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A rapid method for myoglobin radioimmunoanalysis as a diagnostic tool in myocardial infarction
Clinica Chimica Acta, 124 (1982) 235-238
235
Elsevier Biomedical Press CCA 2235
Brief technical
note
A rapid method for myoglobin radioimmunoanalysis as a diagnostic tool in myocardial infarction M&hail A. Grachev *, Leonid E. Matveev, Evgeniy K. Pressman, Victor V. Roschlce ~~~birsk
Institute
of Organie Chemistry Siberian Biuision of the Academy of Sciences of USSR, N~ib~rsk
90 (USSR)
{Received November 2&h, 1981; revision May 4th, 1982)
Introduction Stone et al [1,2] have elaborated a RIA-method for the determination of myoglobin, and found that increase of its concentration in serum is a reliable criterion for the diagnosis of myocardial infarction. However, the procedure proposed was time-consuming - the test took 24-28 h. Subsequently, the time of the analysis has been reduced to 5-6 h [3-51. Recently, a rapid method for the determination of myoglobin has been proposed based upon the use of antiserum i~ob~ized on a powdered carrier [6]. This method takes a little more than 1 h. The procedure according to Roxin et al [6] is fast due to its non-equilibrium character; after the incubation (30 min) the reaction of the antigen with the immobilized antibody still remains far from equilibrium. It is generally believed that non-equilibrium RIA procedures are less convenient than equilibrium ones for practical clinical applications. According to the RIA procedure proposed here, the time saving compared with the established methods [l-5] is achieved by using relatively high concentrations of radioactive myoglobin of moderate specific radioactivity. Under these conditions, the kinetic plateau is reached in 15-20 min. Hence, the total time of the analysis to obtain a standard curve and results for five unknown sera is 55 min. Therefore, the method becomes more useful as a guide in the treatment of my~ardi~ infarction. Materials and methods Human myocardial myoglobin was isolated by the method of Stone et al [l]. The UV-visible spectrum of the preparation was identical to that of metmyoglobin. The extinction coefficient at 510 nm was 9.6 cm-’ - mol. l-‘, in good accord with [7]. Electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate
* To whom correspondence should be addressed. ~-898~/82/~-~/~2.75
0 1982 Elsevier Biomedical Press
236
showed that the purity of the preparation was no less than 95%. Radioactive labelling of myoglobin was performed using the procedure of Bolton and Hunter [8]. The specific radioactivity of [‘251]myoglobin obtained was 1.5 mCi/mg; the maximum binding with excess of antiserum was 98%. To raise anti-myoglobin antiserum, rabbits were immunized by subcutaneous injection of 1 mg myoglobin in complete Freund’s adjuvant; the same amounts of the protein were injected on the 21st, 31st and 41st days after the first immunization. The dilution of the serum raised, which gave 50% binding of 5.5 ng [‘251]myoglobin per 100 ~1, was 1 : 120. The cross-reactivity (interference) of the antiserum with human proteins (hemoglobin, albumin and IgG) was less than 6 . 10p6, lo--‘, and IO-‘, respectively, as determined by the method of Chard [9]. To prepare stock solutions of antiserum, of [‘*‘I]myoglobin and of myoglobin standard, the corresponding concentrated preparations were diluted by buffer A (0.05 mol/l sodium phosphate, 0.01 mol/l EDTA, 1% NaN,, 0.08 mol/l NaCl, 1% bovine serum albumin, pH 7.3). To prepare myoglobin standard working solutions, standard stock solution (1 g/l) was first prepared in buffer A, and the concentration of myoglobin checked spectrophotometrically. The working solutions were prepared by dilution of the stock solution with buffer A; they were used to prepare the standard curve. Analytical procedure Typically, it was necessary to analyze a series of 3-5 patients’ sera and to obtain a standard curve for this series, all in duplicate. Hence, some 26-30 tubes had to be treated in one batch. The solutions of standards and of [ ‘251]myoglobin were dispensed in advance. The tubes of the standard curve contained 100 ~1 of each of the eight myoglobin standards, and 100 pl of [ ‘*‘I]myogiobin (15000 dpm, 5.5 ng). The test-tubes for the patients’ sera contained 100 ~1 of the same [ ‘251]myoglobin solution. Such tubes, prepared in advance, were stored no longer than 1 week at 4°C. When sera arrived, their loo-p.1 aliquots were added to the sample tubes, and 100 ~1 of antiserum (1: 120) was added to all the tubes with standards and samples. After thorough mixing, the tubes were kept for 10 min at 25°C (zero time = moment of addition of antiserum to the first tube). Subsequently, 200 pl of 5% bovine serum immunoglobulin G solution in 0.05 mol/l sodium phosphate, pH 7.5, and 500 ~1 of 22% aqueous solution of polyethylene glycol (molecular mass 6000, trade name PEG- 115) were dispensed into each tube by means of a mechanical diluter (LKB, Sweden). The tubes were thoroughly shaken, centrifuged for 10 min at 2000 X g, and the supernatants decanted. The radioactivities of the tubes with the precipitates were counted with a Mini-Gamma counter (LKB, Sweden); the counting time was 1 min. The total time taken by this procedure, starting from the moment of arrival of sera and ending with the calculation of the results, was 50-60 min. Results and discussion We studied the kinetics under the assay conditions.
