CIinica Chimica Acta, 209 fi992j 197-207 0 1992 Elsevier Science Publishers B.V. All rights reserved. ~-~98~~2~~5.~
197
Short Communication
An ELISA metkod to measure human rny~gl~~in in urine Alan W. Hodsona, Andrew W. Skillena and Nicholas B. Argentb aDepartment of Clinical Biochemistry, University of Newcastle upon Tyne, NE.2 4HH and bDepartment of Renal Medicine, University of Newcastle, Newcastle upon Tyne, NE2 4HH (UK)
(Received 4 November 1991; revision received 20 April 1992; accepted 28 April 1992)
Key words: Urine rnyo~ob;n~ Sandwich ELISA assay; My~ardial
infarction; Renal disease
Following thrombolytic therapy of patients with acute myocardial infarction, a transient proteinuria was detected [I]. As the myoglobin concentration in plasma, increases after infarction, reaching a peak within 2-3 h [2] and as myoglobin is suspected of being nephrotoxic [3] an increase of this protein in the glomerular filtrate may lead to tubular damage. Therefore an investigation was instigated to examine the conc~tratio~ of myoglobin and retinol binding protein in urine of patients following myo~ardial infarction. For this purpose a sensitive and specific ELISA method for dete~ining urinary myoglobin was developed that is based on a method used by others to determine plasma myoglobin [4]. The current paper describes this method. Material and Methods A polyclonal rabbit antibody to human myoglobin, a horseradish peroxidaseconjugated antibody and purified human myoglobin were bought from Dako Ltd. (High Wycombe, Bucks, UK). An alkaline phosphatase labelled sheep anti-rabbit IgG was bought from Sera-fab Ltd. (Crawley Down, Sussex, UK). Polyoxyethylenesorbitan monoraurate (Tween 20), 5-bromo~hloroindoxyl phosphate and Correspondence to: Afan W. Hodson, Department of ClinicaI Biochemistry, University Tyne, NE2 4HH, UK.
ofNewcastleupon
198
Nitro Blue tetrazolium were bought from Sigma Ltd. (Poole, Dorset, UK). Chicken serum was obtained from Flow Laboratories (Rickmansworth, Herts, UK). Chlorhexidine in alcohol was obtained from Pearce Laboratories (Leeds, Yorkshire, UK). Visking tubing and all other chemicals of ANALAR quality when obtainable, were bought from BDH Ltd. (Poole, Dorset, UK). Hybond-C nitrocellulose hybridization membrane, 0.45 pm, was bought from Amersham Ltd. (Amersham, Bucks, UK). Flat-bottomed microtiter plates, type Immulon-4, were bought from Dynatech Laboratories Ltd. (Billingshurst, Sussex, UK).
Microtiter plates were coated with antibody by incubation at 4*C for 16 h with 6 &ml antibody in borate buffer, pH 8.5 (10 mmol/l boric acid, 2.5 mmol/l sodium borate and 7.5 mmohl sodium chloride), 200 ~1 being applied to each well. Temperature variation was minimized by placing the plate in a small enclosed box in a cold room at 4°C. The plate wells were washed once with 200 11;lof PBS-Tween reagent, pH 7.4, which is 2.7 mmol/l potassium chloride, 1.5 mmolll potassium dihydrogen phosphate, 8 mmol/l disodium hydrogen phosphate and 137 mmol/l sodium chloride containing 500 ~1 of Tween 20 per litre. A stock myoglobin standard of 1024 pl was prepared by diluting a purified preparation containing 2 pglml in PBS reagent and 100 ml chicken serum per litre and the portions stored at -20°C until required. A series of working standards were prepared by diluting the stock standard solution of myoglobin with a PBS-diluent reagent containing 100 ml of chicken serum per litre PBS Tween. Urine samples were usually diluted 15, I:50 and I:100 with this PBS-diluent reagent. However, some urine samples were assayed undiluted in which case 100 ~1 of chicken serum were added per 900 ~1 of urine. Standards were assayed in triplicate and urine samples in duplicate by adding 100 ~1 to wells, placing the microtiter plate in a small closed container and incubating for 2 h at room temperature. The plates were washed twice with 200 ~1 of PBS-Tween reagent delivered with a multi pipette and blotted briefly by inversion on paper-towelling between ejection of antigen solution and washes. After washing, 150 ~1 of PBS Tween reagent cont~ning 0.6 &ml of horseradish ~roxida~-conju~ted rabbit antibody to human myoglobin was added to each welI. The plate was then placed in a small closed container and incubated for 1 h at room temperature. After incubation, the plates were washed 6 times as previously described with the plate being rotated horizontally through 180°C after each wash. To each well 100 pl of substrate consisting of 3.7 mmol/l o-phenylenediamine and 5 mmol/l hydrogen peroxide in 34.7 mmolfl citric acid, 66.7 mmol/l disodium hydrogen phosphate buffer, pH 5.0, was then added. Colour was allowed to develop for 30 min at room temperature and the reaction then stopped by addition of 100 ~1 of 2.5 mol/l H2S04 with the absorbance being measured at 495 nm with a Titertek Multiscan MCC/340 plate reader (Flow Laboratories). A sample blank was obtained using diluted chick serum and a colour reagent blank was obtained by omitting sample, standard and antibody.
