AU703944B2 - Ultrasensitive process for assaying cardiac troponin I - Google Patents

Ultrasensitive process for assaying cardiac troponin I Download PDF

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AU703944B2
AU703944B2 AU46251/96A AU4625196A AU703944B2 AU 703944 B2 AU703944 B2 AU 703944B2 AU 46251/96 A AU46251/96 A AU 46251/96A AU 4625196 A AU4625196 A AU 4625196A AU 703944 B2 AU703944 B2 AU 703944B2
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cardiac troponin
monoclonal antibody
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troponin
chemiluminescent substrate
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Charles Calzolari
Odile Flecheux
Bernard Pau
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Bio Rad Innovations SAS
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Abstract

The present invention relates to a ultrasensitive process for assaying cardiac troponine I by means of chemiluminescence.

Description

WO 96/22535 PCT/FR96/00095 1 ULTRASENSITIVE PROCESS FOR ASSAYING CARDIAC TROPONIN I The present invention relates to an ultrasensitive process for assaying cardiac troponin I by means of chemiluminescence.
Myocardial infarction (MIF) still represents a medical emergency and remains the major cause of death and of cardiovascular morbidity worldwide.
In all cases, the treatment introduced is designed to protect the myocardial tissue from irreversible necrosis. New therapeutic means such as thrombolytic or first line angioplasty make it possible nowadays to rapidly restore coronary circulation so as to limit the size of the infarcted region and to reduce the mortality and morbidity rates.
These therapeutic tools are all the more effective the earlier they are used after the onset of clinical manifestations. Clinicians faced with this pathology should be able to have increasingly efficient diagnostic tools.
The assay of serum proteins and in particular of enzymes has become a decisive component for the diagnosis of myocardial infarction and for assessing the success or failure of a reperfusion.
It is known that troponin is a myofibrillar protein complex consisting of 3 proteins, troponins C, I and T.
Cardiac troponin I is a contractile protein which is exclusively present in the cardiac muscle [Wilkinson J. M. et al., Nature (1978), 271, p. 31-35; Wade R. et al., Genomics (1990), p. 346-357].
Its physiological role is to inhibit, in the absence of calcium, the ATPase activity of the actinmyosin complex and therefore to prevent muscle contraction [Perry S. Biochem. Soc. Trans (1979), 7, p.
593-617].
During infarction due to the obstruction of one of the arteries supplying the heart (coronary artery), REPLACEMENT SHEET (RULE 26) 2 an ischaemia appears followed by a myocardial necrosis which results in a destruction of the cardiac cells and the release of cardiac troponin I into the blood. Troponin I therefore represents a marker which is very specific for myocardial infarction.
Thus, the assay of troponin I has recently been recommended for the early diagnosis of myocardial infarction [Am. Heart J. (1981), 110, p. 1334-1344; Molecular Immunology (1992), 29 p. 271-278].
Furthermore, it is now accepted that in certain patients, unstable angina which represents a critical stage of myocardial ischaemia may be associated with a high risk of onset of myocardial infarction and of sudden death. Post-mortem studies on this category of patients has shown that the appearance of these complications was very often preceded by cardiac microinfarctions [Davies M.J. et al., Circulation (1985), 71, p. 699-708].
The assay of cardiac troponin I therefore appears to be particularly advantageous in the detection of at risk subjects, in patients with unstable angina, more especially for the diagnosis of microinfarctions.
Moreover, the assay of troponin I may be envisaged during the monitoring of a thrombolytic treatment to confirm the success or failure of reperfusion, as also during postoperative monitoring of cardiac surgery operations (coronary bypass, angioplasty and the like). The assay of troponin I also finds application in the diagnosis of cardiac graft rejections after transplantation and the monitoring of cardiotoxic therapies using, for example, anthracyclines.
Immunoenzymatic methods are already known which allow the determination of troponin I. Larue C. et al., [Mol. Immunol. (1992), 29(2), p. 271-278; Clin. Chem.
