of Monoclonal Antibodies for an Assay of Cardiac Troponin-l and. Preliminary. Results in Suspected. Cases of Myocardial Infarction. Geza. S. Bodor,. Sharon.
CLIN. CHEM. 38/11,
(1992)
2203-2214
Development Preliminary
of Monoclonal Antibodies for an Assay of Cardiac Results in Suspected Cases of Myocardial Infarction
Geza
Sharon
To
S. Bodor,
improve
the
Porter,
specificity
Yvonne
Landt,
of biochemical
lgG monoclonai
troponin-l (cTnI) animal (canine,
antibodies
and tested bovine, and
against
them rabbit)
Jack
markers
myocardial infarction (Ml), we have developed monocional “sandwich” enzyme immunoassay sure cardiac troponin-i (cTnl) in serum. We
eight
and
of
a double to meaproduced
human
and anti-
bodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTni-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinitystripped cTni-free human sera to cover the range 0-100 p..gIL for cTni. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit ofthe reference
range (nonparametric 95% interval) was established as 3.1 g/L by measuring cTni concentration in s#{233}ra of 159 hospitalized patients without evidence of cardiac disease. Purified human skeietal Tnl up to 1 0 000 jg/L did not affect the assay (calculated cross-reactivity 3 SD from its respective mean (138 rule); if two observations exceeded 2 SD from the mean (228 rule); or if at least two of the observations were at least 2 SD on opposite sides of their mean rule) (24). Samples containing more cThI than the highest-concentration calibrator were diluted with cTnI-free human serum and reassayed. 2206
CLINICAL
CHEMISTRY,
Vol. 38, No. 11, 1992
Assay as the
of the cTnl
Performance
reproducibility. standard
for each
error
concentration
feasibility studies. utive assays were Imprecision was pipetted calculated
Immunoassay
antibodies
Analytical
The
mean
of the
mean
of the
absorbance (SE)
was
12 calibrators
Absorbance readings used to assess assay
studies. into the cTnI
ELISA as well calculated during
the
from 12 consecreproducibility.
Each set of the five QC samples microtiter plates in duplicate. The
concentration
of the
individual
QC ma-
terial was utilized to estimate the within-run, betweenrun, and total imprecision of the ELISA (25). The data collected from 16 consecutive plates during the feasibility studies were included in the calculation. Minimum
detectable
readings
of the
We
dose.
cThI-free
human
used serum
32
absorbance
to calculate
the
mean (SD) absorbance of the zero standard. This was converted to concentration by use of the average doseresponse curve determined at the same time. Influence ofpurified sTnI. Sera from five patients with proven MI containing cTnI were supplemented
known with
(measured) amounts various concentrations
purified measured
sTiil; the apparent cThI concentration in both the supplemented and
mented
samples
in the
same
concentrations in the two lated. The log of the ratio t-test for each concentration difference reactivity unable sTnI.
from could to
Retrospective
zero not
dissolve
Clinical
assay.
The
(log 1.0 = be calculated sufficiently
Evaluation
Samples were collected two phases to evaluate cThI ELISA. The first
ratio
sets of samples was tested via of sTnI added
of of
was then nonsuppleofthe
cTnI
was calcua two-sided to check for
0.0). Percent crossbecause we were
high
of cTnl
concentrations
of
ELISA
from hospitalized patients in the clinical performance of the phase included 30 consecutive
patients, all of them admitted to Barnes Hospital cardiac care unit and later diagnosed to have had an MI or not. Ten patients had an acute MI and 20 patients did not. The diagnosis was based on the presence of at least two of the following three criteria: acute chest pain, evolving Q wave on electrocardiogram, and results for serial CK-MB measurement (26). Studies with these patients served two purposes: to establish whether the cTnI immunoassay could identify patients with MI in the cardiac intensive-care unit, and to estimate the early time course of cTnI release in the serum after an MI. For this latter purpose, we collected serial samples from the 10 patients who were diagnosed to have had an MI. Samples for CK-MB analysis were collected by venipuncture hourly during the first 6 h after the onset of symptoms, every 2 h for the next 6 h, and every 12 h afterwards, as long as the patient remained at the cardiac care unit. Informed consent was obtained from all 10 patients before enrolling them in the study. The frequent sampling was necessary to ensure reliable comparison of the early time course of CK-MB and cThI after an MI. After analysis for CK-
MB, the serum samples were frozen at -80 #{176}C until the cTnI immunoassay could be performed. The second phase ofthe evaluation included all serum samples (not just those from the cardiac care unit) that were submitted to the Barnes Hospital clinical chemistry laboratory for CK-MB 10 and October 5, 1990. frozen at -80 #{176}C until performed. Total CK and measured by the routine tal. Total CK was measured con, Tarrytown, NY) or Success, NY) analyzers, noassay (Baxter, McGaw were reviewed for patients
analysis between September The samples were then stored cTnI immunoassay could be CK-MB concentrations were assays used at Barnes Hospiwith the RA-1000 (Technithe AU-5000 (Olympus, Lake CK-MB by the Stratus immuPark, IL). The medical records in whom the discharge diag-
sults are specificity.
