Journal of Clinical Laboratory Analysis 6:7-11 (1992)
Measurement of Serum Myoglobin by a Turbidimetric Latex Agglutination Method Yoshinori Uji,’ Hiroaki Okabe,’ Hiroyuki Sugiuchi,’ and Sakari Sekine2 ‘Department of Laboratoty Medicine, Kumamoto University Medical School, Kumamoto and ’Department of Clinical Chemistry and Immunological Reagents, Denka Seiken Co., Ltd., Kabuki-cho, Tokyo, Japan We evaluated an immunoturbidimetric quantitation for serum myoglobin by the latex agglutinationmethod using an automated biochemical analyzer. This method is rapid, specific, accurate, precise, and has wide dynamic range. The total assay time is 10 min and is performed at 37°C with continuous monitoring at 570 nm. The assay results were compared with radioisotopic immunoassay results and showed a good correlation coefficient, r = 0.99; Y = 0.98x 9.3;N = 79. Sera from healthy adults has a myoglobinconcentration inthe range of 15-80 ng/ml(N =362). Sex- and age-related differences were observed. The
+
Key words:
acute myocardialinfarction, muscle dystrophy, automated imrnunoturbidimetric latex agglutination method
INTRODUCTION The most commonly employed diagnostic methods currently used in the recognition of myocardial ischemia and infarctions are electrocardiographic and serum enzyme assays (1-3). However, there is considerable variation in serum enzyme levels in patients with acute myocardial infarction. In addition, non-specific electrocardiographic changes may occur which are difficult to interpret in the absence of clear-cut changes in serum enzymes. Myoglobin is a porphyrin-containing protein of 17 KD, which is localized in the cytosol of skeletal and cardiac muscle. An increase in serum myoglobin level reflects the severity of cardiac muscle injury. Myoglobin is rapidly removed from the blood, filtered through the glomerular membrane of kidney, and excreted in the urine. The measurement of serum myoglobin is useful to help evaluate the diagnosis and extent of cardiac muscle damage. In the past, myoglobin has been measured by counter-immunoelectrophoresis (4), complement fixation ( 5 ) , and RIA ( 6 ) . The present communication describes the evaluation of rapid, simple, and fully automated immunoturbidimetric quantitation by a latex agglutination technique. 0 1992 Wiley-Liss, Inc.
serum myoglobin levels in males and elderly people showed higher concentration than in females and younger people. The peak elevation of serum myoglobin compared with other cardiac markers was observed within 6 hours after onset of chest pain as well as the CK-isoform ratio (MM3/MM,). All of the serum from 21 patients with definite acute myocardial infarction showed increased serum myoglobin levels (100-1200 ng/ml) upon admission and within 6 hours. The results suggest that assays for serum myoglobin levels are helpful in the early diagnosis of acute myocardial necrosis.
MATERIALS AND METHODS
Reagents Myoglobin standards (human myoglobin) and anti-human myoglobin rabbit IgG antibody were purchasd from DAKO A / S (Denmark). The anti-human cardiac myoglobin rabbit IgG antibody used showed two bands on PAGE and the reactivity with purified skeletal human myoglobin, and also showed the same results except serum sample with the antibody by immunoblotting analysis (7,8). But less than 3% of three kinds of unknown higher molecular weight substances which were reacted with anti-cardiac myoglobin rabbit IgG antibody in the human serum were observed, as shown in Figure 1. Latex suspension (particle size 0.12 pm) was purchased from Sekisui Chemicals (Tokyo, Japan) and coated with antihuman myoglobin-rabbit IgG by the method of Cambiaso et al. (9). Other chemicals were of reagent grade.
Received February 6, 1991; accepted August 6, 1991 Address reprint requests to Yoshinori Uji, Ph.D., Department of Laboratory Medicine, Kumamoto University Medical School 1 - 1 - 1 , Honjo, Kumamoto, 860, Japan.
8
Ujietal.
.M
a
B 500
114
214
314
1
Dilution ratio Fig. 2. Calibration curve for myoglobin.
1
2
3
4
5
6
7
Fig. 1. Immunoblotting analysis of anti-myoglobin antibody with serum, cardiac muscle, extracts, and skeletal muscle extracts. A: 1) Molecular weight markers (BIO-RAD, USA). 2) Human cardiac muscle extracts. 3) Immunoblotting analysis of human cardiac muscle extracts. 4) Human skeletal muscle extracts. 5) Immunoblotting analysis of human skeletal extracts. 6) Human serum. 7) Immunoblotting analysis of human serum.