For polyacrylamide gel electrophoresis
[5] we cast gels, 150 mm x 130 mm x 2
199
mm containing 150 g/l acrylamide and 3.9 g/l his-a~rylamide in a running gel buffer consisting of 0.5 mol/l Tris-HCI at pH 8.7 at 25”C, 4.4 mmol/l ammonium persulphate, 10 pmol/l TEMED and with or without 3.5 mmol/l SDS. After polymerisation, a stacking gel 150 mm x 20 mm x 2 mm was cast containing 80 g/l acylamide and 2.13 g/l bis-acrylamide in a buffer consisting of 125 mmol/l Tris-HCl, pH 6.8 at 25*C, 4.4 mmolll ammonium persulphate, 10 pmol/l TEMED and with or without 3.5 mmol/l SDS. Samples were diluted with equal parts of stacking gel buffer, 0.125 mol/l Tris-HCl at pH 6.8, containing 2.2 mol/l glycerol, 45 ml/l saturated aqueous Bromophenol Blue solution, 20 mmol/l EDTA and with or without 350 rnmol/l SDS. We carried out electrophoresis with an electrode buffer consisting of 25.3 mmol/l Tris, 192 mmol/l glycine, with or without 3.5 mm0111SDS. Western plotting After electrophoresis the gel was soaked for 30 min in transfer buffer consisting of 25 mmol/l Tris and 192 mmolll glycine and 5 mol/l methanol and the separated proteins transferred by electrophoresis onto nitro cellulose sheet at 4°C at 10 V overnight, then at 30 V for 1 h using the transfer buffer. After transfer el~trophoresis nitrocellulose membrane was blocked by soaking in 100 ml PBS-Tween for 1 h and washed once with the same reagent. Antibody reagent containing 1 ~1 of rabbit IgG anti-myoglobin in 1 ml was then used to soak the nitrocellulose sheet. After 2 h at room temperature the sheet was washed twice with loo-ml lots of PBS-Tween and then soaked in a solution of labelled antibody containing 5 pg/lOO ml anti-rabbit IgG conjugated with alkaline phosphatase in the same buffer. Thereafter the staining technique was carried out according to the technique of Blake et al. [6]. Protein staining After electrophoresis the gels were soaked in dye made by dissolving 250 mgJ1 Coomassie Brilliant Blue R-250 in a solvent consisting of 1.7 molll acetic acid and 4.4 mol/l ethanol (industrial methylated spirit 99%) in water. The gels were stained for l-2 h, then washed with several changes of dye solvent until a clear background to the protein bands was obtained.
Urine samples were obtained by catheter from patients admitted to the coronary care unit all of whom had received thrombolytic therapy with streptokinase preparations. Urine was also obtained from patients receiving cytotoxic drugs and under investigation for possible renal damage. Random urine samples were collected from healthy persons and samples were also collected from volunteers before and after running a half marathon (13 miles). Samples were preserved by adding to 100 ml of urine 7.5 ml of a preservative solution consisting of 2 mol/l Tris-HCl at pH 7.8 containing 62 mm01 Na aside and 60 nun0111chlorhexidine (0.5% w/v chlorhexidine gluconate in 70% industrial alcohol). Analysis was either carried out immediately or the samples were stored at -20°C. Urinary creatinine was measured using the method of Henry [6] and retinol binding protein by an ELISA method [8].
