Detection Of Myoglobin Research Articles

Direct detection of myoglobin in whole blood using a disposable amperometric immunosensor

An amperometric immunosensor for the rapid detection of myoglobin in whole blood was developed. Due to its rapid kinetics, myoglobin is a useful biochemical marker for the early assessment of acute myocardial infarction (AMI). A one-step indirect sandwich assay was employed using a polyclonal goat anti-human cardiac myoglobin antibody with monoclonal mouse anti-myoglobin and goat anti-mouse IgG conjugated to alkaline phosphatase (AP), as the detecting antibodies. The final sensor required 30 min for incubation. The standard curve was linear between 85 and 925 ng/ml. The intra- and inter-assay coefficients of variation were below 8%. No cross-reactivity of the antibodies was found with other cardiac proteins. The overall performance of the sensor, rapid analysis time, wide working range, good precision and specificity demonstrate its potential usefulness for early assessment of AMI.

The presence of pharmacological substances myoglobin and histamine in venoms

It is well documented that several pharmacological substances are released within the victim's body after snakebite. These substances are also believed to be endogenously present in animals, specifically levels of myoglobin and histamine that are reported to rise after envenomation. However, there is no published data regarding the presence of these substances in venoms per se. This research reports the detection of myoglobin and histamine in snake, scorpion, honeybee, and toad venoms by immunological test. It is unlikely that the rise in levels of myoglobin and histamine is due to that added from the bite, since a single toxin devoid of such components is capable of elevating levels of these substances. Nonetheless, it is likely that the rise in levels of myoglobin and histamine after envenomation is due to the venom or toxin reacting with cells of various organs of the victim. Therefore, this phenomenon can be compared to cancer markers, which are endogeneously present in humans at low levels and elevated in cancerous states.

Project title myoglobin detection and oxidative metabolism

Project title myoglobin detection and oxidative metabolism

Serial use of bedside CKMB/myoglobin device to detect acute myocardial infarction in emergency department chest pain patients

A qualitative bedside device (Spectral Diagnostics, Toronto, Canada) for CKMB and myoglobin (MYOG) detection was evaluated in emergency department (ED) patients with chest pain to determine performance characteristics. At presentation (0 h) and at three hours (3 h), serum was analyzed in the ED with results considered positive if either 0-h or 3-h CKMB or MYOG bands were visible. The results were compared with the diagnosis of myocardial infarction (MI) per hospital discharge diagnosis ( n = 132, 87%) or telephone follow-up ( n = 19; 1 patient lost to follow-up). Of 151 study patients, 30 (20%) were diagnosed with MI; all were admitted to hospital. On electrocardiogram (EKG), 17 (57%) MI patients had ST-segment elevation. At 0-h, 26 of 30 (87%) MI patients were positive for CKMB/MYOG. By 3 h, 21 of 23 (91%) MI patients were positive for CKMB/MYOG; 7 MI patients were already admitted to hospital. Combining 0-h and 3-h results, the device sensitivity for MI was 93% (28/30) with specificity of 54%. Combining device results plus diagnostic EKG, sensitivity was found to be 100% (30/30). If the device result was positive, then the odds ratio for having an ischemic complication was 6.5. We conclude that the CKMB/MYOG device identified most MI patients at ED presentation and 3 h later. Combining device results with EKG detected all MI patients in the ED.

Real time sensing of specific molecular binding using surface plasmon resonance spectroscopy

A new planar surface plasmon resonance sensor with spectral interrogation was constructed and tested. The SPR device was directly compared with a high-end commercial BIAcore 2000 instrument. In immunological applications, we used the SPR-device for the detection of myoglobin and to measure the concentration of staphylokinase in a competitive assay. The lower limits of detection were tested when the binding of Ni 2+-ions on a matrix with nitrilotriacetic acid (NTA) was measured. The tested sensor was comparable in performance to the commercial instrument. Its detection of the plasmon resonance in a spectral mode and its fiber optical connection between sensor and detector unit offer additional advantages for future applications in on-site analysis with a portable detector.

Clinical evaluation of the CARDIAC STATus, a rapid immunochromatographic assay for simultaneous detection of elevated concentrations of CK-MB and myoglobin in whole blood.

