223

Clinica Chimica Acta, 58 ( 1 9 7 5 ) 2 2 3 - - 2 3 2 © Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

CCA 6 7 2 3

QUANTITATION OF CREATINE KINASE ISOENZYMES IN HUMAN TISSUES AND SERA BV AN IMMUNOLOGICAL METHOD

E V A N G E L I A J O C K E R S - W R E T O U * and G E R H A R D P F L E I D E R E R

Lehrstuhl fiir Biochemie, Abteilung Chemie, Ruhr-[hliversitdt Bochum, .1630 Bochum, Postfach 2148 (G.F.R) ( R e c e i v e d J u l y 5, 1 9 7 4 )

Summary Antisera against the crystallized creatine kinase isoenzymes from human skeletal muscle (MM) and from human brain (BB) were produced in rabbits. Both the MM and BB isoenzymes were precipitated quantitatively by their homologous antisera. No cross-reaction was observed. The hybrid MB from human heart muscle could not be Drecipitated completely by either of the twc antisera. In artificial mixtures the concentrations of individual creatine kinP..,e isoenzymes were dete~-*nined from the percentage of non-precipitable activity in the supernatant afte~ reac!ion with each of the antisera. This immunotitration assay was applied to study the quantitative distribution of creatine kinase isoenzymes in extracts of human tissues. The iscenzyme patterns obtained were compared with those determined by electrophoretic analysis. In sera of patients with myocardial infarction, the immunotitration assay allowed the sensitive and rapid quantitation of creatine kinase isoef_,zymes, especially of the "infarct-specific" hybrid MB, even in sera with low total activity. This indicates that the method is of diagnos'Ac value.

Introduction

In 1964 it was demonstrated that human tissues contain thre~ forms of creatine kinase (CK, ATP:creatine phosphotransferase, EC 2.7.3.2) [1]. These consist of two pure isoenzymes, CK-MM and CK-BB, and a hybrid form CK-MB resulting from the combination of the monomers M and B [2]. The BB or "brain t y p e " isoenzyme has an electrophore~ic mobility similar to albumin, whereas the MM or "muscle t y p e " isoenzy'ne behaves, during electropho~esis,

* The results presented here are part of a doctoral thesis at the Ruhr. Universit/it Boehum.

224 like ~-globulins. The hybrid MB exhibits an electrophoretical mobility similar. to a2 - or fl-globulins. There are many reports describing chromatographic [3,4] and electrophoretic techniques [5--9] for the separation and characterisation of CK isoenzymes. Althougn some of these techniques have been widely used in clinical medicine, they are not sensitive enough to quantitate CK isoenzyme patterns in sera and biological fluids with low enzyme activities. Methods utilizing differences in the kinetic properties of the isoenzymes and not requiring timeconsuming electrophoresis have also been proposed for the detection of CK isoenzymes [4,10]. However, they have not yet been used fgr quantitative estimations. The present paper describes a new method for the quautitation of CK isoenzymes based on their different immunological properties. The use of specific antisera against the pure CK-MM and CK-BB isoenzymes from human tissue allows the quantitation of the three isoenzymes in tissue extracts and sera. The assay is very sensitive and gives reproducible results. Similar methods have been reported from this department for isoenzymes of aldolase [11], hexokinase [ 12] and lactate dehydrogenase [ 13]. Expenment~ Creatine kinase activity was measured with an Eppendorf filter photometer at 366 nm in a thermocuvette at 25°C according to Oliver [14] using "Monotest CK-activated" from Boehringer Mannheim. Enzyme activity on cellulose acetate membranes and agarose plates was localized by staining according to Goto and Katsuki [ 5]. CK-MM from human muscle and CK-BB from human brain were purified by a modification of the method of Keutel et al. [15]. The maximum specific activities obtained for the crystallized enzymes were 360 U/mg and 290 U/mg for the muscle and brain e.uzyme, respectively. Protein was determined by a modified biuret method [16]. The purity of the preparations was checked by disc electrophoresis. The hybrid CK-MB extracted from human heart tissue was separated from the MM and BB isoenzymes by column chromatography on DEAE-Sephadex A-50 (Pharmacia, Uppsala). Electrophoresis was performed on cellulose acetate membranes (Macherey, Nagel and Co.) in 0.03 M verona] buffeL" pH 8.6 at ~00 V fo_r one hour. Samples of 1-pl were separated onto several membranes simultaneously. The positions of the CK isoenzyme bands were determined by enzyme-specific staining of one of the membranes. The o~her membranes were cut accordingly and the isoenzymes eluted. The activity of the eluate was t e s ~ d immediately because the hybrid MB is known to be unstable in diluted solutions.

