Radioimmunoassay assay and reagents for MB isoenzyme of CK

Methods and materials for quantitative detection of MB and BB isoenzymes of creatine kinase by radioimmunoassay competitive displacement techniques involving antibodies to human BB isoenzyme which are specific for the B monomer, react with BB CK and cross-react with MB CK, but do not cross-react with MM CK. An acylating agent is employed to radioactively label purified CK isoenzymes used as antigens. Incubation is preferably carried out in a Tris buffer having a pH of about 7.4, in the presence of a suitable organic reducing agent such as mercaptoethanol.

BACKGROUND OF THE INVENTION 
The present invention relates generally to analyses for enzymatic 
substances and, more specifically, to methods and materials for the 
quantitative detection of BB and MB isoenzymes of creatine kinase by 
radioimmunoassay techniques. 
Enzymes are proteinaceous substances produced by living cells. They 
function to bring about or accelerate chemical reactions in an organism 
without themselves undergoing marked chemical alteration in the process. 
One such enzyme, called creatine kinase, catalyzes an energy transfer 
(transphosphorylation) reaction, i.e., creatine+adenosine 
triphosphate.revreaction.creatine phosphate+adenosine diphosphate. 
Creatine kinase enzyme is a dimeric molecule which exists in at least three 
combinative forms (isoenzymes), designated "MM," "MB" and "BB" on the 
basis of monomer combination. The M and B monomers which make up the 
enzymes each have a sulfydryl (SH) group-containing "active" site, the 
integrity of which is essential to catalytic activity. This active site is 
easily susceptible to inactivation upon contact with oxidizing materials. 
Each isoenzyme has a mass of approximately 82,000 daltons. 
Analyses of profiles of creatine kinase isoenzyme (hereafter, "CK") 
activities in human tissue indicate that brain tissue ordinarily contains 
only the BB isoenzyme; skeletal muscle tissue ordinarily only the MM 
isoenzyme; and heart muscle tissue ordinarily both the MM and MB 
isoenzymes. Human plasma from normal subjects includes primarily the MM 
isoenzyme with less than 0.005 I.U. of MB CK per milliliter and no 
detectable quantities of BB CK. 
After cerebral infarction, infection or other forms of cerebral damage, the 
concentration of MM isoenzyme in plasma is often somewhat elevated but the 
BB isoenzyme in plasma is generally not detectable. The lack of detectable 
BB CK is probably related to numerous factors including an effective 
blood/brain barrier. Damage to skeletal muscle by trauma or muscle disease 
such as muscular dystrophy is almost invariably accompanied by elevation 
of concentrations of the MM isoenzyme in plasma. Some very small increases 
in plasma BB activity have been reported in diseased muscle patients and 
immature muscle fibers of regenerating muscle have been proposed as the 
source of the BB in the blood.* Damage to heart muscle ordinarily results 
in elevation of plasma activity of both MM and MB CK. 
FNT *It has been reported that developmentally mature skeletal muscle may 
contain substantial quantities of BB CK which appears to be immunoreactive 
with a BB immune plasma but inactive when analyzed by cellulose acetate 
electrophoresis. [See, Armstrong, et al., J.Biol.Chem., Vol. 252, No. 10, 
3105, 3112 and 3117 (1977). 
Because the only human tissue containing appreciable amounts of MB CK is 
the myocardium, an elevation of MB concentration in blood--to the extent 
that it can be distinguished from increases in MM CK provides a remarkably 
sensitive and specific indicator of myocardial injury. Accordingly, 
analysis of serial changes in plasma MB CK has heretofore been employed to 
estimate the extent of acute myocardial infarction in experimental 
anaimals and human patients. 