included
in the
calculations
of the
cTnI
assay
Patients with cardiac disease other than MI (non-MI cardiac group). The non-MI cardiac group included all subjects in whom any acute or chronic cardiac disease was indicated in their medical records, regardless of the cause ofhospital admission. This group includes chronic ischemia as well as other cardiac pathology, including unstable and variant angina. These patients were separated into two subgroups, based on their discharge diagnosis: those with ischemic cardiac disease and those without ischemic disease. Patients
These cardiac
without
patients pathology
cardiac
had
disease
(noncardiac
group).
CK-MB analysis ordered but no was indicated in their medical rec-
nosis was MI, or whose CK was >200 UIL, or CK-MB >6.7 gfL, or cTnI >3.1 p.gfL. The review ofthe medical records was used to categorize the patients into the various groups described below. The time of onset of chest pain, the means of establishing or excluding the diagnosis of MI, and other relevant clinical information were recorded. Of the 691 patients for whom CK-MB was assayed, we examined the medical records for 371 (54%). All patients for whom CK-MB and cThI could be measured in the same sample were included in the study. The patients were divided into three groups after review of their medical records. Acute myocardial infarction (Migroup). Patients were diagnosed to have had an acute MI on the basis of chest
ords. These subjects served to limit ofthe cTnI ELISA. Some skeletal muscle injury from an results of patients with skeletal sented separately; they served ificity of the cTnI ELISA. Two
pain, electrocardiogram, and CK-MB, as noted above. Several patients had other routes to this diagnosis, such as imaging with “Tc or 201T1 or echocardiography according to the cardiology practice ofour hospital. This group includes patients whose diagnosis was originally established at an outside hospital but who were later transferred to Barnes Hospital for follow-up treatment. The timing ofthe laboratory values for this latter group was recalculated relative to the time of onset of chest pain as time 0, where possible. This allowed calculation of the average time course of changes in serum CK, CK-MB, and cTnI concentrations in 30 patients. This information was also used to assess the time-dependent diagnostic sensitivity of the two markers, CK-MB and cTnI. Six patients had recurrent MI as diagnosed by the concurrent presence of at least two of the following diagnostic signs: new onset of chest pain (n = 6), occurrence of a new Q wave on the electrocardiogram (n = 4), previously unseen complete occlusion ofa coronary artery on angiography (n = 1), and (or) a new increase of CK-MB after chest pain (n = 3). The results for patients with multiple infarctions were excluded from the calcu-
Four fusions yielded eight monoclonal antibodies. All were of the IgG class with K light chains but of various IgG isotypes (five IgG1, one IgG, two IgG2). The specificity of the antibodies as assessed by an ELISA with cThI and sThI is presented in Figure 1. All eight antibodies reacted with purified human cTnI in the presence of ThC (Figure 1, top). The reactivity of two mAbs, 3C5.10 and 1E11.3, was somewhat enhanced when human cThI was immobilized on the plastic surface via ThC (ThC/cTnI-coated plate). One antibody, 5D4.1, did not react with cTnI in the absence of ThC. The remaining five antibodies reacted with cThI independently ofthe presence ofThC. The antibodies gave a different reactivity pattern when tested against human sTnI (Figure 1, bottom). Only three of the antibodies reacted with human sThI. Two of these, 3C5.10 and 7B11.4, reacted with human sTnI independently of the presence or absence of ThC; the other, 5D4.1, reacted with human sThI only when ThC was present. All eight antibodies reacted with canine, bovine, and rabbit cTnI in the same way as they reacted with human cThI. The N-terminal peptide competed with cTnI for one of the antibodies, 1E11.3, in a solid-phase assay. This same antibody reacted with immobilized N-terminal peptide, giving an absorbance reading of 0.388A, more than five times the background reading. These studies confirmed the N-terminal specificity of the 1E11.3 antibody. The antibody specificities were further confirmed by blotting and immunostaining methods (Figure 2). When
lation ofthe are presented
time curves separately.
and
diagnostic The time of could not be established in 19 patients were found unconscious, had had several of worsening chest pain, or were first outside hospital at which the onset recorded. The results for these patients cluded from the time-curve calculations,
sensitivities and onset of an MI because they days or weeks admitted to an of MI was not were also exbut their re-
establish the reference ofthe patients had acute accident or surgery. The muscle injury are proto assess the tissue specadditional patients were
identified who were admitted to Barnes Hospital with an extreme degree ofrhabdomyolysis, as indicated by an increase of serum CK values without cardiac disease. The cTnI assay was performed on routine serum samples used for CK and CK-MB measurements from these two patients.
