ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 8, No. 4Copyright © 1978, Institute for Clinical Science

Radioimmunoassay for Serum Myoglobin

ERNEST C. ADAMS, Ph.D., KATHLEEN M. LAYMAN,and IVARS JAUNAKAIS

Ames Research Laboratory, Division o f Miles Laboratories, Inc.,

Elkhart, IN 46514

ABSTRACT

A column flow-through radioimmune assay for serum myoglobin has been developed. The assay uses antibodies to human or rhesus monkey myoglobin coupled to Sepharose by cyanogen brom ide activation and myoglobin labeled by the Chloramine-T method. The labeled myoglobin is stable over two to three half-lives. Serum myoglobin levels were elevated when serum was taken up to 12 hours after myocardial infarction and gen­erally elevated up to 24 hours. A continued or repeated elevation of serum myoglobin probably indicates continued myocardial damage or new a t­tacks. In the cases of myocardial infarction, levels up to 1340 ng per ml were found.

Introduction

Myoglobin is the oxygen binding pig­ment of muscle. It can be extracted from muscle with water. Myoglobin is readily released into the blood as a result of dam ­age to muscle tissue. Because of its small size and lack of binding to haptoglobin, it is rapidly removed from the blood by the kidney and excreted into the urine. Since myoglobin is similar to hemoglobin in many of its properties and identical in its behavior in peroxidase-like reactions, its distinction from hemoglobin may be dif­ficult. T he m yoglobin con ten t of red muscle, such as skeletal and heart, has been reported to be 2.1 to 3.4 mg per gm wet weight. It is highest in muscle that carries out sustained work with a slow period. White muscle does not contain m yoglobin. M yoglobin from sk e le ta l

m uscle and heart are iden tica l, both chemically and immunologically.10 Thus, myoglobinemia is an indication of muscle damage and not of heart alone. Properly, the assay should be referred to as one for serum m yoglobin and not one for myocardial infarction.

Previous reports from this laboratory have been concerned with the determ i­nation o f m yoglobin in u rine .1 It was shown that im m unochem ical m ethods could be used to distinguish myoglobin from hemoglobin and that myoglobinuria was a diagnostic sign for myocardial in­farction.1,12,13 It would be expected that myoglobinemia would also be a useful sign. Since myoglobin is rapidly excreted in the urine, the amount in the serum fol­lowing muscle damage is less than that in urine. The methods for urine myoglobin requ ire sensitiv ities in the /¿g per ml

3300091-7370/78/0700-0330 $01.80 © Institute for Clinical Science, Inc.

RADIOIMMUNOASSAY FO R SERUM M YOGLOBIN 3 3 1

range while the serum methods require sen s itiv itie s in the nanogram range. Kagen has reported the use of a comple­ment fixation m ethod for assay of serum myoglobin.11 Three groups8,15,16 have re­ported the determination of serum myo­globin by radioimmune assay methods. All three groups used an equilibrium with antibody, labeled antigen and unlabeled antigen, followed by some means of sep­aration of free from bound antigen such as am m onium su lfa te p rec ip ita tio n , polyethylene glycol precipitation or dou­ble antibody precipitation. Recently a commercial assay kit* based on double antibody has been placed on the market. Previously this laboratory reported on radioimmune assay methods using fixed antibodies in a column flow-through for­mat.5,7 This m ethod has been extended to serum myoglobin.

Principle

The crude myoglobin is purified for immunization, for labeling and for stand­ards by electrophoresis on acrylamide gel. Gamma globulin from specific anti­sera to human or monkey myoglobin is coupled to cyanogen bromide activated Sepharose to give a fixed antibody. The antibody-Sepharose, m ixed w ith plain Sepharose, is used to make a chromato­graphic column. W hen a serum contain­ing myoglobin is passed through the col­umn, some of the sites of the antibody are occupied. The remaining sites are availa­ble for binding when the labeled myo­globin is poured through the column. This is a non-equilibrium saturation method.

Reagents

P r e p a r a t io n o f C r u d e M y o g l o b in

Myoglobin is isolated from human or rhesus monkey muscle according to the selective precipitation m ethod of Lugin-

buhl.14 Hemoglobin and proteins other than m yoglobin are p rec ip ita ted from extracts of muscle at pH 8 with 80 percent am m onium sulfate saturation. U nder these conditions, the myoglobin remains in solution. Myoglobin is precipitated at pH 7 with 100 percent ammonium sulfate saturation. The precipitate is collected on 0.45 micron filters.!