Preparation of antisera Antibodies to the purified CK-MM and CK-BB isoenzymes were prepared in rabbits. Primary injection of 4 mg of the pure enzymes in complete Freund's adjuvant into the hind foot pads was followed by two injections of ~he same dose at two weeks interval given intradermally and subcutaneously. The ani-

225 mals were bled from the ear vein 6--7 days after an intravenous boost with 4 mg of the enzyme in saline. The antisera against the muscle and the brain isoenzyme (desi~.ated anti-CK-MM and anti-CK-BB) were pooled. They were kept at 60°C for 5 min to inactivate the CK activity in the rabbit serum. A two-fold concentration of the antisera was obtained by filtration through a selectron ultramembrane (Schleicher and Schbll, Dassel, G.F.R.). lmmunoinhibition

a n d i m m u n o t i t r a t i o n assays

Samples of 100 pl of each isoenzyme containing 296 mU CK-MM, 91 mU CK-MB and 96 mU CK-BB, respectively, were mixed with increasing volumes of the antiserum being studied (0.01--0.2 n,1, as can he seen from the legends to Figs 3 and 4). Tris/HC1 buffer 0.05 M {pH 8.00), containing 0.001 M EDTA and 0.02 M 2-mercaptoethanol (for enzyme stabilization) was added if necessary to keep the volume constanL (final volume 0.3 ml). The samples were incubated for 30 min at room temperature and 30 min at 4°C. A similar set was incubated as a control with normal rabbit serum treated like the antisera. Residual activity in the reaction mixture after incubation was expressed as a percentage of control activity and referred to as immunoinhibition, lmmunotitration was given as percentage of control activity after incubation and centrifugation (Eppendorf centrifuge 3200, 4 min). I m r n u n o t i t r a t i o n assay in tissue e x t r a c t s a n d sera

Extracts were prepared from frozen tissue specimens (--20 ° C) obtained by autopsy 12--24 h after death. They were first homogenized in an Ultra-Turrax (Ikawerk, Staufen, G.F.R.) and then in a Potter-Elvejhem apparatus with Tris/ HC1 buffer, 0.05 M (pH 8.00), containing 0.01 M KCL, 0.001 M EDTA and 0.02 M 2-mercaptoeth~riol (! : 2 or 1 : 3, w/v). The homogenates were centrifuged for 20 min at 40 060 × g (Sor~all RC2B). Aliquots of 50 pl of the supernatant or an appropriate dilution c(~ntaining about 50 m U C K activity were incubated for 30 min at room temperat:are and 30 min at 4°C in parallel with: (a) 50 pl buffer (homogenization buffer without KCI), (b) 50 pl normal rabbit serum, (c) 50 pl anti-CK-MM serum, (d) 50 pl anti-CK-BB serum, and (e) 50 pl of a mixture of both antisera. The residual activity was tested in the supernatants after centrifugation. The activity of the sample incubated with buffer (a) did not differ from the control (b) proving that normal serum did not affect the CK activity. Sample(e) was used as a check and should not contain activity after centrifugation. S~ra obtained from the Bergmannsheil hospital, Bochum, were analyzed a few hours after b]ood collection. The creatine kinase was activated by incubating the sample for 30--60 min with 2-mercaptoethanol (final concentration 0.02 M). The immunotitration assay was performed with aliquots containing up to 50 m U C K activity as described for the tissue extracts. Results

Antigenic differences between creatine kinase MM and creatine kinase BB

226

C :?

- ..

Fig. 1. D e m o n s t r a t i o n o f tile i m m u n o l o g i c a l i n d e p e n d e n c e o f C K - M M and CK-BB by t h e double-dlffusiora t e c h n i q u e in 1% a g a r o s e gel. (1) C K - B B , ( 2 ) C K - M M , ( 3 ) a n t i o C K - B B - s e r u m a n d ( 4 ) a n t i - C K - M M - s e r u m . T h e u n d i s t u r b e d c r o s s i n g o f t h e p r e c i p i t a t i o n lines i n d i c a t e s t h a t t h e r e is n o s e r o l o g i c a l r e l a t i o n s h i p b e t w e e n the t w o i s o e n z y m e s . Fig. 2. D e m o n s t r a t i o n o f t h e i m m u n o l o g i c a l r e l a t i o n s h i p b e t w e e n t h e h y b r i d C K - M B ( 1 ) a n d t h e pure i ¢ o e n z y m c s C K - M M ( 3 ) a n d C K - B B (5). Wells ( 2 ) a n d ( 4 ) c o n t a i n t h e c o r r e s p o n d i n g a n t i s e r a a n t i - C K - M M (2) a n d a n t i - C K - B B (4). W h e r e a s t h e s p u r f o r m a t i o n b e t w e e n C K - M B a n d C K - M M is well p r o n o u n c e d the s p u r b e t w e e n C K - M B a n d C K - B B is v e r y f a i n t .