(1993), 39/6, p. 972-979] describe a process for sandwich-type immunoenzymatic assay which uses, for the enzymatic visualization, the chromogenic substrate REPLACEMENT SHEET (RULE 26) 3 tetramethylbenzidine (TMB)-H 2 0 2 After the visualization, the absorbance is read at 450 nm and the detection limit, according to the authors, is 0.2 pg/l.
Another immunoenzymometric procedure for troponin I described by Bodor et al. makes it possible to detect the latter at concentrations of the order of to 3.1 ig/l [Bodor et al., Clin. Chem. (1992), 38/11, p. 2203-2214; Adams J. et al., Circulation (1993), 88(1), p. 101-106]. This assay uses, for the enzymatic visualization, a chromogenic substrate for alkaline phosphatase, p-nitrophenylphosphate, and the measurement is performed at 405 nm.
Using these assays, it has been possible to demonstrate the presence of troponin I in the plasma of patients about 4 hours after the onset of clinical manifestations of myocardial infarction [Adams J. et al., Circulation (1993), 88(1), p. 101-106].
However, the sensitivity of these immunoenzymometric assays is considerably less than that obtained by assays based on methods which are very sophisticated and difficult to use, for example assays based on the measurement of radioactivity [Am. Heart Journal, (June 1987), 113(6), p. 1333-1334].
It is also known that cyclic derivatives of hydrazines, especially diacylhydrazines, may be used as chemiluminescence reagents [Roswell et al., Methods Enzymol. (1978), 57, p. 409-423]. The use of these reagents has been envisaged or carried out during certain immunoenzymatic assays [Thrope G.H.G. et al., Clin.
Chem. (1985), 31, p. 1335-1341; Thrope G.H.G. et al., Medors. Enzymol. (1988), 133, p. 331-353] (Application WO 88/00695). Moreover, chemiluminescence detection methods are known which use the enzymatic decomposition of derivatives of 1,2-dioxetane.
However, it is known that the use of these reagents requires intensive research studies since persons skilled in the art are confronted with many limiting factors for the development of sensitive assays REPLACEMENT SHEET (RULE 26) 4 (affinity of the different assay components and in particular of the antigen/antibody system, enzymatic marker, substrate, principle of detection and the like). Indeed, a suitable choice of the different reagents and assay conditions is required. It is evident that it is not possible to imagine a sensitive and specific assay when the measurement background (background noise or blank background) is high or when the signal/noise ratio for the measurement is too small. Now precisely, this measurement background depends on the different reagents and assay conditions chosen.
It has now been found that it is possible, using a certain type of anti-cardiac troponin I monoclonal antibody and of suitable substrates for the enzymatic visualization, to carry out the chemiluminescent assay of troponin I with a very high sensitivity.
Indeed, by applying the process of the invention, it is possible to obtain a sensitivity of the order of 2 to ng/, this represents a sensitivity 40 to about 1500 times greater than those of the most sensitive immunoenzymometric procedures for troponin I known so far.
The present invention relates to a process for a sandwich-type immunoenzymatic assay allowing the quantitative assay of cardiac troponin I, wherein the substrates used for the enzymatic visualization are chemiluminescent substrates, selected from a large number of chemiluminescent substrates, in particular derivatives of a diacylhydrazine or of 1,2-dioxetane.
The subject of the present invention is more precisely a process for a sandwich-type immunoenzymatic assay of cardiac troponin I: which uses two anti-cardiac troponin monoclonal antibodies of which one is coupled with an enzymatic marker, which antibodies have either naturally or through cooperation, a high affinity for cardiac troponin I, and therefore an equilibrium dissociation constant equal to or less than 10- 8 M, preferably equal to or less than 10- 9
M,
REPLACEMENT SHEET (RULE 26) 0~ F and which uses for the enzymatic visualization a chemiluminescent substrate which is either a derivative of a diacylhydrazine or a derivative of 1,2-dioxetane.