E l e c t r o p h o r e s i s a n d E l e c t r o e l u t i o n o f M y o g l o b in

The myoglobin is further purified by electrophoresis on acrylamide gel which removes album in and other serum pro­tein contaminants.1 To obtain material for immunization, the portion of gel contain­ing the myoglobin bands is electroeluted. Colored elu ted fractions are pooled and the absorbance in the Soret band region is measured. After the elution, the myo­globin is in the ferro form; at a concentra­tion of one mg per ml, this has an absor­bance of 5.8 at 422 nm. From this value and the volume of elute, the total amount of m yoglobin is calculated. T he elu te which contains buffer material as well as myoglobin is lyophilized. For immuniza­tion, the myoglobin can be either human or monkey.

F or m yoglob in for lab e lin g or for s tan d a rd s, on ly the da rkest e lec- trophoresced band is used. This m ust be from human myoglobin if an assay with maximum sensitivity is desired. A plug containing the dark band is cut from the gel and inserted into a hole cut out of a new 5 percent acrylamide gel and the myoglobin is electroeluted. The elute is concentrated approximately ten times in the 15 ml cell* using a UM-2 membrane. This usually gives a solution of myoglo­bin containing about 2.5 mg per ml. The concentration is calculated from the ab­sorbance before concentration.

* Nuclear Medical Systems, Inc., Newport Beach, CA.

f M illipore* Amicon®.

3 3 2 ADAMS, LAYMAN AND JAUNAKAIS

I m m u n iz a t io n a n d P r o c e s s in g o f An t is e r a

For immunization, castrated male goats aged 12 to 16 months are used. For the initial injection, w eight of lyophilized antigen calculated to contain 45 fig of myoglobin is dissolved in 0.5 ml saline (0.85 percent) and 0.5 ml Freund’s com­plete adjuvantf added. The mixture is emulsified in the micro cup of an Om- nim ixer| until the mixture is thick (ice is held around the cup). The em ulsified m ixture is in jected into the four dew claws of the goat.

For th e bo o ste r in jec tio n s , the lyophilized antigen (containing 45 fig of myoglobin) is dissolved in 0.5 ml saline. Half of this solution is stored in the re­frigerator for the second of the divided pair o f boosters. To the rem ainder is added 0.25 ml of Freund’s incomplete ad­juvant. This is em ulsified in the Om­nimixer. The emulsion is injected I.M. in four sites, one in each quarter of the goat. After waiting 48 hours, the rem ainder of the antigen stored in the refrigerator is made up and injected in the same way. The schedule for initial injection, booster injections, test bleedings and final b leed­ing is the same as in the earlier reference1 except that there is a waiting period be­tween the second and third pair of boos­ters in order to allow the antibody titer to fall near zero.

The antisera are checked for titer and specificity by immunodiffusion (Ouchter- lony) as described.1 The media for im­munodiffusion now being used in the au­thors’ laboratory is 1 percen t agarose (electrophoretic grade)§ and 3 percent polyethylene glycol 400011 in B-2 buf­fer,** pH 8.6, ionic strength 0.025. An agar cutter with a 4 mm diam eter center w ell and 3 mm diam eter outer wells and

I Difco. t Sorval.§ Nutritional Biochemicals. 1 Fisher.** Harleco.

the centers of the center w ell and outer wells separated by 8 mm has been found convenient, although other arrangements may be used. The antisera should give a good precipitin line against a myoglobin solution or a urine containing myoglobin at levels of 40 to 50 fig per ml and no line against human album in at levels of 1000, 100, and 10 fig per ml or against human serum, f t

P r e p a r a t io n o f A n t ib o d y — Se p h a r o s e

The isolation of gamma globulin from the antisera by ammonium sulfate pre­cipitation is essentially the m ethod used by Campbell and Garvey.3 A final solu­tion of two ml contains the gamma globu­lin which was originally in a 10 ml volume of antiserum.

Two ml of the gamma globulin solution are mixed with 10.2 ml of cold citrate buf­fer (0.2M, pH 6.5). Three grams of cyano­gen brom ide activated Sepharose are m ixed w ith 20 m l cold 0.001N h y ­drochloric acid for ten minutes. The acti­vated Sepharose is washed on a coarse sintered glass filter with 600 ml 0.001N hydrochloric acid for a period of five m inutes; the mixing and washing must be done within a total of 15 minutes. Imm e­d ia te ly afte r w ashing , the ac tiv a ted Sepharose is placed in a tube, the gamma globulin solution immediately added and end-over-end mixing is carried out au­tomatically overnight in the refrigerator.

t t Antisera to human myoglobin are supplied commercially by the Research Products Division of Miles Laboratories (Elkhart, IN), by Cappel Laboratories (Downingtown, PA), and by JCL Clin­ical Research Corp. (Knoxville, TN). Antisera from these sources give good precip itin lines on immunodiffusion and immunoelectrophoresis and are suitable for hemagglutination inhibition deter­mination of urinary myoglobin. They may be adequate for the radioimmune assay, but this can only be decided by trial as their behavior in the other assays does not necessarily predict the suita­bility for radioimmune assay. Just as any antisera, they must be checked for binding to the antibody column and for displacement by the standards.