Assay Methods Twenty microliters of the serum was added to 160 p1 of 0.17 moVL of glycine buffer containing 1.O% BSA, 0.1 moVL of NaCl, 0.1% of NaN3, and 50 mmol/L of EDTA-2Na at pH 7.4 for 5 min. One hundred microliters of myoglobin coated on latex particles solution [lo0 pg of IgG on latex particleimg, 0.166 mg of latex particles in 100 pl of 0.17 M glycine buffer (pH 7.3)containingO.l%ofBSA,O.l MNaCl,0.1%NaN3] was poured into the incubation mixture for 2 min at 37°C and measured at 570 nm with a Hitachi 717 analyzer (Tokyo, Japan). RIA myoglobin assay kits were purchased from Daiichi radioisotopic institute (Tokyo, Japan). The reagent for assay of human ventricular myosin light chain- 1 by the EIA method (10) and CK MM isoform kits using specific monoclonal antibodies (1 1) were obtained from Yamasa Shouyu Co. Ltd. (Tokyo, Japan) and Unitica Ltd. (Kyoto, Japan), respectively.
SERUM AND BLOOD SPECIMENS Normal sera were obtained from a Kumamoto Blood Bank or from Tokyo Metropolitan Geriatric Hospital and Kumamoto University Hospital. None of the normal subjects reported any clinical history or indications of myoglobinemia. Their ages ranged from birth to 85 years.
Venous blood was also collected from 2 1 patients on admission and at 4 hour intervals for 3 to 5 days. Each specimen was stored at - 70°C for rnyoglobin estimation.
RESULTS AND DISCUSSION Linearity A typical standard curve showed linearity over a range of 10- 1,200 ng/ml, which is greater than the upper normal serum level. There are several reports which used the latex agglutination method for serum myoglobin. These method does not require dilution between the myoglobin levels of 30 and 650 ngiml (12,13). Our method showed a wide dynamic range. The present assay did not show linearity at levels of myoglobin over 1,500ngiml. At levels over 2,000 ngiml of myoglobin (Fig. 2), a soluble antigen-antibody complex effect with excess antigen was observed.
Reproducibility of the Assay The within-run precision (CV) of the assay was 2.4% at low concentrations (mean ? SD; 35.4 ? 0.85 ngiml; N = 10) and 1.0% at high concentrations of myoglobin (170 k 1.70 nglml) sera. The between-assay CV was less than 3.2% for 10 days with the same sample specimen.
Recovery Study After addition of known amounts of standard myoglobin to patients’ sera, the recovery studies showed recovery of 96- 107%. Two additional experiments showed similar results (Table 1).
Interfering Substance Studies Interferences by various substances (bilirubin, hemoglobin, ascorbic acid, glucose, citrate, disodium EDTA, oxalate, hep-
Myoglobin Measurement by lmmunoturbidimetricMethod
TABLE 2. Age and Sex Differences of Serum Myoglobin in Healthv Subiects
TABLE 1. Recovery Tests of the Proposed Method
Serum
Added value (ng/ml)
Measured value (ngiml)
Expected value (ngiml)
Recovery (%)
0 20.0 40.0
52.6 74.0 93.6 133.2 125.8 145.0 165.2 206.8
52.6 72.6 92.6 132.6 125.8 145.8 165.8 205.8
100.0 107.0 101.0 100.8 100.0 96.0 98.5 101.3
A
80.0 0 20.0 40.0 80.0
B
Age (yr)
arin, reumatoid factor) with the myoglobin measurement were not observed.
Correlation Between lmmunoturbidimetricMethod and RIA The immunoturbidimetric method (Y) and RIA (X) were compared with 79 patients' sera. The coefficient was 0.995 and the regression analysis demonstrated Y = 0.98 X + 9.3 (Fig. 3).
Reference Range Of 364 sera from healthy subjects under 50 years of age, myoglobin values ranged from 18.4 to 78.9 ng/ml in males and 15.1 to 63.6 ng/ml in females. Serum myoglobin levels in males were markedly higher than those in females in most of the age groups (Table 2). The 50-60-year age group and >80-year age group were not significantly different by Student's t-test. However, the total mean serum myoglobin concentrations in males were higher than in females.
"1 I
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4000
-
.A.
v
N=79
W
1 0 .
g
+9.3
2 m r=O. 995
W
a l 0 v)
e 1000-
a
0 c L
0
1000
2000
3000
4000
9
5@00
Radioi munoassay (ng/ml)
Fig. 3. Correlation between proposed method and radioimmunoassay method.