Indeed, the present invention relates to a process for a sandwich-type quantitative immunoenzymatic assay of cardiac troponin I, wherein the samples to be assayed or the standards are brought into contact with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and with another anticardiac troponin I monoclonal antibody, these monoclonal antibodies, either naturally or through a cooperation effect, having an equilibrium dissociation constant for cardiac troponin I equal to or less than 10 M, preferably equal to or less than 10- 9 M, and in that the enzymatic visualization is then performed by addition of a selected chemiluminescent substrate and in that the light signal obtained is then directly read.
The process of the invention may be carried out either using a manual system (diagnostic assay kit) or using an automated system. Depending on the embodiment, it is possible to carry out either an assay in one stage or an assay in two stages.
For the assay in one stage, the samples to be assayed or the standards are brought into contact simultaneously with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and with a second anti-cardiac troponin I monoclonal antibody optionally bound to a solid support. After incubation and washing, the enzymatic visualization is performed.
This is carried out according to the invention by adding a chemiluminescent substrate derived from a diacylhydrazine or from 1,2-dioxetane. The quantity of cardiac troponin I present in the samples to be assayed or the standards is evaluated by direct reading of the light signal obtained.
REPLACEMENT SHEET (RULE 26) 6 For the assay in two stages, the samples to be assayed or the standards are brought into contact, either first with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and then, after incubation, with a second anti-cardiac troponin I monoclonal antibody optionally bound to a solid support and then another incubation is performed, or first with an anti-cardiac troponin I monoclonal antibody optionally bound to a solid support and then, after incubation and an optional wash, with a second anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and then another incubation is performed.
In both cases, an optional wash is carried out, followed by enzymatic visualization which is performed, according to the invention, by addition of a chemiluminescent substrate derived from a diacylhydrazine or from 1,2-dioxetane. The quantity of cardiac troponin I present in the samples to be assayed or the standards is evaluated by direct reading of the light signal obtained.
For the assay in two stages, preferably, the samples to be assayed and the standards are brought into contact first with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and then, after incubation, with a second anti-cardiac troponin I monoclonal antibody bound to a solid support and then incubated again.
As anti-cardiac troponin I monoclonal antibody, use may be made of any monoclonal antibody having a high affinity for cardiac troponin I and whose equilibrium dissociation constant is equal to or less than 8 M, preferably equal to or less than 10- 9 M. It is also possible to use any pair of monoclonal antibodies having cooperative binding in relation to cardiac troponin I, this cooperation producing, by way of result, an increase in the affinity of one of the two monoclonal antibodies for cardiac troponin I (equilibrium REPLACEMENT SHEET (RULE 26) 7 dissociation constant I 10 M) when the cardiac troponin I is already bound to the second antibody.
Preferably, as anti-cardiac troponin I monoclonal antibodies, use may be made of the mouse 11E12 and 8E1 monoclonal antibodies described in Clin. Chem.
(1993), 39, p. 972-979. Indeed, surprisingly, it has been observed that the 11E12 monoclonal antibody blocks the release of the cardiac troponin I bound to the 8E1 antibody. This phenomenon was demonstrated using the BIACORETM system from PHARMACIA, and by measuring the kinetic constants for association and dissociation of cardiac troponin I (cTnI) on the 8E1 monoclonal antibody, in the presence or absence of the 11E12 antibody.
The different results are given in Table I.
TABLE I AcM 8E1: cTnI alone AcM 8E1: cTnI+11E12 KINETIC CONSTANT FOR 3.2x10" 3 2.8x10- 4
DISSOCIATION
koff(S-l) KINETIC CONSTANT FOR 5.8x10 5 4.5x10
ASSOCIATION
kon (M-xS 1 EQUILIBRIUM 5.7x10- 9 0.6x10-9 DISSOCIATION CONSTANT koff/kon(M) The results in Table I demonstrate that for the 8E1 antibody, the affinity is 10 times higher if cardiac troponin I is in the presence of the 11E12 monoclonal antibody. More precisely, the equilibrium dissociation constant is 10 times lower (the kinetic constant for association does not vary). Cooperation therefore exists for the binding of the two anti-troponin I monoclonal antibodies in relation to cardiac troponin I. By virtue of this effect of "blocking" the dissociation of the complex, it is possible to carry REPLACEMENT SHEET (RULE 26) 8 out the process for immunoenzymatic assay, which is the subject of the present invention, with this pair of monoclonal antibodies.