Analysis of a plasma sample for MB CK activity is frequently "indirectly" 
secured through prior art assays for enzyme activity. Employing a 
spectrophotometric technique for detecting NADPH developed from creatine 
kinase-catalyzed formation of ATP through coupled enzyme systems, relative 
increases or decreases in blood sample CK activity are predicated upon 
corresponding changes in catalytic activity of the enzyme isolated from 
the sample. The results of such prior art assays are directly dependent on 
several perameters, one of which is the rate of disappearance of the 
isoenzyme activity from plasma. Unfortunately, the factors responsible for 
disappearance of MB activity from the circulation have not been well 
elucidated. It is unclear, for example, whether disappearance of enzyme 
activity is ratelimited by inactivation, denaturation, or some form of 
removal of intact enzyme molecules from circulation. Consequently (and 
apart from the difficulty in distinguishing activity of MM CK from MB CK) 
activity-based determinations are subject to substantial unreliability. 
The prior art provides certain proposals for radioimmunoassay ("RIA") 
analysis of plasma samples for use in quantitative detection of enzymes, 
and specifically creatine kinase isoenzymes, independently of analysis for 
enzyme activity. See, generally, the review article, "The Measurement of 
Enzymes by Radioimmunoassay" by J. Landon, et al. [Ann. Clin. Biochem., 
14, pp. 90-99 (1977)] which recounts the relative superiority of RIA 
techniques to those based on catalytic activity. Simply put, according to 
one such RIA technique a stoichiometric excess of a pure, labelled 
(radioisotopic) material is allowed to associate (e.g., by 
antigen/antibody reaction) with the selected reactive substance such as 
antibody previously exposed to a sample containing an "unknown" quantity 
of enzyme which is unlabelled, but which is capable of a similar 
association. Direct quantitative information concerning the "unknown" 
concentration, as opposed to activity, is obtained on the basis of a count 
of radioactivity of the remaining selected substance which associates with 
the labelled material. 
Nicholson et al., [Proc. Austral. Assoc. Neurologists, Vol. 10, pp. 105-108 
(1973)] described a method involving labelling skeletal muscle MM creatine 
kinase with .sup.125 I and report development of an RIA for the MM 
isoenzyme which assertedly measures "enzymes independently of the 
integrity of the active site." The method employed by Nicholson, et al. 
for labelling human creatine kinase is that described by Hunter, et al. 
[Nature, Vol. 194, pp. 4956 (1962)], employing chloramine-T to directly 
introduce the desired isotope into tyrosyl and histidyl residues of the 
enzyme protein chain. This method, however, has been associated with 
considerable structural changes (i.e., tertiary structure destruction) and 
loss of MM isoenzyme activity through disruption of the 
sulfhydryl-group-containing active site. Because the assay is assertedly 
specific for the M subunit, it invites cross-reaction with MM CK and MB CK 
and is thus incapable of distinguishing elevation of serum MB 
concentration resulting from myocardial infarction from elevation of serum 
MM concentration resulting from skeletal muscle damage or disease. The 
chloramine-T reagent used in the labelling procedure of Nicholson, et al. 
cannot be used for labelling the BB or MB isoenzymes due to their relative 
lability (instability, visa-vis MM) in the presence of the highly 
oxidative --sulfhydryl-group-disrupting--chloramine-T reagent. In sum, the 
Nicholson, et al. proposal has have not provided a useful basis to develop 
an RIA specific for BB and MB isoenzymes. 
A prior art publication of interest to the background of the invention is 
Fang, et al. [Biochem. Biophys. Res. Comm., Vol. 65, pp. 413-419 (1975)] 
which reports that creatine kinase enzyme activity losses from direct 
iodination by prior art chloramine-T, thallic trichloride and 
lactoperoxidase methods may be avoided through use, for iodination, of a 
Bolton-Hunter acylation reagent which conjugates (combines at a free amino 
(NH.sub.2) group of the protein, thus avoiding disruption of the active 
site. The reagent involved was an iodinated compound derived from 
N-succinimidyl-3-(4-hydroxyphenyl propionate). Fang, et al. labelled 
rabbit skeletal muscle MM CK. There is no mention of human MM CK nor was 
there any mention of animal or human MB or BB CK isoenzymes. While the 
reported preservation of enzyme active sites would have suggested that a 
more accurate RIA for MM creatine kinase than that of Nicholson, et al. 
might be secured, the prior art was still without a method for labelling 
the more labile MB and BB CK isoenzymes. Furthermore, the antibody noted 
in Fang, et al. was to rabbit MM CK which offered no specificity for human 
BB or MB CK isoenzymes. The necessary ingredients for a human CK isoenzyme 
RIA with a specific antibody to BB and MB CK and the necessary stabilizing 
conditions for such analysis were yet to be developed. 
