dilute polyethylene glycol. 0.01% sodium azide is
added as preservative. 100 ml.
STORAGE AND STABILITY
Store the test kit in refrigerator (2-8ºC). Reagents are stable until the expiration date shown
on kit labels.
3/9
VI.
SPECIMAN COLLECTION
•
Collect blood by venipuncture into plain tubes avoiding hemolysis. Separate the
serum from the cells by centrifugation. The procedure calls for 100 µl of serum per
assay tube. Grossly lipemic samples and samples contaminated with radioactivity are
unsuitable for use. The samples may be stored under refrigeration (2-8º C) for 24
hours. Samples may be stored frozen (-20º C or lower) for longer periods of time.
Aliquot the sample before freezing to avoid repeated freeze-thaw cycles.
Collect urine sample in a urine specimen container without any preservative. The
specimen may be stored under refrigeration for 24 hours; for longer periods freeze
the specimen at -20º C or lower. All urine samples must be free from gross turbidity,
if required centrifuge or filter before assay.
•
VIII.
TEST PROCEDURE
•
Materials supplied:
Material
Myoglobin Calibrator 1
Myoglobin Calibrator 2
Myoglobin Calibrator 3
Myoglobin Calibrator 4
Myoglobin Calibrator 5
125-I myoglobin reagent
Myoglobin antiserum reagent
Myoglobin precipitating reagent
Myoglobin control level I
Myoglobin control level 2
•
Quantity
One vial
One vial
One vial
One vial
One vial
One vial
One vial
One bottle
One vial
One vial
Catalog No
11-11
11-12
11-13
11-14
11-15
11-30
11-40
11-50
11-21
11-22
Materials required but not supplied:
Gamma counter
Centrifuge capable of > 1500 xg (preferably
refrigerated) Refrigerator (2°-8°C and -20°C)
Vortex mixer
Test tube racks
Distilled or deionized water
Repeating dispenser for 1 ml (optional)
Foam decanting rack
12 x 75 mm disposable plastic or borosilicate test
tubes 100-200 µl precision pipet with disposable tips
Volumetric pipets 1.0 and 10.0 ml
•
Assay Procedure:
All components except the precipitating solution must be at ambient temperature
before use. Keep precipitating reagent in cold.
1. Label and arrange test tubes in duplicate according to the protocol
shown in table l.
2. Pipet 100 µl of the calibrator, control or unknown to the appropriate
4/9
3.
4.
5.
6.
7.
8.
9.
tubes except the total count tubes. To NSB tubes add 300 µl of “0”
calibrator.
Add 100 µl 125-I myoglobin reagent (red color) to every tube.
Add 200 µl myoglobin antiserum (green color) to every tube except the NSB
and total count tubes. Vortex.
Incubate for 30 minutes at room temperature.
Add 1.0 ml of cold precipitating solution (shake the bottle before use) to all tubes
except the total count tubes. Vortex.
Centrifuge all tubes except the total count tubes for 15 minutes at 1500 x g,
preferably at 2-8ºC.
Using a foam decanting rack decant (or aspirate) all tubes except the total count
tubes. Let the tubes stand inverted on absorbent paper for 30-60 seconds. Tap the
tubes gently and blot the rims to remove all residual droplets.
Count each tube for 1 minute.
Table 1 Assay Protocol
Calibrator/
Table #
Control/
125-I Myoglobin
µl
Unknown
1,2
3,4
5,6
7,8
9,10
11,12
13,14
15,16
17,18
•
•
•
•
reagent (red color)
µl
Myoglobin
antiserum reagent
(green color)
µl
PEG-goat
anti-rabbit IgG
ml
Total Count
NSB, calibrator 1 300
100
Maximum binding 100
(B0) calibrator 1
100
200
1
Calibrator 2
Calibrator 3
Calibrator 4
Calibratror 5
Control 1
Control 2
100
100
100
100
100
100
200
200
200
200
200
200
1
1
1
1
1
1
100
100
100
100
100
100
1
Pipet 100 µl calibrator, control, or unknown
Add 100 µl (125-I) Myoglobin reagent
Add 200 µl antiserum, vortex, incubate for 30 min, at room temperature
Add 1 ml cold precipitating reagent, vortex, centrifuge, decant, and count
Calculation of results:
1. Determine average counts per minute (cpm> for NSB, calibrators, controls and sample
tubes.