RADIOIMMUNOASSAY FO R SERUM MYOGLOBIN 3 3 3

The gamma globulin-Sepharose mix­ture is collected on a coarse sintered glass filter and washed with 100 ml cold 0.2M citrate until the filtered washing has an absorbance at 280 nm less than 0.2. The antibody-Sepharose is washed with 500 ml cold saline (0.85 percent) until the ab­sorbance of the washings at 21.5 nm is less than 0.1. T he an tibody-S epharose is w ashed w ith a sm all am ount of cold saline con tain ing 0.1 p e rcen t sodium azide. The antibody-Sepharose is washed w ith saline (w ith 0.1 percen t sodium azide) and diluted to 25 ml with saline (containing 0.1 percent sodium azide). This is mixed and allowed to stand over­night in the refrigerator. The antibody- Sepharose is made up to 25 percent (v/v) w ith sa line c o n ta in in g 0.1 p e rc e n t sodium azide.

For dilution of the antibody-Sepharose, Sepharose-4B is made up to 25 percent (v/v) concentration with saline contain­ing 0.1 percent sodium azide. For deter­m ination o f the d ilu tion factor, sm all batches of 1:5, 1:10, 1:20, 1:50 and 1:100 of the antibody-Sepharose in the plain Sepharose are prepared . Colum ns are prepared, as to be described, from the di­lu tions and the b in d in g w ith labeled myoglobin is determ ined. The dilution that will bind approximately 30 percent of freshly labeled myoglobin is chosen. A large batch of this dilution is prepared and used for the assay columns.

P r e p a r a t io n o f An t ib o d y C o l u m n s

The polypropylene columns are pre­pared elsewhere for the authors, bu t the barrel from any 3 ml disposable plastic syringe may be used. The porous filter sheet* is cut with a cork "borer to give discs of the diam eter of the inside diame­ter of the column. The discs are soaked in1 percent Brij 35 solution, rinsed twice with water and air dried.

* Vyon®.

Leuer caps are placed tightly on the tips of the columns. Approximately 1 ml of saline (0.85 percent) is placed into each of the columns. With a glass tube, a disc* (smooth side up) is forced down through the saline into the bottom of the column. The diluted antibody-Sepharose is thoroughly mixed with a m agnetic stir­rer. While the mixture is still being stir­red, two ml are removed with a two ml p ipet and rapidly added to one of the col­umns. This is repeated for the other col­um ns. S im u ltaneously colum ns from plain Sepharose are prepared for serum blanks. The packed bed volume of the columns is 0.5 ml. The columns are al­lowed to stand upright for several hours or overnight. A second disc* is forced, with the glass tube, down through the liquid in all columns until it is tightly against the packed Sepharose with no air or liquid space. The Leuer tips are re­moved and the columns allowed to drain. The columns are equilibrated with bicine buffer (0.2M, pH 8.5) by filling each twice with the buffer and allowing the buffer to run through. The columns are prepared in large numbers ahead of time and stored in the refrigerator.

P r e p a r a t io n o f 1251-La b e l e d M y o g l o b in

The radioiodination of myoglobin is a modification of Greenwood et al.® The io d in a tio n is pe rfo rm ed in a glass septum -sealed reaction vial. M icroliter syringes are used in reagent transfers and additions. The Na 1251 in sodium hy­droxide solution is neutralized w ith suffi­cient 0.3M phosphate buffer (pH 7.0) and the desired am ount of radioactivity is added to the reaction vial. Five fig of m yoglobin per mCi of 1251 are added, fo llow ed by su ffic ien t 0.1M ph o s­phate buffer (pH 7.0) to bring the myoglo­bin concentration to approximately 0.27 tig p e r fi\ of to ta l reac tion volum e. T w en ty /u,l o f fresh ly p rep a re d

334 ADAMS, LAYMAN AND JAUNAKAIS

chloram ide-T dissolved in 0.3M phos­phate buffer (pH 7.0) are added to the reaction vial. The concentration of the chloramide-T is such that the final con­centration in the reaction vial is approxi­mately 0.25 fig per fil. After 30 seconds, the radioiodination is quenched by the addition of sodium metabisulfate in 0.3M phosphate buffer (pH 7.0).