BRIEF SUMMARY 
The present invention provides methods and materials for accurate and 
extremely sensitive analysis, by RIA, of enzymatic materials. More 
specifically, the invention provides for quantitative detection of B 
subunit-containing (MB and BB) isoenzymes of creatine kinase and therefore 
permits, for the first time, the accurate determination of serum MB CK 
without either reliance upon kinase activity (ATP and then NADPH 
formation) or substantial interference by the MM isoenzyme as well as the 
accurate determination of tissue or blood BB CK. As such, the invention is 
expected to provide a most useful tool for the early diagnosis of 
myocardial infarction and for disorders which may involve release of BB CK 
into the plasma. 
According to the invention, antibodies to human BB creatine kinase are 
obtained by immunization of rabbits with purified BB CK to provide serum 
containing antibodies which demonstrate specificity for BB and MB 
isoenzymes, but no cross-reactivity with the MM isoenzyme. Pure MB CK from 
myocardial tissue and/or BB CK from brain tissue is labelled with .sup.125 
I through use of a Bolton-Hunter acylating agent [e.g., 
N-succinimidyl-3-(4-hydroxyphenyl propionate)] to provide a reagent for 
competitive displacement binding RIA to ascertain concentration of MB or 
BB of a plasma or tissue sample. Incubation of BB antibody with labelled 
and unlabelled enzyme (antigen) is preferably carried out in an aqueous 
buffer comprising Tris at a concentration of from about 1.2 to about 2.0 
M, and preferably 1.6 M, having a pH of from about 7.0 to about 8.0 and 
preferably 7.4. A preferred buffer also includes a suitable organic 
reducing agent such as mercaptoethanol in a concentration of from about 
10.0 to about 30.0 mM, and preferably 20.0 mM, as well as standard agents 
such as gamma globulin and serum albumin for the prevention of 
non-specific binding. The buffer is believed to have substantial utility 
in preserving active site and tertiary structure as well as in retarding 
both undesirable "polymerization" caused by multiple reactions between 
antigen and antibody moieties and dissociation of reacted moieties. 
Separations from unbound (unreacted) reactants are carried out using 
ammonium sulfate to precipitate antibody associated with labelled and 
unlabelled antigen, because the unreacted materials have a substantially 
lower mass. 
For purposes of indicating the background and/or illustrating the state of 
the art pertaining to the invention, applicant specifically incorporates 
by reference herein the disclosures of his jointly-authored publication 
entitled "Radioimmunoassay for Creatine Kinase Isoenzymes" appearing in 
Science, Vol. 194, pp. 855-857 (November, 1976), as well as the disclosure 
of the above-mentioned Landon, et al. article.

DETAILED DESCRIPTION 
The following Examples illustrate practice of the invention and more 
specifically relate to (a) isolation of purified human CK enzymes for use 
in providing labelled enzymes; (b) labelling of CK isoenzymes; (c) 
preparation of antibodies to BB CK; (d) analysis of binding affinity and 
specificity of the antibodies; (e) general RIA procedures; (f) 
determination of MB CK in a plasma sample; and (g) methods and material 
for a typical clinical analysis for MB CK in a plasma sample. 
EXAMPLE 1 
Isolation of Purified Human CK Isoenzymes 
MM and MB isoenzymes were prepared from human myocardium and BB from human 
brain obtained at necropsy within six hours of death. Homogenates from 
human myocardium and brain were prepared as follows. The fresh tissue was 
trimmed of fat, cut into small pieces with scissors and passed through a 
pre-cooled meat grinder. Ground tissue was homogenized in a Waring blender 
containing 20 g/ml of 0.5 M Tris-HCl [Tris(hydroxymethyl) aminoethane 
hydrochloride], pH 7.4, and 0.001 M 2-mercaptoethanol. All preparative 
procedures were performed at 0.degree.-4.degree. C. 