2. Calculate percent tracer bound <%B> for each calibrators, controls and sample relative
to the maximum binding (BO, zero stands) tubes as follows:
% B/BO =
pm <standard, control or unknown> - cpm (NSB)
cpm (zero standard) - cpm <NSB)
x 100
5/9
Using Logit-Log graph paper, plot percent bound on the vertical axis against concentration
on the horizontal axis for each of the calibrators and draw a best fit straight line through the
points.
Alternatively, the data can be plotted on a linear-log graph paper by plotting percent bound
on the vertical axis against concentration on the horizontal axis for each of the calibrators
and draw a best fit straight line through the points. Myoglobin concentrations for the
unknowns can then be estimated from the line by interpolation.
•
Quality Control
The reliability of test results should be monitored by the routine use of control reagents of
known myoglobin concentrations. Two quality control pools are supplied with the kit and
should be analyzed with each assay. The results should be charted from assay to assay and
the overall performance checked periodically.
IX.
LIMITATIONS
1. Samples with values greater than 500 ng/ml should be diluted with 0 calibrator and
reassayed.
2. Slight hemolysis and/or lipemia do not interfere with the assay.
3. Do not use acidified urine samples. Do not use if the sample exhibits significant
bacterial growth.
4. Since serum myoglobin values return to normal sooner than enzymes in acute
myocardial infarction, late blood sampling (12-24 hours following hospital admission)
may show abnormal values in about 50% with those with acute myocardial
infarctions.
Typical Standard Curve Data
TUBE
NO.
COUNTS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
34840
33616
590
678
14835
15299
12351
12288
7615
7388
4561
4461
2766
2542
AVG.
%CV B/BO
COUNTS
TC
TC
34228
1.79 TC
NSB
634
6.94 NSB
BO
15067
1.54 BO
.820
12319
.26 .816
.505
7501
1.51 .490
.303
4511
1.11 .296
.184
2654
4.22 .169
AVG. CONC. CALC.
B/BO (NG/ML) CONC.
AVG.
CONC.
%DIFF
TC
NSB
BO
.818
.498
.299
.176
0.00
0.00
25.00
25.00
100.00
100.00
250.00
250.00
500.00
500.00
23.82
24.49
104.15
110.54
242.71
250.42
465.01
514.59
24.15
107.34
246.56
489.8
-4.7
-2.0
4.1
10.5
-2.9
0.2
-7.0
2.9
Logit (B/BO)
%BO = 44.02
ED (20%) = 418.77
SLOPE = -1.011
%NSB = 1.85
ED (50%) = 106.40
INTERCEPT = 4.722
ED (80%) = 27.04
Correlation Coefficient = -0.9988
6/9
Typical Standard Curve Data
25
X.
100
250
500
PERFORMANCE CHARACTERISTICS
•Precision:
The reliability of the myoglobin RIA test procedure was assessed by examining its
reproducibility on samples selected to represent a range of myoglobin levels.
a. Intraassay:
Within assay variation was determined from 10 single determination of myoglobin in
four different sample pools. Data are summarized below:
No. of
determinations
(single)
Mean value
(ng/ml)
10
10
10
10
19.71
53.58
266.2
390.80
SD
(ng/ml)
1.6
3.0
3.1
11.6
% CV
8.2
5.6
1.2
2.9
b.
Interassay:
Between assay variation was determined from five to six different assays run in 8-10 single
determination of myoglobin on four different sample pools. Data are summarized below:
No. of
determinations
5
6
6
5
Mean value
(ng/ml)
19.93
52.89
267.81
399.64
SD
(ng/ml)
0.67
1.65
2.07
9.21
% CV
3.36
3.12
0.77
2.31
7/9
• Sensitivity:
Ten “0” calibrator (maximum binding) tubes were processed along with a calibration curve.
Mean and standard deviation were calculated for the counts per minute of the ten “0” tubes.