Results Characterization
of mAbs
CLINICAL
CHEMISTRY,
Vol. 38, No. 1 1, 1992
2207
1.500 Immobilized
0 C
a
Ag:
cTn
+
cml
Table I . Summary
of Antibody
Specificity
AntIgen 1.000-
I I
-U U)
-a
InC
0.500-
4:
0.. ‘0
Human
AntIbody
cTnI
Human
sTnI
I N.n i RNRH
cTnI
canIne
+
2B1.9
Canh*
cml
+
5D4.1
a) (1 C
3C5.10
+
1 El 1.3
+
7B11.4 3D11.11
+ +
+
1012.6
+
+
2F6.6
+
+
+. i445 aRe
0
+
+
+
+ +
+
+
O.400; , /445 nm 85% between 4 and 12 h post-MI, that of cPa! was >75% during the same time. The sensitivity of both markers reached 93% 12 h postMI. The sensitivity of CK-MB declined rapidly 48 h post-MI, becoming 0% after the fifth day. On the other hand, cPa! was positive in --70% of the samples for as long as 7 days. Samples collected >7 days post-MI had no increases ofCK-MB, but above-normal cPa! was found as long as 205 h (8.5 days) post-MI, though only sporadic data were available this late in the course of MI. When the overall diagnostic sensitivity ofCK-MB and cPa! was considered without regard to the onset of symptoms, similar values were obtained. For this analysis, we used the data from all 49 patients. CK-MB was increased in 40 of the patients in at least one sample; cPa! was increased in 42. Six additional patients (data not included in the above calculations) had a second MI during the sample collection period, as described above. The onset of the second MI was established to be an average of 143 h (range: 52-288 h) after the occurrence ofthe first MI. cPa! was undetectable in three ofthese patients and was between 2.9 and 34.5 p.gfL in the other three patients in the last sample drawn before the onset of the second MI. cPa! concentrations in subsequent samples were increased
Table 6. Time-Dependent
Sensitivity
of Ml Markersa
Hours
CK-MB
>6.7
Resutts shown are
a
0-4
4-S
8-12
12-24
24-48
48-72
72-96
44
78 (7/9)
93
86
40
20
(8/18)
92 (12/13)
77 (10/13)
67 (6/9)
(25/29) 83
(10/25) 60
(5/25)
17 (3/18)
(13/14) 93 (13/14)
(24/29)
(15/25)
t9/L
cTnl >3.1 g/L
nt of positl
Table 7. CardIac
Markers
e samples
In Non-MI
per category
Cardiac
(and no. of ve
Patients
CK-MB
Patients
with chronic
cardiac Median,
=
% of patients
43.5
u/d 32.1
posftivea
9.6
21.6
u/d
% of samples positiveb Patients without chronic isctiemic cardiac disease (n = 352)
6.1
Median, g/L Maximum found, cg/L % of patients % of samples u/d a
=
of patients
one sample tested. b Percent of samples
>6.7
g/L
Patient
all
six
patients,
with
of
CK-MB
cTnI
5.2
u/d
79.4
48800
279.4
21.0
55400
187.3
48 900
100.3
1 027
2.7
813
2.2
26.0 129.5
10.3
16.8
9.1
10.5
140.0 Patient
or cTnl >3.1 zg/L..
CK, U/I
9500
u/d 20.9
in at least
h 1
526
peak
concentrations
ranging
from 5.4 to 55.7 pgfL, but an increase in CK-MB was seen in only three of the six patients. Non-MI cardiac group. Four hundred and seventyseven patients were identified to have acute or chronic cardiac disease; 125 (26%) had chronic ischemic cardiac disease. Of the 1396 samples submitted from these patients for CK-MB analysis, 1165 samples could also be analyzed for cPa!. About 10% ofthe patients in both groups had increased CK-MB results; 16.8% of the patients with ischemic cardiac disease and 21.6% of the patients with nonischemic cardiac disease had abovenormal cPa! concentrations (Table 7). Acute skeletal muscle injury. Of the 159 patients without cardiac disease (control group), 40 had acute skeletal muscle injury as the result of an accident or major surgery. Median CK, CK-MB, and cPa! values of these 40 patients were 508 UIL, 2.2 p.gfL, and