Male (ngiml)
Female (ng/ml)
M:F
81-
13.6-74.7(N=25) 22.2-77.8(N=20) 13.0-78.8(N= 38) 16.5-81.8(N=41) 16.7-80.8(N=40) 26.8-107.7(N=20) 18.0-108.3(N=38) 9.4-156.0(N=35)
20.4-61.1(N= 37) 21.2-60.3(N= 26) 22.3-60.4(N= 51) 19.2-64.0(N= 38) 19.2-74.6(N= 82) 21.3-98.0(N=94) 0.0-95.5(N=27) 24.0-132.0(N=49)
P
Measurement of Serum Myoglobin by a Turbidimetric Latex Agglutination Method Yoshinori Uji,’ Hiroaki Okabe,’ Hiroyuki Sugiuchi,’ and Sakari Sekine2 ‘Department of Laboratoty Medicine, Kumamoto University Medical School, Kumamoto and ’Department of Clinical Chemistry and Immunological Reagents, Denka Seiken Co., Ltd., Kabuki-cho, Tokyo, Japan We evaluated an immunoturbidimetric quantitation for serum myoglobin by the latex agglutinationmethod using an automated biochemical analyzer. This method is rapid, specific, accurate, precise, and has wide dynamic range. The total assay time is 10 min and is performed at 37°C with continuous monitoring at 570 nm. The assay results were compared with radioisotopic immunoassay results and showed a good correlation coefficient, r = 0.99; Y = 0.98x 9.3;N = 79. Sera from healthy adults has a myoglobinconcentration inthe range of 15-80 ng/ml(N =362). Sex- and age-related differences were observed. The
+
Key words:
acute myocardialinfarction, muscle dystrophy, automated imrnunoturbidimetric latex agglutination method
INTRODUCTION The most commonly employed diagnostic methods currently used in the recognition of myocardial ischemia and infarctions are electrocardiographic and serum enzyme assays (1-3). However, there is considerable variation in serum enzyme levels in patients with acute myocardial infarction. In addition, non-specific electrocardiographic changes may occur which are difficult to interpret in the absence of clear-cut changes in serum enzymes. Myoglobin is a porphyrin-containing protein of 17 KD, which is localized in the cytosol of skeletal and cardiac muscle. An increase in serum myoglobin level reflects the severity of cardiac muscle injury. Myoglobin is rapidly removed from the blood, filtered through the glomerular membrane of kidney, and excreted in the urine. The measurement of serum myoglobin is useful to help evaluate the diagnosis and extent of cardiac muscle damage. In the past, myoglobin has been measured by counter-immunoelectrophoresis (4), complement fixation ( 5 ) , and RIA ( 6 ) . The present communication describes the evaluation of rapid, simple, and fully automated immunoturbidimetric quantitation by a latex agglutination technique. 0 1992 Wiley-Liss, Inc.
serum myoglobin levels in males and elderly people showed higher concentration than in females and younger people. The peak elevation of serum myoglobin compared with other cardiac markers was observed within 6 hours after onset of chest pain as well as the CK-isoform ratio (MM3/MM,). All of the serum from 21 patients with definite acute myocardial infarction showed increased serum myoglobin levels (100-1200 ng/ml) upon admission and within 6 hours. The results suggest that assays for serum myoglobin levels are helpful in the early diagnosis of acute myocardial necrosis.
MATERIALS AND METHODS
Reagents Myoglobin standards (human myoglobin) and anti-human myoglobin rabbit IgG antibody were purchasd from DAKO A / S (Denmark). The anti-human cardiac myoglobin rabbit IgG antibody used showed two bands on PAGE and the reactivity with purified skeletal human myoglobin, and also showed the same results except serum sample with the antibody by immunoblotting analysis (7,8). But less than 3% of three kinds of unknown higher molecular weight substances which were reacted with anti-cardiac myoglobin rabbit IgG antibody in the human serum were observed, as shown in Figure 1. Latex suspension (particle size 0.12 pm) was purchased from Sekisui Chemicals (Tokyo, Japan) and coated with antihuman myoglobin-rabbit IgG by the method of Cambiaso et al. (9). Other chemicals were of reagent grade.
Received February 6, 1991; accepted August 6, 1991 Address reprint requests to Yoshinori Uji, Ph.D., Department of Laboratory Medicine, Kumamoto University Medical School 1 - 1 - 1 , Honjo, Kumamoto, 860, Japan.
8
Ujietal.
.M
a
B 500
114
214
314
1
Dilution ratio Fig. 2. Calibration curve for myoglobin.
1
2
3
4
5
6
7
Fig. 1. Immunoblotting analysis of anti-myoglobin antibody with serum, cardiac muscle, extracts, and skeletal muscle extracts. A: 1) Molecular weight markers (BIO-RAD, USA). 2) Human cardiac muscle extracts. 3) Immunoblotting analysis of human cardiac muscle extracts. 4) Human skeletal muscle extracts. 5) Immunoblotting analysis of human skeletal extracts. 6) Human serum. 7) Immunoblotting analysis of human serum.