The myocardial homogenate was centrifuged at 31,000 g for 15 minutes and 
the supernatant fraction filtered through 8 layers of cheesecloth. 
Ninety-five percent ethanol was added to the supernatant in dropwise 
fashion until the final concentration was 50%, and the mixture was allowed 
to stand while slowly stirring at 4.degree. C. for 30 minutes. The 
precipitated material was removed by centrifugation and the supernatant 
fraction decanted. Again ethanol was added in stepwise fashion until a 
final concentration of 70% was obtained. The mixture was allowed to stand 
for 30 minutes and the resulting precipitate was recovered and saved. With 
the use of a homogenizer, the precipitated pellet was suspended in 
homogenizing medium equal in volume to 50% of the original homogenate. 
After centrifugation at 31,000 g, the pellet from this resuspended mixture 
was discarded and the supernatant fraction saved. To separate the MM CK, 
NaCl, 5 M, was added with rapid stirring to a final concentration of 0.05 
M. DEAE Sephadex A-50 was then added (10 slurry for 48 mg of protein) and 
the mixture was stirred for 30 minutes. This suspension was filtered with 
a Buchner funnel lined with Whatman's No. 1 filter paper, and the filtrate 
was dialyzed against several changes of 0.01 M glycine, NaOH, pH 9.0, 
prior to freeze drying of the MM fraction. The MB CK present in the 
homogenate was adsorbed to the DEAE Sephadex previously added. 
Accordingly, after the MM filtrate had been obtained, the DEAE Sephadex 
was retained in the Buchner funnel and washed five times with 0.05 M 
Tris-HCl, pH 7.4, 0.05 M NaCl, and 0.001 2-mercaptoethanol. After the DEAE 
Sephadex had been washed, MB CK was eluted with Tris-HCl, 0.05 M, pH 7.4 
containing 0.3 M NaCl. The eluate was dialyzed with glycine buffer, and 
then freeze-dried to obtain the MB fraction. To further enrich the MM and 
MB fractions, further ethanol fractionation and column chromatography was 
performed followed by dialysis, freeze-drying and storage at 
0.degree.-4.degree. C. 
CK was extracted from brain in the same fashion as from myocardium with the 
following exceptions. The final concentration of ethanol was 60% rather 
than 50%. After filtration to remove any potential MM fraction, Sephadex 
retained in the Buchner funnel was washed five times with Tris-HCl buffer 
containing 0.1 NaCl, and subsequently washed once with buffer containing 
0.3 NaCl. BB CK remained adsorbed to Sephadex under these conditions and 
was then eluted with Tris buffer containing 0.4 M NaCl dialyzed against 
0.01 M glycine, NaOH, pH 9.0, and freeze-dried. 
Polyacrylamide gel electrophoresis [per Anido, et al., Am. J. Clin. Path., 
Vol. 61, p. 599 (1974)] of the human preparations indicated that each 
isoenzyme was obtained in a sample devoid of activity attributable to 
other isoenzymes in the initial extract. MM CK averaged 392 IU/mg of 
protein, MB 46 IU/mg, and BB 110 IU/mg. Specific activity of the 
isoenzymes was increased by more than one hundred-fold over that present 
in the initial extract and analysis by SDS gel electrophoresis [per Weber, 
et al., J. Biol. Chem., Vol. 244, p. 4406 (1969)] with staining for 
protein showed only one faint contaminating band of the MM, no 
contaminating bands of BB, and two very faint contaminating bands of the 
MB, indicating that the preparations were probably more than 90% pure. 