Ri?SUIQ
&wc.@eity testing of rnyo~~obi~antibody To test the specificity of the capture antibody a sample of urine with a high protein content, a purified preparation of myoglobin and a preparation of haemoglobin were subjected to native polyacrylamide gel electrophoresis. After electrophoresis, half of the gel was subjected to Western blotting and (the cellulose nitrate membrane incubated with the rabbit anti-myoglobin anti~~m, followed by the anti-IgC conjugated with alkaline phosphatase and then stained for enzyme activity) and the other half was stained for protein with Coomassie Blue stain. The results of Westernblotting are shown in Fig. 1 with the purilied myoglobin preparation showing two main components and several very minor components. The main components were in the same position as those visualised by Coomassie Blue staining and the urine sample showed these with approximately the same intensity of staining. With haemoglobin staining was very faint but the positions occupied by the haemoglobin components were located in the original gel by their intrinsic red colour and contirmed by the reaction obtained when Multistix (Miles Ltd., Stoke Poges, Slough, UK) were inserted into the gel in these positions. A urine sample, the purified myoglobin preparation and a series of protein markers were subj~~ to denaturation with mer~aptoethano1 and SDS prior to SDS-PAGE in a 15% acrylamide gel. After eI~trophoresis half of the gel was stained for protein with Coomassie Blue and the other part of the gel containing similar samples was subsequently subjected to Western blotting as before. With the Western blot both the urine sample and the purified myoglobin showed only a single band
Fig. 1. Detection of rn~og~ob~~after Western blotting and trea~nt with myoglobin antibody and conjugated anti-rabbit I@ antibody. (aa) Haemoefobin, 100 fig; (bf urine; (c) purified myoglobin 0.1 pg. Ekctrophoresis was carried out for 210 tin at an average of 175 V and 30 mA.
Fig. 2. Detection of myoglobin after SDS electrophoresis, Western blotting and treatment with myoglobin antibody and conjugated anti-rabbit IgG antibody. Electrophoresis was carried out for 240 min at average 160 V and 28 mA. A: Coomassie Blue staining of part of a gel. Containing in (a) protein marker 1, cytochrome c (mol. wt. 12,500 Da); 2, chymotrypsin (mol. wt. 25,000 Da); 3, aldolase (mol. wt. 40,000 Da); 4, ovalbumin (mol. wt. 45,000 Da); 5, catalase (mol. wt. 60,000 Da); 6, albumin (mol. wt. 68,000 Da); 7, phosphorylase (mol. wt. 94,000 Da). (b) Purified myoglobin; (c) urine. B: Nitrocellulose sheet stained for phosphatase activity after treatment of a part of gel. Containing in (a) purified myoglobin and (b) urine.
at a position the same as the band visualised by Coomassie Blue (Fig. 2). This single component migrated between the chymotrypsin and the cytochrome C markers. Plotting of the distance of migration against molecular weight of the protein markers (Fig. 3) allowed the molecular weight of this component to be estimated as 17,000 Da which is the recognised molecular size of myoglobin [9]. Detection limit and working range A typical standard curve is shown in Fig. 3. The lower detection limit was found to be 0.1 &l. When urine containing high concentrations of myoglobin was diluted with the PBS-chick serum diluent to give a series of concentrations within the work-
202
~oglobin
pg/I (log scale)
Fig. 3. Typicat shape of calibration for the measurement of myoglobin with ELBA.