of the binding The binding
of [‘251]myoglobin with the antiserum at zero time was equal to zero (after
237
subtraction of non-specific binding which was less than 5% in all the experiments) Hence, addition of IgG and polyethylene glycol stops the binding reaction very rapidly. The kinetic plateau was reached within approximately 15 min. Standard curves obtained with different incubation times are shown in Fig. 1. It is seen that these curves do not greatly differ from each other in scope or form. Intra-assay coefficient of variation for a control normal serum in 10 measurements was 4.9% (28.3 2 1.4 pg/l). The sensitivity of the assay is better than 10 pg,/l. The accuracy, as demonstrated by the recovery test, was better than 97%. Myoglobin was found in sera of 26 healthy donors studied; its content was 11.6 2 4.6 E.cg/l (average-C 2 SD) and varied between 6 and 28 pg,/l. Fig. 2 shows two typical curves for myoglobin concentration in sera of patients with myocardial infarction. The diagnoses were confirmed according to the criteria of the World Health Organization. Curve A corresponds to a case of a single infarction of the front wall, curve B to multiple micro-infarctions followed by a second attack and propagation of the zone of necrosis. These data are in accord with those of earlier studies [l-S]. A number of workers has shown before [9,10], and it is also seen in Fig. 2, that increase of the level of myo~ob~ begins within l-3 h after the onset of chest pain. Hence, studies of the changes of myoglobin concentration in sera taken at short time intervals directly
hy/, 50
40
30
20
10
, 0
0
8
16
32
6S
128
256
512yq
10
20
30
LO h
Fig. 1. Standard curves at different incubation times. El El, 60 min and 30 min: 0 0, 15 mm; l 0,6 min. Ordinate, B/T, where B is radioactivity of the immune precipitate obtained with given sample minus radioactivity of control (reaction mixture without antiserum); T, total radioactivity of the reaction mixture. Fig. 2. Changes of myoglobin concentration in sera of two patients suffering from myocardial infarction. A, single infarction of the front wall; 3, microinfarctions followed by second attack {moment of second chest pain is shown by arrow); ------, average normal level; ordinate, concentration of myoglobin (pg/l); abscissa, time after onset of chest pain (h).