EXAMPLE 2 
Radioactive Labelling of CK Isoenzymes 
Radioiodine (.sup.125 I) was utilized to radioactively label CK isoenzymes 
for subsequent use in a competitive displacement radioimmunoassay. To 
avoid exposing the enzymes to oxidizing agents and contaminants in the 
radioiodine, the .sup.125 I was first incorporated into N-succinimidyl 
ester 3-(4-hydroxyphenyl propionate) which in turn was reacted with amino 
groups on the CK isoenzyme protein. Radioiodination was performed by the 
method of Bolton and Hunter, supra, carried out at room temperature 
(23.degree. C.) N-succinimidyl 3-(4-hydroxyphenyl propionate) (0.3 mcg) 
was treated with 5 millicuries (10-20 ul) of Na.sup.125 I, 50 mcg of 
chloramine-T and 10 .mu.l of 0.25 M phosphate buffer, pH 7.5. The reaction 
was immediately terminated by the addition of 120 mcg of sodium 
metabisulfite in 10 .mu.l of 0.50 M phosphate buffer, pH 7.5 containing 
200 mcg of KI. The iodinated product was extracted into benzene (0.300 
ml.times.2 portions) and recovered by evaporation of the solvent under 
vacuum. The addition of dimethylformamide (5 .mu.l) before adding the 
benzene was necessary for full extraction of the ester into the solvent. 
The residue was used to label CK isoenzymes. The labelled residue was 
combined with 2-8 mg of MM, MB or BB CK in 1-2 ml of 0.01 M Na-borate 
buffer, pH 8.5. After gently shaking the reaction, for four hours at 
4.degree. C., the labelled isoenzymes were then dialyzed against the same 
buffer containing 0.0005 M 2-mercaptoethanol. Radioactivity per mcg of 
labelled CK isoenzymes averaged 200,000 cmp for MM CK and MB CK and 
100,000 cpm for BB CK. The maximum loss of enzyme activity resulting from 
labelling and dialysis was less than 5% for each isoenzyme preparation. 
EXAMPLE 3 
Preparation of Antibodies to CK Isoenzymes 
Utilizing the purified human MM and BB CK mixed with equal volumes of 
Freund's complete adjuvant, antibodies to CK isoenzymes were induced in 
rabbits. Initially, the rabbits were injected subcutaneously with 1 mg of 
immunogen (0.25 mg/foot pad). Subsequently, they were injected with 0.25 
mg weekly for three weeks. All animals were given booster injections of 
0.1 mg in complete adjuvant at monthly intervals thereafter. Ten days 
after each booster injection, the animals were bled and their serum 
analyzed for antibody activity. Ouchterlony agarose plates, prepared with 
BB antiserum exhibited single precipitant lines to BB and MB antigen but 
no precipitant line to MM. Plates prepared with MM antiserum exhibited a 
single precipitant line to both MB and MM but none to BB. Thus, antibodies 
to BB CK reacted with BB CK and also cross-reacted with MB but did not 
cross-react with MM indicating that it was specific for the B subunit. 
Similarly, MM antibodies were specific for the M subunit. 
EXAMPLE 4 CL Binding Affinity and Specificity of Antiserum 
The binding affinity and specificity of the BB and MM antibodies (rabbit 
antiserum of Example 3) was determined over a wide range of concentrations 
of the antibody by diluting the appropriate antiserum over a range of 1:15 
to 1:1000. All determinations performed in duplicate were carried out in 
12.times.75 mm glass tubes containing 1.6 M Tris buffer, pH 7.6, (200 ul) 
2% bovine serum albumin (100 .mu.l), 0.020 M mercaptoethanol (10 .mu.l), 5 
picograms of rabbit gamma globulin (50 .mu.l). As noted earlier, the gamma 
globulin and serum albumin are believed to minimize nonspecific binding 
and the high concentration of Tris and 2-mercaptoethanol is believed to 
protect the sulfhydryl groups of the isoenzymes and prevent dissociation 
into monomers. To this mixture was added the appropriate dilution of 
antiserum in volumes ranging from 100 .mu.l to 5 .mu.l (dilutions 
performed with normal rabbit serum). .sup.125 I-labelled MM (0.1 mcg), MB 
(0.1 mcg), and BB CK (0.2 mcg) were added such that approximately 25,000 
cpm were present in each tube. The total volume was kept constant at 500 
.mu.l with necessary adjustments being made with Tris buffer. The 
solutions were then incubated and gently shaken at 4.degree. C. for six 
hours. Appropriate controls were incubated containing normal rabbit serum 
rather than rabbit antiserum. 