ing range of the assay, the results parallel those obtained with dilutions of the calibrant. Imprecision
For urine diluted approx. 1:1,000 with PBS-diluent, the coefficient of variation for intra-batch assay was 8% at a mean concentration of 100 pgil (n = 10). The coeffrcient of variation for inter-batch assay was 10% at a mean ~on~ntration of 50 pg/l (n = 11). Interference
To determine possible interference, myoglobin was measured in the presence of a relatively high concentration of human haemoglobin. Standard curves were constructed using 100 ~1 samples of a series of myoglobin preparations ranging from 1 to 512 &l and the same samples, containing 50 mg4 of haemoglobin, were assayed concurrently. There was no significant difference between the two sets of results. Recovery
Purified myoglobin was added to 31 samples of urine to give an increase in each of 10 &l; chicken serum was also added so that each sample contained 900 ~1 of urine with 100 ~1 serum. The initial concentration of 23 of these urines was < 1 &l and the mean recovery was 105%. With the remaining samples, the initial myoglobin concentration ranged from 1.3-128 &l and with these the mean recovery was 101% with a range of 73- 113%. Normal range
Of the 20 normal urine samples assayed, only one contained > 1 pg/i myoglobin.
203 TABLE I Urinary protein myoglobin and retinol binding protein and serum CK activity of patients admitted to coronary care unit Protein (mgfl)
Myoglobin (Pgil)
Urinary retinol binding protein (mgfl)
Serum creatine kinase (units/l)
MI
900 440 550 780 1,280 1,020 1,125
13,330 1,050 5,600 3,850 7,500 268 33,750
350 31 128 60 43 50 20
1,033 544 1,259 3,447 439 812 2,340
No MI
690 700 1,170 1,040
95 18 188 8
216 46 170 13
33 17 66 70
All results are the peak values observed within 24 h of admission.
With two subjects urinary myoglobin was measured before and after running a half marathon. Before running myoglobin was not detected but afterwards 2 &l myoglobin was found in the urine of one subject and 10 pg/l myoglobin was found in the urine of the other. Myoglobin after myocardial infarction
Urine samples were collected from patients admitted to a Coronary Care Unit. Samples were taken at the intervals after treatment with streptokinase. The results of urinary myoglobin measurement are shown in Table I with the patients divided into those whose serum creatine kinase showed the characteristic changes expected in myocardial infarction and those whose serum creatine kinase did not increase and where infarction was thought not to have occurred. All these patients showed an increase in urinary myoglobin with the increases being significantly higher in those showing evidence of infarction, Correlation between retinol binding protein and myoglobin in urine
Of the 100 urine samples obtained from patients being ‘investigated for renal tubular damage, 45 had retinal-binding protein RBP leveis within the normal range, (
197
Short Communication
An ELISA metkod to measure human rny~gl~~in in urine Alan W. Hodsona, Andrew W. Skillena and Nicholas B. Argentb aDepartment of Clinical Biochemistry, University of Newcastle upon Tyne, NE.2 4HH and bDepartment of Renal Medicine, University of Newcastle, Newcastle upon Tyne, NE2 4HH (UK)
(Received 4 November 1991; revision received 20 April 1992; accepted 28 April 1992)
Key words: Urine rnyo~ob;n~ Sandwich ELISA assay; My~ardial
infarction; Renal disease
Following thrombolytic therapy of patients with acute myocardial infarction, a transient proteinuria was detected [I]. As the myoglobin concentration in plasma, increases after infarction, reaching a peak within 2-3 h [2] and as myoglobin is suspected of being nephrotoxic [3] an increase of this protein in the glomerular filtrate may lead to tubular damage. Therefore an investigation was instigated to examine the conc~tratio~ of myoglobin and retinol binding protein in urine of patients following myo~ardial infarction. For this purpose a sensitive and specific ELISA method for dete~ining urinary myoglobin was developed that is based on a method used by others to determine plasma myoglobin [4]. The current paper describes this method. Material and Methods A polyclonal rabbit antibody to human myoglobin, a horseradish peroxidaseconjugated antibody and purified human myoglobin were bought from Dako Ltd. (High Wycombe, Bucks, UK). An alkaline phosphatase labelled sheep anti-rabbit IgG was bought from Sera-fab Ltd. (Crawley Down, Sussex, UK). Polyoxyethylenesorbitan monoraurate (Tween 20), 5-bromo~hloroindoxyl phosphate and Correspondence to: Afan W. Hodson, Department of ClinicaI Biochemistry, University Tyne, NE2 4HH, UK.
ofNewcastleupon
198
Nitro Blue tetrazolium were bought from Sigma Ltd. (Poole, Dorset, UK). Chicken serum was obtained from Flow Laboratories (Rickmansworth, Herts, UK). Chlorhexidine in alcohol was obtained from Pearce Laboratories (Leeds, Yorkshire, UK). Visking tubing and all other chemicals of ANALAR quality when obtainable, were bought from BDH Ltd. (Poole, Dorset, UK). Hybond-C nitrocellulose hybridization membrane, 0.45 pm, was bought from Amersham Ltd. (Amersham, Bucks, UK). Flat-bottomed microtiter plates, type Immulon-4, were bought from Dynatech Laboratories Ltd. (Billingshurst, Sussex, UK).