from chicken and rabbit have been found by complement fixation and Ouchterlony techniques [17,18]. In agreement with these findings, antibodies against the crystallized human muscle CK-MM do not crossreact with the human brain CK-BB and vice versa (Fig. 1). The hybrid enzyme CK-MB reacts with both antisera as shown in Fig. 2. Antigen-binding titres (units of CK bound per ml of antiserum) were calculated from the maximum of the precipitin curve performed according to the Heidelberger technique as modified by Schultze and Schwick [19]. A titer of 54.2 units (150/tg) CK-MM per ml anti-CK-MM-serum and 29.2 units (100 pg) CK-BB per ml anti-CK-BB-serum was obtained. Figs 3 and 4 demonstrate the reaction of anti-CK-MM and anti-CK-BB serum with the three creatine kinase isoenzymes. As can be seen from Fig. 3a, anti-CK-MM ~ntibodies inhibited the muscle enzyme completely, which is in agreement with earlier reports for the rabbit muscle enzynJe [ 13,20]. They did not affect the activity of the brain enzyme. The activity of the hybrid enzyme MB, however, was inhibited by 80%. Af*.er centrifugation 10% of the activity remained in the supernatant (Fig. 3b). In contrast to the reaction of CK-MM with anti-CK-MM serum, the BB isoenzyme was not completely inhibited by its homologous antiserum but the residual activity could be removed by centrifugatimi. The MM isoenzyme was not affected by the anti-CK-BB-serum (Fig. 4a). The inhibition of the hybrid enzyme MB ~vith anti-CK-BB antibodies was similar to that obtained with the anti-CK-MM-serum (Fig. 4b). Thus, each of the pure isoenzymes could be removed completely by precipitation with its homologous antiserum, whereas the hybrid enzyme MB could not be totally precipitated by either of the

227 ~IOO .~_ -~ e o

=MM

>. 1 0 0 - e ) - i e - ~ "~ Q "~ 8 0 (J

0

60

"~

~ 2o

40 20' --o--

t

O.O5 ----~ ml

onti

0.1 - CX- MM -

0.2

0.05

serum

-

--~-

~BB

..............

O.1 cnti-CK-BB

mi

.~,oo

o.e2 serum

IO0

8o

'~ o

o

~

"o

80

60

'~ 40 a: ~ 2O

llg

~--~__

o

t

-,o-

0.05 0.1 ----i,. rn I ( 3 n t i - C K - M M - s e r u m

0.2

t

t

0.05 mt

•

•

O.i onti-CK-BB

0.2 serum

Fig. 3. R e a c t i o n o f t h e a n t i - C K - M M - s e m m w i t h (a) the c r e a t i n e k i n a s c i s o e n z y m e s C K - M M ( 2 9 6 m U ) and C K - B B ( 9 4 m U ) , a n d ( b ) w i t h t h e h y b r i d C K - M B ( 9 1 m U ) . l m m u n o i n h i b i t i o n and i m m u n o t i t r a t i o n a s s a y s w e r e d o n e as d e s c r i b e d in t h e e x p e r i m e n t a l s e c t i o n . T h e r e s i d u a l a c t i v i t y is e x p r e s s e d as p e r c e n t a g e o f control after incubation (immunoinhibition assay, o . . . . . . o), a f t e r i n c u b a t i o n and c e n t r i f u g a t i o n (immunotitration assay. • "~). Fig. 4. R e a c t i o n o f t h e a n t i - C K - B B - s e r u m w i t h ( a ) t h e c r e a t i n e k i n a s e i s o e n z y m e C K - M M ( 2 9 6 m U and CKoBB ( 9 4 m U ) , a n d (b) w i t h t h e h y b r i d C K - M B (91 m U ) . I m m u n o i n h i b i t i o n and i m m u n o t i t r a t i o n a s s a y s w e r e d o n e as d e s c r i b e d in t h e e x p e r i m e n t a l s e c t i o n . T h e residual a c t i v i t y i~ e x p l ' e s s e d as p e r c e n t a g e o f control after incubation (im-nunolrhibition assay, c). . . . . . c ) , a f t e r i n c u b a t i o n and c c n t r i f u g a t i o n (immunotitration assay. • 4)).