The radioiodination m ixture is then purified on a 1.0 x 30 cm Biogel P 10 colum n which was previously eq u ili­brated with 0.1M, pH 8.1 tris-hydroxy- ethyl aminomethane buffer containing 3 percent bovine serum album in and 0.2 percent sodium azide. E lution from the column is performed with 0.1M, pH 8.1 tris-hydroxy-ethylam inom ethane buffer containing 0.2 percent bovine albumin. The protein fractions are subsequently purified on a 1.5 x 50 cm Biogel P 30 column by elution w ith the 0.2 percent bovine serum album in in tris buffer. The fractions that show optimum immunolog­ical activity are saved. The specific activ­ity is determ ined by trichloracetic acid precipitation of a small am ount of the radioiodination mixture. The specific ac­tivity of the preparation was 76 /xCi per fig of myoglobin. By altering the ratios, preparations with lower specific activity can be obtained. Preparations with as low specific activity as 14 fi Ci per fig have been successfully used in the assay. For use, the labeled myoglobin is diluted in bicine buffer (0.2M, pH 8.5) so that 0.5 ml contains approxim ately 30,000 cts per min.

P r e p a r a t io n o f St a n d a r d s

For standards, the concentrate, con­taining 1.8 to 2.7 mg per ml, of the eluted material from the single band of myoglo­bin is used. The actual amount is deter­m ined from the Soret band extinction be­fore concentration, as described under the Preparation. This is d iluted 0.01 ml w ith 1 ml norm al goat serum (non- immune) to give the working dilution.

The working dilution is d iluted 0.1 ml with 0.9 ml normal goat serum. This dilu­tion is serially d iluted with equal vol­umes of normal goat serum through seven tubes to give the other standards. Thus, if the original myoglobin concentrate con­tained 2.525 mg per ml, the working dilu­tion would contain 25 fig per ml, and the standards would contain, 2500, 1250, 625, 312, 156, 78, 39 and 19.5 ng per ml, re­spectively.

R a d io im m u n o a s s a y f o r Se r u m M y o g l o b in

The Leuer tips should be on the col­umns at this point. Sufficient antibody columns for the blank, the standards the unknowns and a plain Sepharose column for the serum blank are used. Into each column is placed 0.4 ml of bicine buffer (0.2M, pH 8.5). This may be done ahead of time if both Leuer tips and caps are placed on the columns.) Precisely 0.1 ml of normal goat serum, a standard or an unknown is added to the antibody col­umns. The antibody column to which the normal goat serum is added serves as the blank, i.e., there is no unlabeled antigen and this column will have the greatest binding.

For the serum blank, 0.1 ml of normal goat serum is ad d ed to the p lain Sepharose column; this will measure the binding of the label to Sepharose. The contents of the columns are mixed by tapping. The L euer tips are rem oved from the columns and the columns al­lowed to drain. Thirty m inutes after re­moval of the tips, the tips are replaced and 0.5 ml of the diluted labeled myoglo­bin is added to each column. The tips are again removed and the columns allowed to drain. Thirty m inutes later, the col­umns are washed by filling the columns twice with bicine buffer. The drain ends of the columns are w iped with a tissue and the tip caps are replaced. The col­umns are counted in a well counter. For statistical values, the columns should be

RADIOIMMUNOASSAY FO R SERUM M YOGLOBIN 335

counted long enough to give counts of 10,000, but reasonable accuracy is ob­tained with counts of one minute.

Calculation

The counts per m inute rem aining in the p lain Sepharose colum n (colum n blank) are subtracted from the counts re­maining in each of the antibody columns. The net counts per minute rem aining in the standard columns and unknown col­umns are divided by the net counts re­maining in the blank antibody column (that with no unlabeled myoglobin) and the results expressed as percentage of the latter. When the column blank is small, its value may be ignored without intro­ducing much error.

The percentage values for the stand­ards are p lo tted agaist the m yoglobin concentration in ng per ml on log-logit paper. An example of a standard curve is given in figure 1. The myoglobin concen­tration of the unknowns is read from the standard curve.