Microtiter plates were coated with antibody by incubation at 4*C for 16 h with 6 &ml antibody in borate buffer, pH 8.5 (10 mmol/l boric acid, 2.5 mmol/l sodium borate and 7.5 mmohl sodium chloride), 200 ~1 being applied to each well. Temperature variation was minimized by placing the plate in a small enclosed box in a cold room at 4°C. The plate wells were washed once with 200 11;lof PBS-Tween reagent, pH 7.4, which is 2.7 mmol/l potassium chloride, 1.5 mmolll potassium dihydrogen phosphate, 8 mmol/l disodium hydrogen phosphate and 137 mmol/l sodium chloride containing 500 ~1 of Tween 20 per litre. A stock myoglobin standard of 1024 pl was prepared by diluting a purified preparation containing 2 pglml in PBS reagent and 100 ml chicken serum per litre and the portions stored at -20°C until required. A series of working standards were prepared by diluting the stock standard solution of myoglobin with a PBS-diluent reagent containing 100 ml of chicken serum per litre PBS Tween. Urine samples were usually diluted 15, I:50 and I:100 with this PBS-diluent reagent. However, some urine samples were assayed undiluted in which case 100 ~1 of chicken serum were added per 900 ~1 of urine. Standards were assayed in triplicate and urine samples in duplicate by adding 100 ~1 to wells, placing the microtiter plate in a small closed container and incubating for 2 h at room temperature. The plates were washed twice with 200 ~1 of PBS-Tween reagent delivered with a multi pipette and blotted briefly by inversion on paper-towelling between ejection of antigen solution and washes. After washing, 150 ~1 of PBS Tween reagent cont~ning 0.6 &ml of horseradish ~roxida~-conju~ted rabbit antibody to human myoglobin was added to each welI. The plate was then placed in a small closed container and incubated for 1 h at room temperature. After incubation, the plates were washed 6 times as previously described with the plate being rotated horizontally through 180°C after each wash. To each well 100 pl of substrate consisting of 3.7 mmol/l o-phenylenediamine and 5 mmol/l hydrogen peroxide in 34.7 mmolfl citric acid, 66.7 mmol/l disodium hydrogen phosphate buffer, pH 5.0, was then added. Colour was allowed to develop for 30 min at room temperature and the reaction then stopped by addition of 100 ~1 of 2.5 mol/l H2S04 with the absorbance being measured at 495 nm with a Titertek Multiscan MCC/340 plate reader (Flow Laboratories). A sample blank was obtained using diluted chick serum and a colour reagent blank was obtained by omitting sample, standard and antibody.