Assay Methods Twenty microliters of the serum was added to 160 p1 of 0.17 moVL of glycine buffer containing 1.O% BSA, 0.1 moVL of NaCl, 0.1% of NaN3, and 50 mmol/L of EDTA-2Na at pH 7.4 for 5 min. One hundred microliters of myoglobin coated on latex particles solution [lo0 pg of IgG on latex particleimg, 0.166 mg of latex particles in 100 pl of 0.17 M glycine buffer (pH 7.3)containingO.l%ofBSA,O.l MNaCl,0.1%NaN3] was poured into the incubation mixture for 2 min at 37°C and measured at 570 nm with a Hitachi 717 analyzer (Tokyo, Japan). RIA myoglobin assay kits were purchased from Daiichi radioisotopic institute (Tokyo, Japan). The reagent for assay of human ventricular myosin light chain- 1 by the EIA method (10) and CK MM isoform kits using specific monoclonal antibodies (1 1) were obtained from Yamasa Shouyu Co. Ltd. (Tokyo, Japan) and Unitica Ltd. (Kyoto, Japan), respectively.
SERUM AND BLOOD SPECIMENS Normal sera were obtained from a Kumamoto Blood Bank or from Tokyo Metropolitan Geriatric Hospital and Kumamoto University Hospital. None of the normal subjects reported any clinical history or indications of myoglobinemia. Their ages ranged from birth to 85 years.
Venous blood was also collected from 2 1 patients on admission and at 4 hour intervals for 3 to 5 days. Each specimen was stored at - 70°C for rnyoglobin estimation.
RESULTS AND DISCUSSION Linearity A typical standard curve showed linearity over a range of 10- 1,200 ng/ml, which is greater than the upper normal serum level. There are several reports which used the latex agglutination method for serum myoglobin. These method does not require dilution between the myoglobin levels of 30 and 650 ngiml (12,13). Our method showed a wide dynamic range. The present assay did not show linearity at levels of myoglobin over 1,500ngiml. At levels over 2,000 ngiml of myoglobin (Fig. 2), a soluble antigen-antibody complex effect with excess antigen was observed.
Reproducibility of the Assay The within-run precision (CV) of the assay was 2.4% at low concentrations (mean ? SD; 35.4 ? 0.85 ngiml; N = 10) and 1.0% at high concentrations of myoglobin (170 k 1.70 nglml) sera. The between-assay CV was less than 3.2% for 10 days with the same sample specimen.
Recovery Study After addition of known amounts of standard myoglobin to patients’ sera, the recovery studies showed recovery of 96- 107%. Two additional experiments showed similar results (Table 1).
Interfering Substance Studies Interferences by various substances (bilirubin, hemoglobin, ascorbic acid, glucose, citrate, disodium EDTA, oxalate, hep-
Myoglobin Measurement by lmmunoturbidimetricMethod
TABLE 2. Age and Sex Differences of Serum Myoglobin in Healthv Subiects
TABLE 1. Recovery Tests of the Proposed Method
Serum
Added value (ng/ml)
Measured value (ngiml)
Expected value (ngiml)
Recovery (%)
0 20.0 40.0
52.6 74.0 93.6 133.2 125.8 145.0 165.2 206.8
52.6 72.6 92.6 132.6 125.8 145.8 165.8 205.8
100.0 107.0 101.0 100.8 100.0 96.0 98.5 101.3
A
80.0 0 20.0 40.0 80.0
B
Age (yr)
arin, reumatoid factor) with the myoglobin measurement were not observed.
Correlation Between lmmunoturbidimetricMethod and RIA The immunoturbidimetric method (Y) and RIA (X) were compared with 79 patients' sera. The coefficient was 0.995 and the regression analysis demonstrated Y = 0.98 X + 9.3 (Fig. 3).
Reference Range Of 364 sera from healthy subjects under 50 years of age, myoglobin values ranged from 18.4 to 78.9 ng/ml in males and 15.1 to 63.6 ng/ml in females. Serum myoglobin levels in males were markedly higher than those in females in most of the age groups (Table 2). The 50-60-year age group and >80-year age group were not significantly different by Student's t-test. However, the total mean serum myoglobin concentrations in males were higher than in females.
"1 I
J /
4000
-
.A.
v
N=79
W
1 0 .
g
+9.3
2 m r=O. 995
W
a l 0 v)
e 1000-
a
0 c L
0
1000
2000
3000
4000
9
5@00
Radioi munoassay (ng/ml)
Fig. 3. Correlation between proposed method and radioimmunoassay method.
Male (ngiml)
Female (ng/ml)
M:F
81-
13.6-74.7(N=25) 22.2-77.8(N=20) 13.0-78.8(N= 38) 16.5-81.8(N=41) 16.7-80.8(N=40) 26.8-107.7(N=20) 18.0-108.3(N=38) 9.4-156.0(N=35)
20.4-61.1(N= 37) 21.2-60.3(N= 26) 22.3-60.4(N= 51) 19.2-64.0(N= 38) 19.2-74.6(N= 82) 21.3-98.0(N=94) 0.0-95.5(N=27) 24.0-132.0(N=49)
P