A more detailed experim ental proce­dure may be found elsew here.17

Results

An t i g e n L a b e l in g

In figure 2 is shown the distribution of molecular weights of the labeled material when chromatographed on Biogel P-30. The hum p at the front of the curve proba­bly represents label bound to albumin, while the m ain peak is myoglobin. The m yoglobin was c a lcu la ted to have a specific activity of 76 and 60 microcuries per ju.g, respectively, for the two prepara­tions. In figure 3 is shown an autoradio­graph of the labeled myoglobin immuno- electrophoresced against an antiserum to human myoglobin and an antiserum to monkey myoglobin. In figure 4 is shown, by the small increase of small molecules over a period of more than two half-lives, the stability of the labeled myoglobin. The stability of the label was also shown by the fact that the binding of antibody decreased only in the same ratio as the activity over the same period. This stabil­ity is in contrast to the instability in the lite ra tu re8,16 for ch loram ine-T -labeled myoglobin.

F igure 1. Standard curve for radioim m une assay of myoglobin. The label bound in the pres­ence of a standard is plotted as a percentage of the label bound in the absence of a standard against nanograms of myoglobin on log-logit paper.

90

_______ I_______ I__________I_______ I__________I_______ I_______ I_10 20 40 100 200 500 1000 2000

NANOGRAMS PER MILLILITER

336 ADAMS, LAYMAN AND JAUNAKAIS

VO LU M E

F ig u r e 2. Fractionation of I 125 myoglobin on Bio-Gel P-30. The solid line is for a preparation with a specific activity of 60 microcuries per microgram. The dotted line is for a preparation with a specific activity of 76 microcuries per microgram.

M yoglobin was also labeled by the B o lton -H un ter m ethod in our own laboratories and by another.* These pre­parations also bound to the antibody and could be used in a radioimmune assay,

New England Nuclear, Boston, MA.

but the m inimal detectab le level was greater and the sensitivity was less.

Se l e c t io n o f An t is e r u m a n d R a t io o f A n t ib o d y -Se p h a r o s e t o S e p h a r o s e

An antiserum is selected on its ability to bind the label and also on the competition of the unlabeled protein with the label for the binding sites. In table I, it is shown that when the antisera to human myoglo­bin 220, 111, and 129 are attached to Sepharose, the label is bound but un­labeled myoglobin competes poorly with the label in the case of 220.

In table II is shown the binding of the label to several ratios o f an tibody- Sepharose to Sepharose for one of the antisera used in the preparation of this paper. The ratio giving about 30 percent binding with freshly labeled myoglobin was chosen.

S p e c ie s C r o s s R e a c t io n s

The antisera to the two species of myo­globin, the label on the myoglobin of the two species, and the inhibition by un­labeled m yoglobin of the two species were studied in all the possible combina­tions. In figure 5 it is shown that when the antibody to monkey myoglobin bound labeled human myoglobin, human myo­globin and monkey myoglobin competed equally. When the antibody to human myoglobin bound labeled monkey myo­globin, human and monkey myoglobin competed equally. However, when the antibody to monkey m yoglobin bound lab e led m onkey m yoglobin , hum an myoglobin was a poorer competer than monkey myoglobin. W hen the antibody to

ANTI HUMAN MYOGLOBIN

LABELED MYOGLOBIN

ANTI MONKEY MYOGLOBIN

F ig u r e 3. Autoradio­graph of 1 125 myoglobin. The labeled myoglobin was electrophoresced on agarose-poly-ethylene gly­col and diffused against antisera to human myo­globin and monkey myo­globin.

RADIOIMMUNOASSAY F O R SERUM MYOGLOBIN 3 3 7

human myoglobin bound labeled human m yoglobin, m onkey m yoglobin was a poorer competer than human myoglobin. It was also found that horse heart myoglo­bin and sperm whale myoglobin did not compete for e ither the human or monkey m yoglob in lev e l in e ith e r system . Likewise, ne ither hum an nor m onkey m yoglobin com peted with whale myo­globin in a system using antibody to whale myoglobin and labeled whale myoglobin.

R e a c t io n T i m e s : O p t im a l Vs . P r a c t ic a l

The binding of labeled myoglobin to an antibody-Sepharose colum n increases with time up to 16 hours. The competition of un labeled myoglobin increases with time up to four hours. This would suggest that an optimal sensitivity would be ob­tained w ith four-hour exposure to the standards and unknowns followed by a 16-hour exposure to the labeled myoglo­bin. Since a half-hour exposure to the un­labeled myoglobin and a half-hour expo­sure to the lab e led m yoglobin gives adequate sensitivity, these times were chosen for practical reasons.

R e c o v e r y o f A d d e d M y o g l o b in

Myoglobin was added to serum to give calculated levels of 866, 458, 117 and 65 ng per ml. D uplicate assays for these levels were 840 and 788, 460 and 395,120 and 122,78 and 78 ngper ml, respectively.