For polyacrylamide gel electrophoresis
[5] we cast gels, 150 mm x 130 mm x 2
199
mm containing 150 g/l acrylamide and 3.9 g/l his-a~rylamide in a running gel buffer consisting of 0.5 mol/l Tris-HCI at pH 8.7 at 25”C, 4.4 mmol/l ammonium persulphate, 10 pmol/l TEMED and with or without 3.5 mmol/l SDS. After polymerisation, a stacking gel 150 mm x 20 mm x 2 mm was cast containing 80 g/l acylamide and 2.13 g/l bis-acrylamide in a buffer consisting of 125 mmol/l Tris-HCl, pH 6.8 at 25*C, 4.4 mmolll ammonium persulphate, 10 pmol/l TEMED and with or without 3.5 mmol/l SDS. Samples were diluted with equal parts of stacking gel buffer, 0.125 mol/l Tris-HCl at pH 6.8, containing 2.2 mol/l glycerol, 45 ml/l saturated aqueous Bromophenol Blue solution, 20 mmol/l EDTA and with or without 350 rnmol/l SDS. We carried out electrophoresis with an electrode buffer consisting of 25.3 mmol/l Tris, 192 mmol/l glycine, with or without 3.5 mm0111SDS. Western plotting After electrophoresis the gel was soaked for 30 min in transfer buffer consisting of 25 mmol/l Tris and 192 mmolll glycine and 5 mol/l methanol and the separated proteins transferred by electrophoresis onto nitro cellulose sheet at 4°C at 10 V overnight, then at 30 V for 1 h using the transfer buffer. After transfer el~trophoresis nitrocellulose membrane was blocked by soaking in 100 ml PBS-Tween for 1 h and washed once with the same reagent. Antibody reagent containing 1 ~1 of rabbit IgG anti-myoglobin in 1 ml was then used to soak the nitrocellulose sheet. After 2 h at room temperature the sheet was washed twice with loo-ml lots of PBS-Tween and then soaked in a solution of labelled antibody containing 5 pg/lOO ml anti-rabbit IgG conjugated with alkaline phosphatase in the same buffer. Thereafter the staining technique was carried out according to the technique of Blake et al. [6]. Protein staining After electrophoresis the gels were soaked in dye made by dissolving 250 mgJ1 Coomassie Brilliant Blue R-250 in a solvent consisting of 1.7 molll acetic acid and 4.4 mol/l ethanol (industrial methylated spirit 99%) in water. The gels were stained for l-2 h, then washed with several changes of dye solvent until a clear background to the protein bands was obtained.
Urine samples were obtained by catheter from patients admitted to the coronary care unit all of whom had received thrombolytic therapy with streptokinase preparations. Urine was also obtained from patients receiving cytotoxic drugs and under investigation for possible renal damage. Random urine samples were collected from healthy persons and samples were also collected from volunteers before and after running a half marathon (13 miles). Samples were preserved by adding to 100 ml of urine 7.5 ml of a preservative solution consisting of 2 mol/l Tris-HCl at pH 7.8 containing 62 mm01 Na aside and 60 nun0111chlorhexidine (0.5% w/v chlorhexidine gluconate in 70% industrial alcohol). Analysis was either carried out immediately or the samples were stored at -20°C. Urinary creatinine was measured using the method of Henry [6] and retinol binding protein by an ELISA method [8].
Ri?SUIQ
&wc.@eity testing of rnyo~~obi~antibody To test the specificity of the capture antibody a sample of urine with a high protein content, a purified preparation of myoglobin and a preparation of haemoglobin were subjected to native polyacrylamide gel electrophoresis. After electrophoresis, half of the gel was subjected to Western blotting and (the cellulose nitrate membrane incubated with the rabbit anti-myoglobin anti~~m, followed by the anti-IgC conjugated with alkaline phosphatase and then stained for enzyme activity) and the other half was stained for protein with Coomassie Blue stain. The results of Westernblotting are shown in Fig. 1 with the purilied myoglobin preparation showing two main components and several very minor components. The main components were in the same position as those visualised by Coomassie Blue staining and the urine sample showed these with approximately the same intensity of staining. With haemoglobin staining was very faint but the positions occupied by the haemoglobin components were located in the original gel by their intrinsic red colour and contirmed by the reaction obtained when Multistix (Miles Ltd., Stoke Poges, Slough, UK) were inserted into the gel in these positions. A urine sample, the purified myoglobin preparation and a series of protein markers were subj~~ to denaturation with mer~aptoethano1 and SDS prior to SDS-PAGE in a 15% acrylamide gel. After eI~trophoresis half of the gel was stained for protein with Coomassie Blue and the other part of the gel containing similar samples was subsequently subjected to Western blotting as before. With the Western blot both the urine sample and the purified myoglobin showed only a single band
Fig. 1. Detection of rn~og~ob~~after Western blotting and trea~nt with myoglobin antibody and conjugated anti-rabbit I@ antibody. (aa) Haemoefobin, 100 fig; (bf urine; (c) purified myoglobin 0.1 pg. Ekctrophoresis was carried out for 210 tin at an average of 175 V and 30 mA.