For the formation of the conjugate anti-cardiac troponin I monoclonal antibody-enzymatic marker, either alkaline phosphatase or peroxidase is used as enzymatic marker.
Alkaline phosphatase is used when the chemiluminescent substrate is a derivative of 1,2-dioxetane and peroxidase when the chemiluminescent substrate is a derivative of a diacylhydrazine.
It is possible to use especially the monoclonal antibodies 8E1 or 11E12 to prepare the conjugates with an enzymatic marker. Preferably, conjugates of the 11E12 monoclonal antibody with peroxidase and conjugates of the 8E1 monoclonal antibody with alkaline phosphatase are prepared.
According to the embodiment, it is possible to consider as solid support either polystyrene tubes on which the anti-cardiac troponin I antibody is bound (case of the diagnostic kit), or particles which are magnetic (especially ferric) or otherwise, on which the anti-cardiac troponin I monoclonal antibody is bound (automated assay).
The anti-troponin I monoclonal antibody-enzymatic marker conjugates are prepared according to known methods Histochem. Cytochem. (1974), 22, p. 1084- 10911.
The preparation of the anti-troponin I monoclonal antibodies bound, for example, by covalent bonds to a solid phase is also carried out according to methods described in the literature Immunological Methods, (1979), 31, p. 231-236].
During the bringing of the samples to be assayed or of the standards into contact with an anticardiac troponin I monoclonal antibody and/or an anticardiac troponin I monoclonal antibody coupled to an enzymatic marker, the pH of the reaction medium should REPLACEMENT SHEET (RULE 26) 9 be preferably slightly acidic. The pH should be in particular between 5 and 6, more precisely between 5.4 and 5.7. To adjust the pH, it is possible to use different suitable buffers or solutions of weak organic acids, for example succinic acid solutions or buffers having a pH of 5.2 to 5.4.
The incubation between the plasma or the serum and the anti-troponin I antibodies or the standards is carried out between 20 0 C and 370C and the incubation time may vary between 5 and 20 minutes.
After incubation, one or more washes are carried out at a temperature of 20 to 37°C, preferably at a temperature less than 10 0 C. For the washes, a phosphate buffer solution pH 6.8 or a Tris buffer solution pH8 is preferably used.
As indicated above, as luminescent substrate, either a diacylhydrazine derivative, more especially luminol [5-amino-2,3-dihydro-1,4-naphthalazinedione], or a 1,2-dioxetane derivative, more especially the lumigen PPD [sodium salt of 4-methoxy-4-(3-phosphatephenyl)spiro(1,2-dioxetane-3-adamantane-2')]. Luminescent substrates containing luminol are available commercially such as, for example, the luminol ECL-immunoassay signal reagent RPN 190 marketed by Amersham or the luminol BM Chemiluminescence ELISA reagent 1582950 marketed by Boehringer Mannheim. Substrates which contain a mixture of luminol and 4-iodophenol are preferred. Luminescent substrates containing lumigen PPD are also available commercially such as, for example, Lumi-Phos 530 marketed by Analytical Luminescence Laboratory. This reagent contains lumigen PPD and a chemiluminescence promoter which is a surfactant derived from fluorescein.
Enzymatic visualization is also performed at a temperature of between 20 0 C and 370C. The reading of the chemiluminescence is carried out preferably after about 2-10 minutes.
REPLACEMENT SHEET (RULE 26) 10 To prepare the calibration series for a quantitative assay, standard solutions (standards) of purified human cardiac troponin I are used [Larue C. et al., Clin. Chem. (1993), 39, p. 972-979].