Once the principles of the m ethod were established, sera from two patients with diagnosed myocardial infarction were as­sayed. The values ranged from 334 to 1340 ng per ml. The serum with a level of 1340 ng per ml when diluted 1:5 was assayed as 250 ng per ml.

Sera from 11 patients with heart disease other than myocardial infarction were as­sayed for myoglobin. Three patients had values that would be considered abnor­mal. These three patients had heart en­largem en t, congestive h ea rt fa ilu re ,

(ODD)

VOLUME IN MILLILITERS

F ig u r e 4. Stability of labeled myoglobin. The increase in small molecules after two to three half-lives is shown by passing the label compound through Bio-Gel P-30.

pulmonary congestion and pneum onia. There was no evidence of acute myocar­d ia l infarction, although the patients had infarction in the past. The other eight pa­tients had values in the normal range.

S e r u m L e v e l s f o r N o r m a l Su b j e c t s a n d H o s p i t a l iz e d P a t ie n t s W it h o u t M y o c a r d ia l I n f a r c t io n

Sera taken at random from ten labora­tory personnel were assayed for myoglo­bin. The mean value found was 26 ± 26 ng

TABLE I

Competition of Human Myoglobin with I125 Labeled Human Myoglobin for Binding

Sites of Antibody to Human Myoglobin

Anti­serum

Ratio of Antibody- Sepharose to

Sepharoseng per ml

Human Myoglobin

Percentof

Blank

220 1:10 610 76.53129 1:4 625 36.82111 1:4 250 69.55

3 3 8 ADAMS, LAYMAN AND JAUNAKAIS

TABLE II

Binding of Label with Ratios of Antibody-Sepharose to Sepharose

RatioCounts per Minute Added to Column

Counts per Minute Remaining on Column After Two Washes

PercentLabel

Bound

1:20 35,670 21,030 601:50 26,120 9,210 351:100 34,280 8,050 25

per ml with a range of 0 to 69 ng per ml. The scatter is shown in figure 6. Ninety- six sera from patients diagnosed as not having myocardial infarction had a mean value of 32 ± 8 ng per ml with a range from0 to 99 nanograms per ml. The scatter for these sera is also shown in figure 6.

S e r u m M y o g l o b in L e v e l s f o r P a t ie n t s A d m it t e d f o r M y o c a r d ia l I n f a r c t io n o r t o R u l e O u t M y o c a r d ia l I n f a r c t io n

Sera from 45 patients who were adm it­ted to a coronary care unit for myocardial infarction or to rule out myocardial infarc­tion were assayed for m yoglobin. The

scatter of the values for the myocardial infarction is shown in figure 6 along with the normals and hosp ita lized w ithout myocardial infarction. N ineteen of the 24 patients diagnosed as having myocardial infarction had at least one elevated serum myoglobin. For two of the five patients without elevated serum myoglobin, the serum enzymes (total CPK, SGOT, LDH) were not elevated, the urine myoglobin was negative and the ECG was question­able; however, the diagnosis was myocar­dial infarction.

For the other three patients without elevated serum myoglobin, it is likely that the critical sera were not available. Values as high as 1120 ng per ml were found with most being in the 300 to 500 ng per ml range. W hen the tim e of myocardial in­farction could be established by an objec­tive sign such as onset of chest pain, it was shown that the serum myoglobin level rose in the first six hours, peaked in about 12 hours and fell to normal w ithin 24 hours. In some cases the serum myoglobin level rem ained elevated, indicating con­tinued damage to the heart. In other cases

50

40

54

3 -

ANTIBODY LABEL INH IB ITO R

(1) M O N KEY M O N KEY M O N K E Y

(2) M O N KEY M O N KEY H U M A N

(3) M O N K E Y H U M A N M O NKEY

14) M O N K E Y H U M A N H U M A N

(5) H U M A N H U M A N M O N KEY

|6) H U M A N H U M A N H U M A N

(7) H U M A N M O N KEY M O N KEY

(8| H U M A N M O N K E Y M O NKEY

F ig u r e 5. E ffec t of species used for immuno­gen, label and antigen.

RADIOIMMUNOASSAY FO R SERUM M YOGLOBIN 3 3 9

the level peaked, fell to normal and then later rose again, probably indicating a new attack. Sometimes, there were elevated levels of myoglobin in the serum drawn on admission. This suggested that the in­farction had occurred some time earlier and was generally confirmed by the clini­cal history.

N ineteen of the 21 patients diagnosed as not having myocardial infarction had no elevated serum myoglobin level. Two of the 21 patients had at least one elevated myoglobin level. These two patients also had urinary myoglobin.