Fig. 2. Detection of myoglobin after SDS electrophoresis, Western blotting and treatment with myoglobin antibody and conjugated anti-rabbit IgG antibody. Electrophoresis was carried out for 240 min at average 160 V and 28 mA. A: Coomassie Blue staining of part of a gel. Containing in (a) protein marker 1, cytochrome c (mol. wt. 12,500 Da); 2, chymotrypsin (mol. wt. 25,000 Da); 3, aldolase (mol. wt. 40,000 Da); 4, ovalbumin (mol. wt. 45,000 Da); 5, catalase (mol. wt. 60,000 Da); 6, albumin (mol. wt. 68,000 Da); 7, phosphorylase (mol. wt. 94,000 Da). (b) Purified myoglobin; (c) urine. B: Nitrocellulose sheet stained for phosphatase activity after treatment of a part of gel. Containing in (a) purified myoglobin and (b) urine.
at a position the same as the band visualised by Coomassie Blue (Fig. 2). This single component migrated between the chymotrypsin and the cytochrome C markers. Plotting of the distance of migration against molecular weight of the protein markers (Fig. 3) allowed the molecular weight of this component to be estimated as 17,000 Da which is the recognised molecular size of myoglobin [9]. Detection limit and working range A typical standard curve is shown in Fig. 3. The lower detection limit was found to be 0.1 &l. When urine containing high concentrations of myoglobin was diluted with the PBS-chick serum diluent to give a series of concentrations within the work-
202
~oglobin
pg/I (log scale)
Fig. 3. Typicat shape of calibration for the measurement of myoglobin with ELBA.
ing range of the assay, the results parallel those obtained with dilutions of the calibrant. Imprecision
For urine diluted approx. 1:1,000 with PBS-diluent, the coefficient of variation for intra-batch assay was 8% at a mean concentration of 100 pgil (n = 10). The coeffrcient of variation for inter-batch assay was 10% at a mean ~on~ntration of 50 pg/l (n = 11). Interference
To determine possible interference, myoglobin was measured in the presence of a relatively high concentration of human haemoglobin. Standard curves were constructed using 100 ~1 samples of a series of myoglobin preparations ranging from 1 to 512 &l and the same samples, containing 50 mg4 of haemoglobin, were assayed concurrently. There was no significant difference between the two sets of results. Recovery
Purified myoglobin was added to 31 samples of urine to give an increase in each of 10 &l; chicken serum was also added so that each sample contained 900 ~1 of urine with 100 ~1 serum. The initial concentration of 23 of these urines was < 1 &l and the mean recovery was 105%. With the remaining samples, the initial myoglobin concentration ranged from 1.3-128 &l and with these the mean recovery was 101% with a range of 73- 113%. Normal range
Of the 20 normal urine samples assayed, only one contained > 1 pg/i myoglobin.
203 TABLE I Urinary protein myoglobin and retinol binding protein and serum CK activity of patients admitted to coronary care unit Protein (mgfl)
Myoglobin (Pgil)
Urinary retinol binding protein (mgfl)
Serum creatine kinase (units/l)
MI
900 440 550 780 1,280 1,020 1,125
13,330 1,050 5,600 3,850 7,500 268 33,750
350 31 128 60 43 50 20
1,033 544 1,259 3,447 439 812 2,340
No MI
690 700 1,170 1,040
95 18 188 8
216 46 170 13
33 17 66 70
All results are the peak values observed within 24 h of admission.
With two subjects urinary myoglobin was measured before and after running a half marathon. Before running myoglobin was not detected but afterwards 2 &l myoglobin was found in the urine of one subject and 10 pg/l myoglobin was found in the urine of the other. Myoglobin after myocardial infarction
Urine samples were collected from patients admitted to a Coronary Care Unit. Samples were taken at the intervals after treatment with streptokinase. The results of urinary myoglobin measurement are shown in Table I with the patients divided into those whose serum creatine kinase showed the characteristic changes expected in myocardial infarction and those whose serum creatine kinase did not increase and where infarction was thought not to have occurred. All these patients showed an increase in urinary myoglobin with the increases being significantly higher in those showing evidence of infarction, Correlation between retinol binding protein and myoglobin in urine
Of the 100 urine samples obtained from patients being ‘investigated for renal tubular damage, 45 had retinal-binding protein RBP leveis within the normal range, (