238
after the first attack may give valuable information for clinicians. A prerequisite to this end is the rapid character of the assay - the data must be obtained in the shortest possible time. The procedure presented above yields results in 50-60 mm after the arrival of the specimen. Acceleration of the assay was achieved because the concentrations of the reagents (myoglobin and antibodies) were high: 20-30 times greater than those used by other workers [3-61. The sensitivity of the method was less, but it is still sufficient to differentiate between normal and pathological sera. Another reason for the acceleration is ?he use of a high concentration of carrier IgG, which makes it possible to avoid incubation during the pr~ipitation of the immune complex with polyethylene glycol. Acknowledgements The authors thank Dr. G.V. Shishkin and Mr. A. Gall for the synthesis of the Bolton-Hunter N-hydroxysuccinimide ester, and Prof. Yu.P. Nikitin of the Siberian Division of the Academy of Medical Sciences, and Dr. M.B. Altman of the Central Hospital of the Siberian Division of the Academy of Sciences for the sera from patients with confirmed myocardial infarction. References 1 Stone MJ, Willerson JT, Gomez-Sanchez CE, Waterman MR. Radioimmunoassay of myoglobin in human serum. Results in patients with acute myocardial infarction. J Clin Invest 1975; 56: 1334- 1339. 2 Stone MJ. Waterman MR, Harimoto D et al. Serum myoglobin level as diagnostic test in patients with acute myocardial infarction. Br Heart J 1977; 39: 375-380. 3 Rosano TG, Kenny MA. A radioimmunoassay for human serum myoglobin: method development and normal values. Clin Chem 1977; 23: 69-75. 4 Miyoshi K, Saito S, Kawai H et al. Radioimmunoassay for human myoglobin: methods and results in patients with skeletal muscle or myocardial disorders. J Lab Clin Med 1978; 92: 342-35 1. 5 Ndrregaard-Hansen K, Lindo KE, Ludvigsen CV, Ntirgaard-Pedersen B. Serum myoglobin compared with creatine kinase in patients with acute myocardial infarction. Acta Med Stand 1980; 207: 265-270. 6 Roxin L-E, Venge P, Wide L. A fast and sensitive radioimmunoassay of human myoglobin for use in the early diagnosis of heart infarction. Clin Chim Acta 1980; 107: 129-134. 7 Yamazaki I, Yokota K, Shikama C. Preparation of crystalline ox~yogiobin from horse heart. J Biol Chem 1964; 239: 4151-4155. 8 Bolton AE, Hunter WH. The labelling of proteins to high specific radioactivities by conjugation to a ‘2SI-containing acylating agent. Biochem J 1973; 133: 529-539. 9 Chard T. An introduction to radioimmunoassay and related techniques. Amsterdam: Elsevier/North Holland Biomedical Press, 1978: 178. 10 Cathtline M, Bouget J, Aubr&e A, Daubert C, Gouffault J, LeGall JY. Etude de la myoglobinemie et de son tvolution au tours de l’infarctus du myocarde. Clin Chim Acta 1980; 105: 31 l-315. 11 Aldor E, Kainz W, Kahn P, Heeger H. Kritische Bewertung der Serummyoglobinbestimmung in der Infarktdiagnostik. Intensivmed 1980; 17: 106- 109. I2 Desbuquois B, Aurbach GD. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol 1971; 33: 732-738.
235
Elsevier Biomedical Press CCA 2235
Brief technical
note
A rapid method for myoglobin radioimmunoanalysis as a diagnostic tool in myocardial infarction M&hail A. Grachev *, Leonid E. Matveev, Evgeniy K. Pressman, Victor V. Roschlce ~~~birsk
Institute
of Organie Chemistry Siberian Biuision of the Academy of Sciences of USSR, N~ib~rsk
90 (USSR)
{Received November 2&h, 1981; revision May 4th, 1982)
Introduction Stone et al [1,2] have elaborated a RIA-method for the determination of myoglobin, and found that increase of its concentration in serum is a reliable criterion for the diagnosis of myocardial infarction. However, the procedure proposed was time-consuming - the test took 24-28 h. Subsequently, the time of the analysis has been reduced to 5-6 h [3-51. Recently, a rapid method for the determination of myoglobin has been proposed based upon the use of antiserum i~ob~ized on a powdered carrier [6]. This method takes a little more than 1 h. The procedure according to Roxin et al [6] is fast due to its non-equilibrium character; after the incubation (30 min) the reaction of the antigen with the immobilized antibody still remains far from equilibrium. It is generally believed that non-equilibrium RIA procedures are less convenient than equilibrium ones for practical clinical applications. According to the RIA procedure proposed here, the time saving compared with the established methods [l-5] is achieved by using relatively high concentrations of radioactive myoglobin of moderate specific radioactivity. Under these conditions, the kinetic plateau is reached in 15-20 min. Hence, the total time of the analysis to obtain a standard curve and results for five unknown sera is 55 min. Therefore, the method becomes more useful as a guide in the treatment of my~ardi~ infarction. Materials and methods Human myocardial myoglobin was isolated by the method of Stone et al [l]. The UV-visible spectrum of the preparation was identical to that of metmyoglobin. The extinction coefficient at 510 nm was 9.6 cm-’ - mol. l-‘, in good accord with [7]. Electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulphate
* To whom correspondence should be addressed. ~-898~/82/~-~/~2.75
0 1982 Elsevier Biomedical Press
236