Following the incubation period, separation of free from antibody bound 
labelled CK was accomplished by the addition of cold saturated ammonium 
sulphate with a final concentration of 44%, and allowing the solution to 
sit at room temperature for 15 minutes. The solution was then centrifuged 
at 2,000 g for 20 minutes, the supernatant decanted, and the pellet washed 
with 50% ammonium sulphate (400 .mu.l) and again centrifuged. The pellets 
were counted in a gamma counter (Micromedic Systems, Inc.) until a minimum 
of 10,000 counts were obtained. The .sup.125 I counts present in the 
pellet expressed as a percent of the total number of counts initially 
present represent percent binding. To optimize conditions for any possible 
cross-reactivity between the BB antibody and MM CK and vice versa for the 
MM antibody, determinations were done in which BB antiserum diluted only 
1:15 was incubated with 4 mcg of .sup.125 I MM and MM antiserum in a 
dilution of 1:15 with 4 mcg of .sup.125 I BB. 
Results of binding experiments using serial dilutions of BB antiserum from 
1:15 to 1:1000 incubated with iodinated MM, MB and BB CK are shown in FIG. 
1. Ninety-three percent of the .sup.125 I BB was recovered in the pellet 
in dilutions of 1:30, but binding diminished rapidly with only 7% at 
1:1000, demonstrating that binding was dependent on antibody 
concentration. Maximum binding of .sup.125 I MB CK (60%) occurred at 1:15 
dilutions but again binding was dependent on the concentration of antibody 
with only 5% binding at 1:1000 dilution. In contradistinction, .sup.125 I 
MM exhibited no such antibody concentration dependent binding and at all 
dilutions was the same being between 3-5% which is the same as that of 
control (normal rabbit serum). These results demonstrated the antibody is 
specific for the B sub-unit rather than the molecule as a whole. 
Results of binding with MM antiserum showed 94% binding of .sup.125 I MM 
and 56% binding of .sup.125 I MB and dilutions of 1:30. Again binding was 
dependent on the concentration of antibody and returned to control levels 
at a dilution of 1:1000. No specific binding was seen between the MM 
antiserum and .sup.125 I BB. The MM antiserum exhibited specificity for 
the M subunit but no cross-reactivity with the B subunit. 
EXAMPLE 5 
General Procedure for Radioimmunoassay 
To develop a competitive displacement radioimmunoassay for plasma MB CK, BB 
antiserum was used and the specificity of the BB antiserum for B subunits 
further established by comparing the ability of unlabelled BB, MB and MM 
CK to inhibit .sup.125 I BB binding. The reaction was performed in the 
same buffer solution used for the binding experiments, but with the 
exceptions that the dilution of antiserum was kept constant at 1:150 and 
the amount of .sup.125 I BB CK was kept constant at 0.2 mcg containing 
approximately 25,000 cpm. The antiserum dilution of 1:150 was chosen since 
this concentration of antibody binds about 50% of the .sup.125 I BB [See 
generally, Parker, C. W., "Radioimmunoassays" in Progress in Clinical 
Pathology, Volume IV (Grune and Stratton, Inc., New York, 1974)]. A known 
amount of unlabelled BB, MB or MM CK was diluted from 1:15 to 1:1000 and 
incubated for six hours with .sup.125 I-labelled BB. Following incubation, 
the ammonium precipitated pellet was washed, centrifuged and counter for 
.sup.125 I radioactivity. To further determine the specificity of 
unlabelled BB or MB to displace .sup.125 I BB binding in the face of MM 
CK, inhibition curves were determined for MM incubated with BB or MB in 
which MM was present in a 25,000-fold excess over that of unlabelled BB or 
MB. 
Unlabelled MB CK competitively displaced labelled BB CK from binding to the 
BB antibody which was dependent on the concentration of MB CK as shown in 
FIG. 2. The inhibition curve is steep between 17-80 pg/ml with 50% 
inhibition at 51 pg and complete inhibition of binding at a concentration 
of 80 pg/ml and above. A similar inhibition curve was seen for unlabelled 
BB which showed 50% inhibition at 26 pg/ml and complete inhibition at 125 
pg/ml and higher. Unlabelled MM CK showed no inhibition of .sup.125 I BB 
binding, even at 5 mcg/ml (2000-fold excess over .sup.125 I BB CK). 