The invention also relates to the use of luminol and the lumigen PPD as chemiluminescent substrates for the sandwich-type immunoenzymatic assay of cardiac troponin I using two anti-cardiac troponin I monoclonal antibodies of which one is coupled with an enzymatic marker, in particular peroxidase, if the substrate is luminol, and alkaline phosphatase, if the substrate is lumigen PPD, which antibodies (either naturally or through cooperation) have, for cardiac troponin I, an equilibrium dissociation constant equal to or less than 10- 8
M.
The subject of the invention is also a kit for assaying cardiac troponin I comprising two anti-cardiac troponin I monoclonal antibodies of which one is coupled with an enzymatic marker, which antibodies, either naturally or through cooperation, have for cardiac troponin I an equilibrium dissociation constant equal to or less than 10 9 M, and a chemiluminescent substrate derived from a diacylhydrazine, preferably luminol or a 1,2-dioxetane derivative, preferably the lumigen PPD.
The following examples, given with no limitation being implied, illustrate the invention.
EXAMPLE 1 Assay of cardiac troponin I with an automated system The automated system used for the immunoenzymatic assay is the Access® immunoassay system, a system marketed by Sanofi Diagnostics Pasteur.
The assay is carried out in the following manner: into the assay well are introduced 50 A 1 of the sample to be assayed, 25 il of a mouse immunoglobulin solution at 4 mg/ml, 10 Al of a 0.1 M succinic acid solution, 50 l of the mouse anti-cardiac troponin I REPLACEMENT SHEET (RULE 26) 11 monoclonal antibody 8El-alkaline phosphatase conjugate (C 5 g/ml).
After incubating for 5 minutes at 37 0 C, 50 p1 of ferric latex beads (Rh6ne Poulenc ref. MI-070/60) are introduced, on which mouse anti-cardiac troponin I monoclonal antibodies 11E12 are bound by covalent bonds.
The mixture is incubated for 36 seconds at 37 0 C, then the ferric latex beads are separated with the aid of a magnetic field. The washing is carried out by means of a Tris buffer solution pH 8 and 200 pl of Lumi-PhosTM 530 substrate are then added.
The visualization is performed at 37 0 C and the luminescence generated by the reaction is measured with a luminometer.
The total analysis time is 15 minutes.
For the calibration series, purified human cardiac troponin I solutions are used.
A calibration series is prepared corresponding to concentrations of between 0 and 50 g/l.
The sensitivity of the process, evaluated by the calibration curve, is 3.5 ng/l.
EXAMPLE II Assay of cardiac troponin I with an immunoenzymatic kit In polystyrene tubes coated with mouse anticardiac troponin I monoclonal antibodies 8E1, 150 il of a succinate buffer solution containing 20 to 0.2% Tween, nonspecific mouse immunoglobulins and 0.1% Kathon, 50 1l of the mouse anti-cardiac troponin I monoclonal antibody 11E12-peroxidase, and 200 1l of sample to be assayed and of a standard solution used for the calibration series.
For the calibration series, human serum containing 0 to 2 Ag/l of purified human cardiac troponin I, is used.
REPLACEMENT SHEET (RULE 26) 12 Incubation is carried out for exactly minutes with horizontal stirring, at room temperature, then the tubes are washed in the following manner: the content of the tubes is poured into a container and the tubes are wiped on an absorbent paper, the washing is carried out by adding 1 ml of 0.1 M phosphate buffer pH 6.8, containing 0.1% Tween and 0.3% Kathon, the temperature being kept at at most 8 0 C. This step is repeated 3 times.
After carrying out the washing and removing any trace of the solution used for the latter, the enzymatic visualization is performed using 300 pl of a mixture consisting of 100 parts of luminol (BM chemiluminescent ELISA reagent Boehringer Mannheim) and 1 part of hydrogen peroxide.
The medium is left at room temperature and the luminescence is measured with a luminometer after 3 minutes. Each tube is read for exactly 30 seconds and exactly 10 seconds.
The sensitivity of this assay is 3 ng/l.