showed that the purity of the preparation was no less than 95%. Radioactive labelling of myoglobin was performed using the procedure of Bolton and Hunter [8]. The specific radioactivity of [‘251]myoglobin obtained was 1.5 mCi/mg; the maximum binding with excess of antiserum was 98%. To raise anti-myoglobin antiserum, rabbits were immunized by subcutaneous injection of 1 mg myoglobin in complete Freund’s adjuvant; the same amounts of the protein were injected on the 21st, 31st and 41st days after the first immunization. The dilution of the serum raised, which gave 50% binding of 5.5 ng [‘251]myoglobin per 100 ~1, was 1 : 120. The cross-reactivity (interference) of the antiserum with human proteins (hemoglobin, albumin and IgG) was less than 6 . 10p6, lo--‘, and IO-‘, respectively, as determined by the method of Chard [9]. To prepare stock solutions of antiserum, of [‘*‘I]myoglobin and of myoglobin standard, the corresponding concentrated preparations were diluted by buffer A (0.05 mol/l sodium phosphate, 0.01 mol/l EDTA, 1% NaN,, 0.08 mol/l NaCl, 1% bovine serum albumin, pH 7.3). To prepare myoglobin standard working solutions, standard stock solution (1 g/l) was first prepared in buffer A, and the concentration of myoglobin checked spectrophotometrically. The working solutions were prepared by dilution of the stock solution with buffer A; they were used to prepare the standard curve. Analytical procedure Typically, it was necessary to analyze a series of 3-5 patients’ sera and to obtain a standard curve for this series, all in duplicate. Hence, some 26-30 tubes had to be treated in one batch. The solutions of standards and of [ ‘251]myoglobin were dispensed in advance. The tubes of the standard curve contained 100 ~1 of each of the eight myoglobin standards, and 100 pl of [ ‘*‘I]myogiobin (15000 dpm, 5.5 ng). The test-tubes for the patients’ sera contained 100 ~1 of the same [ ‘251]myoglobin solution. Such tubes, prepared in advance, were stored no longer than 1 week at 4°C. When sera arrived, their loo-p.1 aliquots were added to the sample tubes, and 100 ~1 of antiserum (1: 120) was added to all the tubes with standards and samples. After thorough mixing, the tubes were kept for 10 min at 25°C (zero time = moment of addition of antiserum to the first tube). Subsequently, 200 pl of 5% bovine serum immunoglobulin G solution in 0.05 mol/l sodium phosphate, pH 7.5, and 500 ~1 of 22% aqueous solution of polyethylene glycol (molecular mass 6000, trade name PEG- 115) were dispensed into each tube by means of a mechanical diluter (LKB, Sweden). The tubes were thoroughly shaken, centrifuged for 10 min at 2000 X g, and the supernatants decanted. The radioactivities of the tubes with the precipitates were counted with a Mini-Gamma counter (LKB, Sweden); the counting time was 1 min. The total time taken by this procedure, starting from the moment of arrival of sera and ending with the calculation of the results, was 50-60 min. Results and discussion We studied the kinetics under the assay conditions.
of the binding The binding
of [‘251]myoglobin with the antiserum at zero time was equal to zero (after
237
subtraction of non-specific binding which was less than 5% in all the experiments) Hence, addition of IgG and polyethylene glycol stops the binding reaction very rapidly. The kinetic plateau was reached within approximately 15 min. Standard curves obtained with different incubation times are shown in Fig. 1. It is seen that these curves do not greatly differ from each other in scope or form. Intra-assay coefficient of variation for a control normal serum in 10 measurements was 4.9% (28.3 2 1.4 pg/l). The sensitivity of the assay is better than 10 pg,/l. The accuracy, as demonstrated by the recovery test, was better than 97%. Myoglobin was found in sera of 26 healthy donors studied; its content was 11.6 2 4.6 E.cg/l (average-C 2 SD) and varied between 6 and 28 pg,/l. Fig. 2 shows two typical curves for myoglobin concentration in sera of patients with myocardial infarction. The diagnoses were confirmed according to the criteria of the World Health Organization. Curve A corresponds to a case of a single infarction of the front wall, curve B to multiple micro-infarctions followed by a second attack and propagation of the zone of necrosis. These data are in accord with those of earlier studies [l-S]. A number of workers has shown before [9,10], and it is also seen in Fig. 2, that increase of the level of myo~ob~ begins within l-3 h after the onset of chest pain. Hence, studies of the changes of myoglobin concentration in sera taken at short time intervals directly
hy/, 50
40
30
20
10
, 0
0
8
16
32
6S
128
256
512yq
10
20
30
LO h
Fig. 1. Standard curves at different incubation times. El El, 60 min and 30 min: 0 0, 15 mm; l 0,6 min. Ordinate, B/T, where B is radioactivity of the immune precipitate obtained with given sample minus radioactivity of control (reaction mixture without antiserum); T, total radioactivity of the reaction mixture. Fig. 2. Changes of myoglobin concentration in sera of two patients suffering from myocardial infarction. A, single infarction of the front wall; 3, microinfarctions followed by second attack {moment of second chest pain is shown by arrow); ------, average normal level; ordinate, concentration of myoglobin (pg/l); abscissa, time after onset of chest pain (h).