Furthermore, the competitive inhibition of unlabelled MB CK was unaltered 
in the presence of high concentrations of MM CK (5000 M excess over that 
of MB CK). Thus, the BB system as a competitive displacement assay for MB 
is extremely sensitive, detecting reliably a concentration of 20 pg/ml 
which in terms of enzymatic activity is 1.times.10.sup.-6 IU/ml. 
Furthermore, the specificity is such that it detects at this level of 
sensitivity even in the presence of a 5000 molar excess of MM. Results of 
radioimmunoassays performed on heat inactivated serum constituted with 
known amounts of MB CK ranging from 20 pg/ml to 2 mcg/ml deviated by less 
than 3% from that expected. 
EXAMPLE 6 
Determination of MB CK in Plasma Samples 
To determine the amount of MB CK present in an unknown sample, a standard 
MB inhibition curve is run with known amounts of unlabelled MB CK ranging 
from 2,000 to 20 picograms (pg)/ml of MB CK. The unlabelled MB CK is 
incubated with a constant amount of antiserum and .sup.125 I BB CK as 
outlined previously under radioimmunoassay procedure. Serial dilutions 
(3-4) of the unknown sample are made and the amount of inhibition 
determined at each dilution and compared to the standard curve from which 
it is possible to calculate the amount of MB CK present expressed as 
ng/ml. To determine the accuracy of the assay, known amounts of human 
unlabelled MB CK were added to heat inactivated serum and serial dilutions 
done and results expected compared to that obtained. Plasma samples were 
obtained from five patients with acute myocardial infarction. At least 15 
samples were obtained serially from each patient over a period of 48 
hours. All samples were performed in duplicate and compared to enzymatic 
activity obtained by a kinetic fluorometric assay previously described in 
Roberts, et al., Am. J. Cardiol., Vol. 33, p. 650 (1974). All 
determinations for total CK enzymatic activity were done according to the 
method of Rosalki, S. B., J. Lab. Clin. Med., Vol. 62, p. 696 (1967). 
Results of samples obtained from five patients with acute myocardial 
infarction exhibited elevated MB CK in all cases. A typical MB CK curve 
from one of the patients is shown in FIG. 3 and demonstrates a high degree 
of correlation between activity-related and RIA analysis findings for a 
situation wherein the MB CK level is markedly elevated. 
EXAMPLE 7 
Typical Clinical Analysis for MB CK in a Plasma Sample 
A. Materials 
1. Five (5) vials, A-1, A-2, A-3, A-4 and A-5, are prepared with each 
containing the same known amount of .sup.125 I-labelled human BB CK. 
2. Four (4) vials marked B-1, B-2, etc., are prepared with each containing 
unlabelled human MB CK with known amounts such that there would be a 100%, 
60%, 40% and 20% inhibition of binding respectively (used to obtain the 
control standard inhibition curve). 
3. Five (5) vials marked C-1, C-2, etc., are prepared with each containing 
the same known amount of human BB antiserum. 
4. Six (6) vials marked D-1, D-2, etc., are prepared with each containing 
the same known amount of RIA-buffer (400 .mu.l) containing 1.6 M Tris, 20 
mM mercaptoethanol or other suitable reducing agent, 5 pg of rabbit gamma 
globin and 0.05% of bovine serum albumin at a pH of 7.4. 
B. Methods 
1. Vials C-1, C-2, C-3, C-4 are mixed with B-1, B-2, B-3, B-4 vials and 
D-1, D-2, D-3 and D-4 vials respectively and incubated for 30 minutes with 
gentle shaking at 4.degree. C. and referred to as AB-1, AB-2, etc. 
2. Following the above incubation steps, vials A-1, A-2, A-3, A-4 are added 
respectively to the appropriate AB vials (1, 2, 3, 4) and incubated for 
six (6) hours at 4.degree. C. with gentle shaking. 