antisera. About 1C% residual activity always remained in the supernatant after centrifugation even with a large excess of the antiserum present. In both cases the remaining activity (10%) could also be precipitated when the complementary antiserum was added to the supernatant. This finding could be explained by assuming that part of the hybrid enzyme dissociates during antibody reaction. The resulting free subunits heterologous to the added antiserum could massociate and remain in the supernatant. After addition of the corresponding antiserum they would be precipitated and thus removed from the reaction mixture. Additional evidence for this assumption was provided by electrophoretical analysis of an incubation mixture of MB enzyme with anti-CK-MM serum after centrifugation. Enzymatic activity was localized in the position of the BB isoenzyme (unpublished observations). On the basis of these results it was possible to apply the immunotitration method to the quantitation of creatine kinase isoenzymes in any given mixture. Samples with an appropriate CK activity were incubated with anti-CK-MM and anti-CK-BB serum, respectively, and centrifuged. From the percentages of residual activities in the supernatants the concentrations of the three isoenzymes were calculated using the following

228 three equations: a=%

residual activity measured after reaction with anti-CK-MM = % BB + 10% of the total MB activity.

(1)

b = % residual activity measured after reaction with anti-CK-BB = % MM + 10% of the total MB activity.

(2) (3)

% MM + % M B + % B B = 1 0 0 % .

Thus M ~ could be calculated using the formula: % M B = ( 1 G 0 - - a - 5)/0.8 and M M and B B according to Equation 1 arid 2. Analysis of defined artificial mixtures of the three isoenzymes proved the reliability of this m e t h o d (Table I). The results obtained in the assays agree well with the theoretical isoenzyme c o m p o s i t i o n o f the samples. Creatine kinase isoenzyme patterns in human tissues The CK isoenzyme patterns obtained by i m m u n o t i t r a t i o n were c o m p a r e d with the patterns f o u n d after electrophoresis on cellulose acetate m e m b r a n e s (Table II). The concentration and isoeilzyme c o m p o s i t i o n of CK in tissue extracts is shown in Table lII. For eacb tissue several samples were tested. Each specimen was assayed in duplicate. The values given in Table III indicate the ranges found for the various tissues. When the total activity was higher than 2 U/g tissue an electrophoretic isoenzyme quantitation was carried out. The variation o f the total CK activity o f tissue specimens, d e m o n s t r a t e d in the first c o l u m n of Table Ill, could be due either to normal variability or to the influence of autolytic processes, although only those samples were used which had been taken within 24 hours after death. Particularly in heart tissue, which contains considerable a m o u n t s of the rather unstable hybrid e n z y m e MB, a broad variation in the percentage of this isoenzyme can be observed. Infant cardiac muscle always contained lower activities of the MB e n z y m e c o m p a r e d to adult heart muscle. When heart extracts were assayed by i m m u n o t i t r a t i o n it was observed that a relatively small fraction of the activity (5--15%) could n o t be precipitated ~ven by a mixture of the two antisera. This was due to a particulate CK-enzyme known to migrate to the c a t h o d e during electrophoresis [2! ]. This additional e n z y m e with CK-activity neither reacts with anti-CK-MM nor with anti-CK-BB TABLE

I

QUANTITATION

OF CK ISOENZYMES

IN A R T I F I C I A L

":,XTURES

BY IMMUNOTITRATION

A. theoretical: B. experimentally obtained isoenzyme composition in %.

]so-

A

B

A

B

A

B

A

B

A

B

60 20 20

62 19 19

20 20 60

21 21 58

60 3~ 10

61 24 15

10 30 60

11 34 55

60 35 5

58 34 8

enzyme

MM MB BB

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229 T A B L E II Q U A N T I T A T I V E D I S T R I B U T I O N O F CK I S O E N Z Y M E S IN S E L E C T E D H U M A N T I S S U E D E T E R M I N E D ( A ) BY T H E E L E C T R O P H O R E T I C A N D (B) BY T H E I M M U N C ) L O G I C A L M E T H O D Tissue

A

Heart Uterus Prostate Thyroid

Diaphragm

B

%MM

%MB

ObBB

%MM

%MB

%BB

72 15 33 78 96

25 24 7 6 2

3 61 60 16 2

71 16 34 79 96

26 20 6 6 2

3 64 60 15 2

zorum under the experimental conoitions used. Small quantities of this particulate enzyme were also found in brain, lung, intestine and thyroid. Skeletal muscle contains ~nly a small proportion of CK.MB and traces of CK-BB. Somewhat higher concentrations of these isoenzymes are found in diaphragm and tongue. Smooth muscles such as bladder and stomach exhibit predominantly the BB isoenzyme and a low content of CK-MM and CK-MB. T A B L E 111 CONCENTRATION .