238
after the first attack may give valuable information for clinicians. A prerequisite to this end is the rapid character of the assay - the data must be obtained in the shortest possible time. The procedure presented above yields results in 50-60 mm after the arrival of the specimen. Acceleration of the assay was achieved because the concentrations of the reagents (myoglobin and antibodies) were high: 20-30 times greater than those used by other workers [3-61. The sensitivity of the method was less, but it is still sufficient to differentiate between normal and pathological sera. Another reason for the acceleration is ?he use of a high concentration of carrier IgG, which makes it possible to avoid incubation during the pr~ipitation of the immune complex with polyethylene glycol. Acknowledgements The authors thank Dr. G.V. Shishkin and Mr. A. Gall for the synthesis of the Bolton-Hunter N-hydroxysuccinimide ester, and Prof. Yu.P. Nikitin of the Siberian Division of the Academy of Medical Sciences, and Dr. M.B. Altman of the Central Hospital of the Siberian Division of the Academy of Sciences for the sera from patients with confirmed myocardial infarction. References 1 Stone MJ, Willerson JT, Gomez-Sanchez CE, Waterman MR. Radioimmunoassay of myoglobin in human serum. Results in patients with acute myocardial infarction. J Clin Invest 1975; 56: 1334- 1339. 2 Stone MJ. Waterman MR, Harimoto D et al. Serum myoglobin level as diagnostic test in patients with acute myocardial infarction. Br Heart J 1977; 39: 375-380. 3 Rosano TG, Kenny MA. A radioimmunoassay for human serum myoglobin: method development and normal values. Clin Chem 1977; 23: 69-75. 4 Miyoshi K, Saito S, Kawai H et al. Radioimmunoassay for human myoglobin: methods and results in patients with skeletal muscle or myocardial disorders. J Lab Clin Med 1978; 92: 342-35 1. 5 Ndrregaard-Hansen K, Lindo KE, Ludvigsen CV, Ntirgaard-Pedersen B. Serum myoglobin compared with creatine kinase in patients with acute myocardial infarction. Acta Med Stand 1980; 207: 265-270. 6 Roxin L-E, Venge P, Wide L. A fast and sensitive radioimmunoassay of human myoglobin for use in the early diagnosis of heart infarction. Clin Chim Acta 1980; 107: 129-134. 7 Yamazaki I, Yokota K, Shikama C. Preparation of crystalline ox~yogiobin from horse heart. J Biol Chem 1964; 239: 4151-4155. 8 Bolton AE, Hunter WH. The labelling of proteins to high specific radioactivities by conjugation to a ‘2SI-containing acylating agent. Biochem J 1973; 133: 529-539. 9 Chard T. An introduction to radioimmunoassay and related techniques. Amsterdam: Elsevier/North Holland Biomedical Press, 1978: 178. 10 Cathtline M, Bouget J, Aubr&e A, Daubert C, Gouffault J, LeGall JY. Etude de la myoglobinemie et de son tvolution au tours de l’infarctus du myocarde. Clin Chim Acta 1980; 105: 31 l-315. 11 Aldor E, Kainz W, Kahn P, Heeger H. Kritische Bewertung der Serummyoglobinbestimmung in der Infarktdiagnostik. Intensivmed 1980; 17: 106- 109. I2 Desbuquois B, Aurbach GD. Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol 1971; 33: 732-738.