C o r r e l a t io n o f S e r u m a n d U r in a r y M y o g l o b in

For 15 of the patients with diagnosed myocardial infarction, it was judged that both serum and urine had been collected at the critical times. The sera was assayed by radioimmune assay and the urines by hemagglutination inhibition. Fourteen of the patients had myoglobin both in serum and urine. One had myoglobin in serum but not in the urine. The sensitivities of the two m ethods are d ifferen t, being about 20 to 40 ng per ml for the radio- immune assay and 0.5 to 1 /xg per ml for the hemagglutination inhibition.

Discussion

N o r m a l a n d A b n o r m a l L e v e l s

Jutzy et al8 reported that none of their subjects w ithout myocardial infarction had serum myoglobin levels higher than 88 ng per ml and that the lowest level from a myocardial infarction patient was 130 ng per ml. An arbitrary level of 100 ng per ml was chosen as the divider betw een normal and abnormal. Serum myoglobin eleva­tion was reported as early as 30 minutes after the infarct. The highest values were found in the first 12 hours and the myo­globin largely disappeared from the serum w ithin 24 hours. These observations are in agreem ent with those of the present

SUBJECTS

F ig u r e 6 . M yoglobin levels for normals, hospitalized patients and patients with myocardial infarct. The solid lines give the means for the first two groups and the points are the values of individual sera for all groups.

authors concerning m yoglobinuria fol­lowing myocardial infarction. Most of the values of Jutzy et a l8 were in the range of 200 to 500 ng per ml although one was 6000 ng per ml.

Stone et al16 reported that levels from normal subjects were all lower than 85 ng per ml, while the mean value when the se ra w ere d raw n from p a tie n ts w ith myocardial infarction w ithin 12 hours after admission was 380 ng per ml. Two of the 20 patients with myocardial infarction did not have myoglobin levels above the

340 ADAMS, LAYMAN AND JAUNAKAIS

Situations Associated with Myoglobinémie States

TABLE I I I

I. TRAUMA AND ISCHEMIC DISEASE Trauma-crush syndrome Arterial ischemia

Extremities Myocardial infarction Pressure necrosis-coma

Surgical procedures Cardiac Orthopedic Vascular

II. EXERTIONAL STATESExertion in otherwise normal individuals Convulsive disorders

III. METABOLIC FACTORSAlcoholic myopathy Anesthetic-associated

Syndromes-malignant Hyperthermia

Defects in carbohydrate metabolism McArdle's disease Phosphofructokinase deficiency Syndrome of abnormal glycosis Defect in lipid metabolism Hypokalemia and potassium depletion states Toxic factors

Heroin user's myoglobinuria Animal venoms Quail eater's disease Haff disease Miscellaneous

IV. MYOSITIS SYNDROMES Dermat ornyo s it i s PolymyositisSystemic lupus erythematosus

V. OTHER FACTORS Infection Tetanus Fever

VI. IDIOPATHIC MYOGLOBINURIAS AND FAMILIAL MYOPATHIES

normal. The chest pains in these two pa­tients had occurred 40 and 23 hours before admission. In the present studies in a series of 10 normal subjects, the values ranged from 0 to 69 ng per ml with a mean value of 26 and a standard deviation of 26. No attempt was made to investigate the activities of the normal subjects. Based on this, the levels for the hospitalized pa­tients as well as the other two studies, the adoption of the 100 ng per ml level by Jutzy et al8 as the dividing line seems rea­sonable. In our first study, in the case of the one patient with myocardial infarction for whom multiple sera were available, the values ranged from 334 to 1340 ng per ml. Sera from other patients with myocar­

dial infarction ranged up to 1400 ng per ml.

Se n s it iv it y

The sensitivity or rather the minimal detectable level in the column m ethod as determ ined from the 90 percent level on the log-logit plot appears to be about 4 ng or 40 ng per ml. Jutzy et al8 claim a sensi­tivity of 1 ng per ml for their method. Stone et al16 claim they can detect as little as 0.5 ng myoglobin. Since 0.02 ml serum is being used in their assays, their claimed sensitivity is 25 ng per ml. The Nuclear Medical System Inc. claims for its kit a sensitivity of 0.5 ng or 5 ng per ml. Inspec­tion of its standard curve would suggest a sensitivity or minimal detection level of 25 ng per ml.