Apparent sensitivity was then calculated from the average of 1, 2, 3, and 4 standard deviations
and was found to be 10 ng/ml.
• Spiking Recovery:
Recovery studies were done by spiking two samples with different concentrations of
myoglobin. The results are summarized below:
Base value
(ng/ml)
A. 26.8
B. 133.1
Amount added
(ng/ml)
12.5
125
250
12.5
125
250
Amount recovered
% Recovered
(ng/ml)
39.3
98
151.8
95.3
276.8
101.8
145.6
104.1
258.1
96.6
383.1
96.6
• Parallelism:
Three samples were serially diluted with the kit’s “0” calibrator. The observed and expected
values are summarized below:
Sample Dilation (ng/ml)
A. 465 (calibrator)
1:2
1:4
1:8
1:16
1:16
1:64
B. 266 (serum sample)
1:2
1:4
1:16
Observed (ng/ml)
465
232.5
133
61.3
29.5
16.5
6.7
266
137.3
67.7
34
Expected (ng/ml)
465
241
120.5
60.3
30.1
15.1
6.3
266
133
66.5
33.3
% Recovered
C. 100 (serum sample)
1:2
100
46.6
100
50
100
93.3
1:4
20.1
25
80.4
1:16
7.4
6.3
100
96.5
110.4
101.7
97.9
109.4
110
100
103.2
101.7
102.3
117
8/9
XI. EXPECTED VALUES
Normal range study was conducted on 64 human serum and 32 human urine samples using
myoglobin double antibody RIA kit. However each laboratory should establish its own normal
values to conform with the characteristics of the population that is being tested.
The following results were obtained with serum samples:
Range: 0-56 ng/ml
Mean: 17.6 ng/ml
The following results were obtained with urine samples:
Range: 0-86 ng/ml
Mean: 7.86 ng/ml
The normal range limit suggested by this study should be regarded as a guideline only. It is
important that each laboratory establishes its own normal range.
XIII. REFERENCES
1. Kagen, L., “Myoglobin: Biochemical, Physiological and Clinical Aspects.” Columbia
University Press, New York 1973
2. Stone, M., Willerson, J., Gomez-Sanchez, C and Waterman, M. J. Clin. Invest. 56,
1334, 1975
3. Stone, M., Waterman, M., Harimoto, D., Murray, G., Wilson, N., Platt, M.,
Blomquist, G and Willerson, J. Br. Heart J. 39, 375, 1977
4. Varki, A., Roby, D., Watts, H, and Zatuchri, J. Am. Heart J. 98, 680, 1978
5. Kubasik, N., Guiney, W., Warren, K, D’Souza, J., Sine, H., Brady, B. Clin. Chem. 24,
2047, 1978.
6. Chatterjee, K Hosp. Pract. 21, 117, 1986
7. Faxon, D. Hosp. Pract. 22, 59, 1987
8. Wu, A., Gornet, T., Harker, C. and Chen, H. Clin. Chem. 35, 1752, 1989
9. Stone, M., Waterman, M., Poliner, L., Templeton, G., Buja, L. and Willerson, J.
Angiology 29, 386, 1981
10. Askmark, H., Osterman, P., Roxin, L.E. and Verge, P. J. Neurol. Neurosurg.
Psychiat. 44, 68, 1981
11. Miyoshi, K., Saito, S., Kawai, H., Kondo, A., Iwasa, M., Hayashi, T. and Yagita, M.
J. Lab. Clin. Medi. 92, 341, 1978
12. Kagaen, L. Arch. Intern. Med. 139, 628, 1979
13. Gilkeson, G., Stone, M., Waterman, M., Ting, R., Gomez-Sanchez, C., Hull, A. and
Willerson, J. Am. Heart J. 95, 70, 1978
14. Hallgren, R., Lundin, L., Roxin, L.E. and Verge, P. Acta Med. Scand. 208, 33,
1960
15. Hallgren, R., Karlson, F.A., Roxin, L.E. and Verge, P. J. Lab. Clin. Med. 91, 146,
1978
16. Roxin, L.E., Cullhed, L., Groth, T. Hallgren, T., and Verge, P. Acta Med. Scand.
215, 417, 1984
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