The values of the net signal/noise ratios for the measurement which are obtained according to the procedure described above and according to the procedure described by Larue C. et al., [Mol. Immunol.
(1992), 29(2), p. 271-278; Clin. Chem. (1993), 39/6, p. 972-979] are given in Table II below.
TABLE II Cardiac troponin Procedure of the Procedure described by I concentration example Larue et al.
100 ng/1 S-N 1200- 95;95 11 S-N 150-502 N 95 N ng/1 S-N 250-95 S-N N -N 95=N1.6 -0 (S=N) N 95 N The values of the signal/noise ratios for the Smeasurement obtained by applying the process of the REPLACEMENT SHEET (RULE 26) P:\OPER\MJC\46251-96.SPE 20/4/98 13 invention prove its high sensitivity and its specificity.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.
e

Claims (20)

1. Process for the sandwich-type immunoenzymatic assay of cardiac troponin I, which uses two anti-cardiac troponin I monoclonal antibodies of which one is coupled with an enzymatic marker, which antibodies either naturally or through cooperation, have for cardiac troponin I an equilibrium dissociation constant equal to or less than 10-8 M, and which uses for the enzymatic visualization a chemiluminescent substrate derived from a diacylhydrazine or from 1,2-dioxetane.
2. A process according to claim 1 wherein the antibodies have, either naturally or through cooperation, for cardiac troponin I an equilibrium dissociation constant equal to or less than 10 9 M.
3. Process according to claim 1 or claim 2 comprising the following steps: 404 a S 6 t *C S S C S. S S S a 5 a a. a 20 b c the samples are brought into contact with an anti- cardiac troponin I monoclonal antibody coupled to an enzymatic marker and with a second anti-cardiac troponin I monoclonal antibody, the enzymatic visualization is performed by addition of a chemiluminescent substrate derived from a diacylhydrazine or from 1,2-dioxetane, the quantity of cardiac troponin I is evaluated by direct reading of the light signal obtained.
4. Process according to any one of claims 1 to 3, comprising the following steps: a the samples to be assayed or the standards are brought 7 P:\OPER\MJC\46251-96.SPE 20/4/98 into contact simultaneously with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and with a second anti-cardiac troponin I monoclonal antibody optionally bound to a solid support, b the reaction mixture is incubated, c then washed, d the enzymatic visualization is performed by addition of a chemiluminescent substrate derived from a 10 diacylhydrazine or derived from 1,2-dioxetane, :e the quantity of cardiac troponin I present in the samples to be assayed or the standards is evaluated by direct reading of the light signal obtained. a. 15
5. Process according to any one of claims 1 to 3, comprising the following steps: a the samples to be assayed or the standards are brought into contact, S- either first with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and then, after incubation with a second anti-cardiac troponin I monoclonal antibody optionally bound to a solid support, and incubated again, or first with an anti-cardiac troponin I monoclonal antibody optionally bound to a solid support and then, after incubation and an optional wash, with a second anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and incubated again, b the reaction mixture is then optionally washed, c the enzymatic visualization is performed by addition P:\QPER\MJC\46251-96.SPE 20/4/98 -16- of a chemiluminescent substrate derived from a diacylhydrazine or derived from 1,2-dioxetane, d the quantity of cardiac troponin I present in the samples to be assayed or the standards is evaluated by direct reading of the light signal obtained.
6. Process according to any one of claims 1 to 5, wherein the chemiluminescent substrate is luminol. 9e 9 10
7. Process according to any one of claims 1 to 5, wherein the chemiluminescent substrate is the lumigen PPD. *999 *999
8. Process according to any one of claims 1 to 5, wherein the anti-troponin I monoclonal antibodies are monoclonal antibodies 15 having the characteristics of the mouse monoclonal antibodies 11E12 and 8E1.
9. Process according to any one of claims 1 to 5, wherein the enzymatic marker coupled to the anti-troponin I monoclonal antibody is alkaline phosphatase or peroxidase.