We studied the performance of the CARDIAC STATus, a new rapid, easy to perform qualitative whole blood bedside test for detection of elevated CK-MB and myoglobin in the emergency room. Blood samples from 182 consecutive patients with chest pain were drawn on admission and at five and seven hours after the onset of symptoms. The CARDIAC STATus tests were performed by coronary care unit nurses and, independently, by a trained laboratory technician. The results were compared with quantitative assays for CK-MB mass and myoglobin. At the end of the study, a second test series using a new lot number of cartridges was performed on the same blood samples because of possible elution buffer contamination. Nurses produced more false negative results than the technician (CK-MB 43 vs. 27 %, p=0.01, myoglobin 31 vs. 13%, p<0.0001), but the technician produced more false positive myoglobin results (9.3 vs. 5.5%, p=0.0001). In the second test series, the nurses produced significantly fewer false negative tests both for CK-MB (19%, p<0.0001) and myoglobin (13%, p=0.0002). The false negative rate for the technician was not different between the first and the second test series. The CARDIAC STATus yields a substantial number of false negative results both for CK-MB and myoglobin when compared to a quantitative assay, and therefore at present has limited value for ruling out an acute myocardial infarction.

Qualitative bedside assay of increased human serum myoglobin by sandwich dot-immunogold filtration for the diagnosis of acute myocardial infarction

We isolated and purified myoglobin (MYO) from human fresh skeletal muscle and prepared monoclonal and polyclonal antibody from it. A sandwich dot-immunogold filtration assay (DIGFA) for the detection of MYO was developed by using affinity purified sheep anti-MYO antibody as the first antibody for coating nitrocellulose membranes (NCMs; the support) and colloidal gold labelled monoclonal antibody (H3) as the second antibody (an indicator). The test can be completed in 3 min without incubation or any equipment. A reddish dot, indicating positivity, is obvious to the naked eye. No interferences from bilirubin, hemoglobin, rheumatoid factors and lipid were found. In order to use undiluted serum, the detection limit was set at 100 μg of MYO/l. Concentrations up to 30 000 μg/l can be measured without getting a “hook” effect. Serum MYO levels in 53 patients with acute myocardial infarction (AMI), 100 healthy individuals, seven patients with chest pain but without myocardial ischemia and in 39 patients with renal insufficiency were measured simultaneously by DIGFA and enzyme-linked immunosorbent assay (ELISA). All serum samples from patients had MYO concentrations above 100 μg/l by ELISA and were positive by DIGFA. Serum creatinine values were related to MYO test results. Healthy individuals had MYO levels below 85 μg/l by ELISA and were negative by DIGFA.

Development of a sensitive ELISA for myoglobin detection in the early diagnosis of acute myocardial infarction.

Development of a sensitive ELISA for myoglobin detection in the early diagnosis of acute myocardial infarction.

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire which is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N=3) was achieved for detection of myoglobin utilizing a 75 μm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550-1350 Da. The advantages of this new design include: (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.

Adaptation of a Quantitative Immunoassay for Urine Myoglobin:Predictor in Detecting Renal Dysfunction

Myoglobinuria, subsequent to rhabdomyolysis, may cause acute renal failure. For this reason, many qualitative and quantitative tests have been developed for the detection of myoglobin in urine. The authors describe the adaptation and optimization of the Stratus II serum myoglobin immunoassay to quantify urine myoglobin. In addition, the assay was used to accurately determine urine myoglobin concentrations in subjects at potential risk for myoglobin-induced renal dysfunction and the results obtained compared to conventional qualitative methods for urine myoglobin. The assay demonstrated with-in run and between-run coefficient of variations (CVs) of 6.2% and 7.2%, respectively, was linear from 0-950 micrograms/L, demonstrated good recovery, and was free from interference by hemoglobin, creatinine, and urea. Specimens were diluted with 0.1 mol/L phosphate buffer, pH 9.0 containing 3% bovine serum albumin before analysis. Myoglobin was assayed on urine obtained from 30 patients suspected of having myoglobinuria. Fifteen of 17 patients with serum creatinine greater than 1.4 mg/dL had myoglobin concentrations greater than 20,000 micrograms/L, whereas the remaining 31 patients with normal serum creatinine had urine myoglobin concentrations of less than 18,000 micrograms/L. If serum creatinine is used as an indicator of renal function, it would appear that accurate measurement of urine myoglobin may facilitate identification of patients with increased susceptibility to myoglobin-induced acute renal failure.

Identification of human skeletal muscle from a tissue fragment by detection of human myoglobin using a double-sandwich ELISA

A method for identifying human skeletal muscle by detection of human myoglobin using a double-sandwich ELISA was developed. When an extract was prepared from 0.1 g skeletal muscle homogenized with 10 ml PBS, this method was able to detect human myoglobin in extracts diluted 10 4-fold. There was no difference in the detection limit between individuals or sites of origin of skeletal muscles. Species specificity was good and no cross reaction occurred with skeletal muscle from other animals except the gorilla. Our method could also discriminate between skeletal muscle and other organs or tissues except the heart. Human myoglobin could be detected in skeletal muscles under the following conditions: putrefied at room temperature for 5 months, dried at room temperature for 11 months, heated at 100 °C for 72 h and immersed in fresh water at room temperature for 6 days. Two practical cases to which this method was applied are presented.