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A N D I S O E N Z Y M E P A T T E R N S O F CK I N H U M A N T I S S U E S .

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Tissue

Skeletal muscle Tongue

Diaphragm Heart (adult) Heart (infant) Aorta (arcusaortae) Kidney Spleen Thyroid

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MM %

MB %

860-.-1310 225-- 292 140 10(~- 2 8 0 78-- 250 3-7 01 O-1 13 .... 34

9(i--100 90-- 99 96 71 .. 9 6 96--100 81-- 88 70--100 65-- 75 6 4 - - 79

0-- 3 1-- 5 4 4--27 0- 4 8--14 0 0 6

O- 1 0-- 5 2 O-- 2 0 3--- 5 0--30 2,rr - 35 I5---30

35-27-56 39 50 34 -~ 5-21 -~ 11-2-3 0 0 0 0

if-H) 0--- 4 2 7 0 2-- :; 2-.-20 5-- 9 7-- 9 3-- 5 2-- 6 0 0 0 0

25--40 18--69 42 54 50 5:,-60 64--93 66--- 73 78--80 89 -93 91--95 100 100 1 O0 100

Adrenal

0---

Lung

2--

Caxotis Artery (aorta ascendens) Liver* Prostate Uterus Pancreas Intestine (mesentery) Bladder Stomach Hypophysis Spinal cord Cerebellum Cerebrum

.

T o t a l CK activity iU/g frozen t,ssue~

2 i 0-73--O-5-14-15-11 23-50-55--

1 9

1 10 9 I 6 35 23 27 87 90

D~Jtribution o f CK i s o c n z y m c s

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Referc, n c e

..........................................

C0 72

39 16 29 13 7

* F r o m 6 specimens assayed only one s h o w e d C K activity.

BB "~ MM, MM, MM, MM, MM.

MB, idB MB MB MB

BB 16l 161 [6] [6, I 0 , 2 2 ] [ 10]

BB [6, 10~ 2 2 l BID [ 2 2 1 BB 16, 10. 22} MM, MB, BB J 2 5 l BB [ 2 2 ] BB [6, 221

B B [22] B B t6) B B [-~I B B [22l BB, M B [22] BB {6, I'0,22] BB [22, 6[

BB [ 1 0 ] BB [6, 10, 22]

230

Although the uterus belongs to the smooth muscle type a considerable percentage of CK-MB was d e ~ c t e d in some specimens. Lung and adrenal can be considered to be the tissues with the greatest variability in CK isoenzyme composition. Three of the 9 lung specimens tested contained the muscle isoenzyme predominantly (up to 72%), three the brain type (up to 69%) and three equal proportions of both isoenzymes. In addition it was found that extracts from different parts o f the same lung exhibited different CK isoenzyme patterns. Creatine kinase i s o e n z y m e patterns in sera Three serum samples from healthy persons, two sera from patients with diseases associated with elevated CK activity and 15 sera from patients with myocardial infarction were assayed for CK isoenzyme composition by the immunotitration method (Table IV). Sera from healthy subjects contained the CK-MM isoenzyme only. Small quantities of CK-BB were found in sera with very high CK activity, e.g. in the serum from a patient with third degree burns and in a case of myocardial infarction. CK-MB was present in all the sera from patients with myocardial infarction. The highest proportion o f CK-MB found was 35%; the lowest, on the third day after myocardial injury, was 3%. Timing is a critical factor in the detection o f the CK-MB-enzyme, which is known to disappear from the serum within two days after the infarction. However, it was still detectable 3--4 days after infarction even when the total CK activity had decreased to values below the normal range of 50 U/1. T A B L E IV CK I S O E N Z Y M E P A T T E R N S IN S E R A

Case No.