Process according to any one of claims 1 to 4, wherein the anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker is a conjugate anti-cardiac troponin I monoclonal antibody peroxidase, the said antibody having the characteristics of the mouse monoclonal antibody 11E12 and the chemiluminescent substrate is a derivative of a diacylhydrazine.
11. Process according to any one of claims 1 to 3 or 5, wherein the anti-cardiac troponin I monoclonal antibody coupled to an P:\OPER\MJC\46251-96.SPE 20/4/98 -17- enzymatic marker is a conjugate anti-cardiac troponin I monoclonal antibody alkaline phosphatase, the said antibody having the characteristics of the mouse monoclonal antibody 8E1 and the chemiluminescent substrate is a derivative of 1,2- dioxetane.
12. Process according to any one of claims 1 to 6, wherein the chemiluminescent substrate is a mixture of luminol and 4- iodophenol.
13. Process according to any one of claims 1 to 5, wherein the samples to be assayed or the standards are brought into contact with an anti-cardiac troponin I monoclonal antibody coupled to an enzymatic marker and optionally to a second anti-troponin I 15 antibody at a slightly acidic pH of between 5 and 6.
14. A process of claim 13 wherein the pH is between 5.4 and S5.7.
15. Process according to any one of claims 1 to 5, wherein the washing solutions used before visualization have a pH equal to 6.8-8 and a temperature of 2 0 to 37 0 C.
16. A process according to claim 15 wherein the washing solutions have a temperature of less than 10 0 C.
17. Use of luminol as chemiluminescent substrate for the sandwich-type immunoenzymatic assay of cardiac troponin I using two anti-cardiac troponin I monoclonal antibodies of which one is coupled with peroxidase, antibodies, either naturally or PAOPER\MJC\46251-96.SPE 10/2199
18- through cooperation, have for cardiac troponin I an equilibrium dissociation constant equal to or less than 10- 8 M. 18. Use of the lumigen PPD as chemiluminescent substrate for the sandwich-type immunoenzymatic assay of cardiac troponin I using two anti-cardiac troponin I monoclonal antibodies of which one is coupled with alkaline phosphatase, which antibodies, either naturally or through cooperation, have for cardiac troponin I an equilibrium dissociation constant equal to or less 10 than 10- 8 M. 0 S
19. Kit when used in the process of claim 1 said kit comprising two anti-cardiac troponin I monoclonal antibodies of which one is coupled with an enzymatic marker, which antibodies, either naturally or through cooperation, have for cardiac troponin I an equilibrium dissociation constant equal to or less than 10- 9 M, 0 and a chemiluminescent substrate derived from a diacylhydrazine or from 1,2-dioxetane. 0
20. A process according to claim 1 substantially as S hereinbefore described with any one of the examples. DATED this 10th day of February, 1999 Pasteur Sanofi Diagnostics by DAVIES COLLISON CAVE Patent Attorneys for the Applicants
AU46251/96A 1995-01-19 1996-01-19 Ultrasensitive process for assaying cardiac troponin I Ceased AU703944B2 (en)

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FR9500597A FR2729759A1 (en) 1995-01-19 1995-01-19 ULTRASENSITIVE DOSING METHOD FOR THE CARDIAC TROPONIN I
FR9500597 1995-01-19
PCT/FR1996/000095 WO1996022535A1 (en) 1995-01-19 1996-01-19 Ultrasensitive process for assaying cardiac troponine i

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WO2013094080A1 (en) * 2011-12-20 2013-06-27 パナソニック株式会社 Recombinant protein capable of specifically and rapidly binding to troponin-i originated from human cardiac muscles
WO2013099043A1 (en) * 2011-12-26 2013-07-04 パナソニック株式会社 Recombinant protein capable of specifically and rapidly binding to troponin-i from human cardiac muscles
CN102735848A (en) * 2012-07-05 2012-10-17 北京源德生物医学工程有限公司 Enzymatic chemiluminescence immunodetection method and reagent kit for human troponin I
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