Dual-label time-resolved fluoroimmunoassay for simultaneous detection of myoglobin and carbonic anhydrase III in serum

We developed a dual-labeled time-resolved fluoroimmunoassay for simultaneous quantification of myoglobin (Mb) and carbonic anhydrase III (CA III) in serum involving polyclonal antibodies and the fluorescent lanthanides europium (Eu3+) and samarium (Sm3+). This solid-phase immunoassay is based on competition between Eu(3+)- or Sm(3+)-labeled antigen and the sample antigen for polyclonal rabbit antibodies. Standards and patients' samples containing antigen inhibit binding of the lanthanide-labeled antigen to the antibody. A second antibody directed against rabbit IgG is coated on a solid phase and binds the IgG-antigen-lanthanide complex, giving rapid and complete separation of antibody-bound and free antigen. The assay requires only one incubation step. An enhancement solution dissociates Eu3+ and Sm3+ ions from the labeled CA III and Mb, respectively, into a solution where they form highly fluorescent chelates. Spectra of the fluorescent chelates in the microtitration-strip wells were run on a time-resolved fluorometer equipped with filters for Eu3+ (613 nm) and Sm3+ (643 nm), the fluorescence from each sample being inversely proportional to the concentration of antigens. The measurement range for both analytes is from 5 to 1500 micrograms/L. The mean within- and between-assay precisions (CV) were 4.6% and 6.2% for CA III and 5.9% and 7.3% for Mb, respectively. Good correlations were obtained with the results of CA III RIA and a commercial myoglobin RIA kit.

Detection of ferryl myoglobin in the isolated ischemic rat heart

Reflectance spectroscopy was utilized to monitor the oxidation states of myoglobin (Mb) in isolated, buffer-perfused rat hearts. Hearts were subjected to 30 min global, no-flow ischemia, followed by reperfusion under anoxic conditions. The addition of Na 2S to the buffer at reperfusion permitted the detection of ferryl myoglobin (MbIV) as its sulfmyoglobin derivative. The accumulation of MbIV was prevented by addition of ascorbic acid (1 mM), ergothioneine (2mM), or desferal (1mM) to the buffer prior to ischemia. Ascorbate and other agents have been previously shown to serve as one-electron reductants of MbIV. We propose that during the early phases of ischemia, deoxymyoglobin is oxidized to MbIV by residual H 2O 2. It also seems reasonable that the peroxidative activity of Mb(IV), during oxygenated reperfusion, might lead to cellular damage if this hypervalent form of Mb is not reduced.

Clinical and laboratory features of South American rattlesnake ( Crotalus durissus terrificus) envenomation in children

The venom of the South American rattlesnake Crotalus durissus terrificus was first reported to have mainly haemolytic and neurotoxic physiopathological activities. Later studies demonstrated the systemic myotoxic action of the venom, characterized by the release of myoglobin from damaged skeletal muscle into serum and urine, and a recent report ruled out the presence of intravascular haemolysis in 3 patients, one child and 2 adults. The present paper describes the clinical-laboratory evolution of 10 children bitten by C. durissus terrificus; 2 developed acute renal failure and one died. The myotoxic activity of the venom was evaluated by measuring serum lactic dehydrogenase, creatine kinase and aspartate aminotransferase, by detection of myoglobin in serum and urine, and by muscle biopsy. Haemolytic activity was evaluated by serial measurements of serum haemoglobin and haptoglobin and by detection of urine haemoglobin. We conclude that the signs and symptoms exhibited by patients bitten by C. durissus terrificus are due only to the myotoxic and neurotoxic action of the venom. The only patients with major morbidity were those who initially received subcutaneous antivenin and did not receive definitive antivenin therapy until later.

Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 μl of standard serum or serum of patients and 200 μl of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37°C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantiti of myoglobin within the range 2–1000 ng/ml serum. 120 samples can be tested in 3 h.

Bovine myoglobin: detection of myoglobin in sera and urine of calves with nutritional myopathy by immunodiffusion method.