N o r m a l sera

Into ×ication 3 rd d e g r e e burns Myocardial infarction

1 2 3

I 2 3(1)* 3 (2) 3 (3) 4 (]) 4 (4) 5 (l) 5 (3) 6 7 8 9 lO 11

CK activity (U/L)

D~tributionofCKisoenzymes

11 " 30 214 8580 139 4O 82"" 416 382 314 47 227 44 169 227 136 118 1050 57

100 100 100 100 B9 85 88 ~ 84 86 80 90 73 97 80 87 90 87 81 82

,MM

%MB

%BB

0 0 0 0 0 15 12 35 16 14 20 lO 27 3 20 13 10 13 16 18

0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0

N u m b e r s in p a r e n t h e s i s i n d i c a t e the h o s p i t a l d a y . ** This s e r u m wa~ lef~ at r o o m t e m p e r a t u r e o v e r n i g h t . T h e or:ginal a c t i v i t y is u n k n o w n .

231

Discussion Electrophoretic studies on the distribution of human creatine kinase isoenzymes in various tissues have been carried out by several investigators [6,10,22]. However, the isoenzyme patterns presented were always determined visually. Quantitative estimations have been reported in the case of fetal muscle only [23]. The distribution of CK isoenzymes in human tissue studied by the immunotitration assay, as described in this paper, differed considerably from those reported previously although the total CK concentrations found were in the same range [6,24]. In earlier papers a very simple distribution of CK isoenzymes in human tissue was suggested. According to these reports, CK-MM and CK-MB occur in skeletal and cardiac muscle only. All the other tissues should contain the CK-BB isoenzyme exclusively. In the present study, however. we could demonstrate that CK isoenzyme patterns are much more complicated. As shown in Table II[, cerebral tissue was the only type which contained one single isoenzyme, the BB enzyme. All the other tissues tested showed two or three isoenzymes, although sometimes in very low concentrations. Conflicting results have been reported in the case of the thyroid gland. Whereas Smith [6] found the CK-BB isoenzyme predominating, Graig [25] demonstrated on starch gel the existence of three isoenzymes in this tissue. Our results showing up to 79% MM, 6% MB and 15--30% BB confirm the pattern suggested by Graig. Previous papers reporting that kidney and spleen contain CK-BB exclusively could not be verified. The muscle type MM is the predominant type of these tissues. Differences between this work and earlier studies presumably depend on the higher sensitivity of the immunochemical technique. Electropt~oretic methods are not sensitive enough to demonstrate the small fractions of CK-MB present in most non-nervous tissue. In addition, the electmphoretic estimation of CK-MM is complicated by the fact that adenylate kinase and other enzymes also react with the staining solution. Therefore, true CK-MM can only be estimated by electrophoresis if the enzyme bands are eluted and tested for activity. The application of this procedure however is restricted to tissue extracts with high total CK activity. Serum CK-MB isoenzyme is established as the most specific and sensitive indicator for acute myocardial ~nfarction [26,27]. Therefore, immunotitration in pathological sera was expected to give a sensitive and rapid assay for this isoenzyme. Electrophoretic techniques allow the demonstration of CK isoenzymes in serum only when the total CK activity is ;ncreased. Quantitation of CK-MB is possible only when the total activity is greater than twice the upper limit of the normal range, even using the most sensitive fluorometric detection method [9]. As shown in Table IV it is possible, by means of the immunotitration method, to detect and quantitate CK-MB in sera even when the total CK activity is not elevated or has already decreased 3--4 days "~ter myocardial infarction. These results differ from previous papers which reported that CK-MB is absent from the serum 48 hours after myocardial injury [81". * N o t e a d d e d i n p r o o f : A p a p e r c o n t a i n i n g additior.~,,l a n a l y t i c a l d a t a a n d c a l c u l a t i o n s c o n c e r n i n g t h e c l u a n t i t a t i o n o f t h e h y b r i d C K - M B in i n f a r c t s e r a is i n p r e p a r a t i o n .

232 The immunotitration technique for the quantitation of CK isoenzymes reveals many advantages as compared to other methods, especially by its high specificity and sensitivity. It is possible by this method to quantitate CK isoenzyme patterns even at low levels of total CK activity. Furthermore, the method is less laborious and time-consuming than electrophoretic analysis. It can be used as a routine method in clinical laboratories and might be o f great value for the diagnosis of myocardial infarction and other clinical investiga. tions. Acknowledgements We are grateful to Dr E. BtShm and Dr H. Baumann from Bergmannsheil Hospital Bochum for obtaining autopsy tissue and pathological sera. Wc :~';sh to acknowledge the technical assistance of Mr K. Grabert and the aid o{ Dr F. Falkenberg and of Dr D.A. Fitzpatrick in the preparation of the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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