In all cases, the sensitivities are defined from standards developed in the ind i­vidual laboratories and not by an outside reference laboratory. If the 100 ng per ml level is accepted as the dividing line, the claim ed extreme sensitivity of these other investigators is not required and the m in­imal detectable level of 40 ng per ml for the column m ethod is acceptable. The column m ethod can cover the range up to and past 2500 ng per ml without dilution.

U t i l i t y

It would be expected that myoglobine- mia would be found in the conditions that result in myoglobinuria. These are listed in table III. Probably a major use of the radioimmunoassay for serum myoglobin would be for patients suspected of having myocardial infarction. With proper tim ing of the blood collection, it would be useful in confirming the diagnosis and possibly in estim ating the extent of myocardial damage. It should be emphasized, how­ever, that the assay is one for serum myo­globin and that conditions o ther than myocardial infarction can result in eleva­tion of the level.

RADIOIMMUNOASSAY FO R SERUM MYOGLOBIN 341

Values obtained for serial sera indicate that myoglobin is released into the serum in a few hours after myocardial infarction and then rapidly d isappears from the serum . C ontinuing elevation of serum m yoglobin probably means continuing myocardial damage while new peaks in levels indicate new attacks. The rise in serum myoglobin following myocardial infarction is at least as early as that of serum CPK, but myoglobin disappears from the serum earlier than CPK and the other enzymes. It is thus important to have the sera at the proper tim e for these studies.

References

1. Adams, E. C.: Differentiation of myoglobin and hemoglobin in biological fluids. Ann. Clin. Lab. Sci. 1 :208-221, 1971.

2. Adams, E. C. and E lliott , T. A.: Urinary myoglobin in myocardial infarction. J. Amer. Med. Assoc. 221:1013-1014, 1970.

3. Campbell, D. H., Garvey, J. S., Crem er , N.E., and Sussdorf , D. H .: Methods in Im­munology. New York, W. A. Benjamin, Inc., 1963, pp. 118-120.

4. CUATRECASAS, P.: Protein purification by affin­ity chromatography. Derivations of agarose and polyacrylam ide beads. J. Biol. Chem. 245:3059-3065, 19707

5. D avis, J. W., Yoder , J. M., and Adams, E. C.: Rapid, column flow-through radioimmunoas­say for insulin. Clin. Chim. Acta 66:379-386,1976.

6. G ree n w o o d , F. C., H unter , W. M., and G lover , J. S.: The preparation of I131-labeled

human growth hormone of high specific radioactivity. Biochem. J. 89:114-123, 1963.

7. J ohnson , P. K., Yod er , J. M., and Adams, E. C.: A rapid, flow-through radioimmunoassay for human chorionic somatomammotropin. Amer. J. Obst. Gyn. 225:45-50, 1976.

8. Jutzy , R. V., Nevatt, G. W., Palmer, F. J., and Nelson , J. C.: Radioimmunoassay of serum myoglobin in acute myocardial infarction. Amer. J. Cardiol. 35:147, 1975.

9. KaGEN, L. J.: Myoglobin. New York, Columbia University Press, 1973, p. 87.

10. KaGEN, L. J. and G u re v ic h , R.: Precipitin reac­tions of anti-human Myoglobin serum with sev­eral human and animal muscle extracts. Im­munology 12:667-673, 1967.

11. Kagen, L. J., Sc h ed id t , S., and Roberts, L.: Myoglobinemia following acute myocardial in­farction. Amer. J. Med. 58:177-182, 1975.

12. K e s s l e r , H. A., L i e b s o n , P. R., M a t - TENHEIMER, H., and ADAMS, E. C.: Acute myocardial infarction diagnosed by myo­globinuria. Arch. Intern. Med. i35:1181-1183,1975.

13. Levine , R. S., Alterm an , U., G ubner , R. S., and Adams, E. C.: Myoglobinuria in myocardial infarction. Amer. J. Med. Science 262:179-183, 1971.

14. LUGINBUHL, W. H.: Immunologic investigation of preparations of human myoglobin. Amer. J. Clin. Path. 38:487-493, 1962.

15. R o s a n a , T. G. and KENNY, M. A.: A radio­immunoassay for human serum myoglobin. Method development and normal values. Clin. Chem. 23:69-75, 1977.

16. St o n e , N. J., W il l e r s o n , J. T., G o m e z - Sanchez, C. E., and Waterman, M. R.: Radio­immunoassay of myoglobin in human serum. Results in patients with acute myocardial in­farction. J. Clin. Invest. 56:1334-1339, 1975.

17. Sunderman, F. W.: Manual of Procedures for the Seminar on Proteins and Proteinopathies. Philadelphia, Institute for Clinical Science,1977, pp. 111-134.