3. Contemporaneously with steps 1 and 2, the C-5 vial is mixed with D-5 and 
100 .mu.l of plasma from the unknown sample and incubated for 30 minutes 
at 4.degree. C. with gentle shaking after which it is added to A-5 vial 
and incubated for six (6) hours as above. 
4. Following the six (6) hour incubation, 100 .mu.l of saturated ammonium 
sulfate is added to all the tubes and incubated with gentle shaking for 15 
minutes at 4.degree. C. 
5. All the tubes are centrifuged at 2000 g for 20 minutes. 
6. Supernatant is discarded from all the tubes and 200 .mu.l of buffer from 
D-6 tube is added for one washing and again supernatant is discarded. 
7. The tubes containing the residue pellets are now put in the gamma 
counter and counted for radioactivity. The amount of binding present in 
vials 1-4 are plotted against the amount of CK present in vials marked 
B-1, B-2, etc. to obtain the standard inhibition reference curve. The 
amount of binding in the unknown plasma sample is placed on the curve and 
from the abscissa the amount of MB CK can be determined. 
The above Examples have described a radioimmunoassay for CK isoenzymes. 
Antibodies were developed for MM and BB CK which are specific for the M 
and B subunits, respectively. Since MB CK, found in the human myocardium, 
contains both subunits, either antibody can be used to detect MB CK. The 
BB antibody reacts with BB and cross-reacts with MB CK, but not MM even 
when present in 5,000 molar excess over than of MB CK, a ratio far greater 
than that seen in plasma after myocardial infarction since MB CK is 
usually 10-15% of total CK activity. Detection of plasma MB CK using the 
BB system is specific for MB. Because BB CK activity is not present in 
normal plasma, even in patients with cerebral disorders or those with 
acute myocardial infarction, displacement binding reflects MB exclusively. 
This was corroborated by the close agreement (See, e.g., FIG. 3) between 
enzymatic activity determined by the kinetic fluorometric method and that 
obtained by the radioimmunoassay in samples obtained from patients with 
acute myocardial infarction. 
The sensitivity provided by the assay (and illustrated in FIG. 2) is 
believed to exceed that of any prior assay by several fold. Present assays 
based on enzymatic activity can barely detect 0.010 IU/ml as opposed to 
the present assay which detects reliably 0.00001 IU/ml. Since mean plasma 
MB CK activity is 0.002 IU/ml, a five-fold increase is necessary for 
detection by enzymatic assays as opposed to the present assay which 
reliably detects any increase above normal. The increased sensitivity, 
coupled with its potential for detection of enzymatically inactive MB CK 
in the circulation should lead to improved estimates of infarct size as 
well as earlier detection of acute myocardial infarction. This is of 
particular importance in view of the recent enthusiasm for protection of 
ischemic myocardium in patients with acute myocardial infarction which 
demands a definitive diagnosis as soon as possible, since agents that can 
potentially decrease infarct size would be more effective if administered 
early. 
The high level of specificity and sensitivity in the B subunit-containing 
CK isoenzyme radioimmunoassay system suggests that a similar approach may 
be useful in differentiation of other clinically important enzymes which 
exist in multiple forms. Studies evaluating the disappearance of other 
enzymes from the circulation have been restricted to determining the loss 
of activity. Because this assay detects the concentration of molecules, 
one can determine the actual rate of isoenzyme protein turnover 
independent of activity. 
The assay should also help to elucidate mechanisms responsible for 
disappearance of individual CK isoenzymes from the circulation as well as 
aid in elucidating the relative importance of inactivation, denaturation, 
or removal of CK molecules under various clinical circumstances. 
Numerous modifications and variations of the invention are expected to 
occur to those skilled in the art upon consideration of the foregoing 
detailed description. It can be anticipated that substantial variations in 
modes of securing BB CK antibodies, in labelling CK isoenzymes and in 
preparing suitable dissociation and polymerization-retarding buffers will 
be made. Consequently, only such limitations as appear in the appended 
claims should be placed upon the invention.