Detection of myoglobin (Mb) in sera and urine of sixteen calves with nutritional myopathy was achieved by immunodiffusion method. Mb were detected in 12 serum samples out of 16 calves and in 9 urine samples out of 13 calves. Concentrations of Mb in serum and urine decreased markedly by treatment and were scarcely detected in any samples after one or two days. The minimal detectable concentration of Mb by immunodiffusion method was 5μg per ml.

Influence of exercise on serum levels of myoglobin measured by radioimmunoassay.

To determine the influence of exercise on serum levels of myoglobin, serum levels of this protein were determined by RIA in 90 healthy men, divided as follows: (1) Basal control (no exercise) 25 cases; (2) Moderate exercise (after subject had been working for 12 h in Medicine Emergency Service) 19 cases, and (3) Intensive exercise: (a) football professional (45-min match) 10 cases; (b) football amateur (45-min match) 10 cases; (c) basketball professional (45-min match) 10 cases, and (d) basketball professional (90-min training) 16 cases. Our results led us to the following conclusions. (1) Moderate exercise, such as the usual daily work, does not modify myoglobin levels; (2) Myoglobin serum levels after exercise increase in nearly all individuals. They are higher in untrained people; (3) There seems to be a correlation between exercise intensity and increase of myoglobin serum levels, and (4) The detection of serum myoglobin by RIA may have a wide field of application for sport medicine.

Serum Myoglobin

Myoglobin is a respiratory protein that is unique to skeletal and cardiac muscle. It is composed of a single polypeptide chain of 153 amino acids, with an iron-containing, heme-prosthetic group, and has a molecular weight of approximately 17,800. Its function is thought to involve the transport of oxygen across the cell, and the modulation of oxygen's availability for cellular reactions of respiration. In the course of muscle disorders or stress, myoglobin may leak from muscle tissue to the circulation, and from there may appear in the urine.¹ Within the past several years, application of sensitive immunologic methods has allowed the detection and quantification of myoglobin in the circulation. Although many types of disorders have been studied, this discussion will highlight three areas, as follows: myocardial disease, genetic disorders of muscle, and acquired skeletal muscle disease. Increased levels of serum myoglobin occur in most, if not all, patients during the

An enzyme-immunoassay for myoglobin in human serum and urine. Method development, normal values and application to acute myocardial infarction

An enzyme-immunoassay has been developed for the detection of myoglobin in human serum and urine which is specific, accurate, precise, and has a sensitivity of 3 ng/ml. When compared with radioimmunoassay, the enzyme-immunoassay gives markedly similar results. Sera from normal adults had a myoglobin concentration in the range 3-65 ng/ml, and 64% of the same group had detectable myoglobinuria (range 3-11.5 ng/ml). All of 8 patients with definite acute myocardial infarction had raised serum myoglobin levels (range 200-1125 ng/ml) either at admission or 4 h later. Myoglobin concentration returned to normal in 6 patients, and in the remaining 2 patients there was evidence of infarct extension. Urinary myoglobin excretion was variable. One patient with possible acute myocardial infarction had elevated serum myoglobin (413 ng/ml 4 h post admission) and 5 patients with no evidence of infarction had normal levels (15-53 ng/ml). The results suggest that detection of serum myoglobin by enzyme-immunoassay may be a valuable test in the early diagnosis of acute myocardial infarction.

Radioimmunoassays of human myoglobin in serum and urine

Two solid-phase radioimmunoassays have been developed for the detection of myoglobin in serum and urine. The sensitivity of the methods is 0.1 and 0.5 microgram/l, respectively, with a coefficient of variation of the respective method of 7-8%. The mean serum concentration of myoglobin in ninety-nine healthy blood donors was 44.3 microgram/l +/- 18.0 microgram/l (SD) with a significant difference (P less than 0.001) between men (50.6 +/- 19.8) and women (35.7 +/- 10.4). Serum myoglobin was positively correlated to age (P less than 0.05), body weight (P less than 0.02), serum creatine kinase (P less than 0.001), and serum creatinine (P less than 0.001) to galactose elimination rate. Serum myoglobin levels were not influenced by exhaustive short time dynamic exercise. The mean urinary excretion of myoglobin in twenty-four healthy students was 1.2 microgram/24 h (range 0.1-4 microgram/24 h). Myoglobin excretion was correlated to excretion of beta 2-microglobulin (P less than 0.02) but not to serum levels of myoglobin. No indications of circulating antibodies to myoglobin were obtained when assaying sixty-seven rheumatoid arthritis and thirteen myastenia gravis sera. Presence of other myoglobin binding substances in serum, which would interfere with the assays also seemed unlikely. Determination of myoglobin in serum by sensitive and specific method might be of clinical value in the diagnosis of diseases involving muscle tissues.