FR2774473A1 - METHOD FOR DETERMINING TROPONINS IN BIOLOGICAL MEDIA TO AVOID INTERFERENCE DUE TO HEPARIN - Google Patents

Abstract

The invention concerns an immunoassay method for troponin I, cardiac or skeletal, T or C and troponin I, T or C mutually associated in the form of IT, IC or TC or in the form of ITC trimer in a biological sample containing heparin, characterised in that the immunoassay is carried out in the presence of polybrene.

Description

- 1 - The present invention relates to a process for the determination of troponins

  in biological media to avoid interference due

with heparin.

  We know that troponin is a myofibrillary protein complex, consisting of three proteins, troponins 1, T and C. This protein complex allows 24- to contribute to the regulation of muscle contraction by the Ca2 ion, by interacting with the myosin and actin. More precisely, it is known that when a nerve impulse arrives at the level of the motor plaque of a muscle, there is [(generation of an action potential which is transmitted to the sarcoplasmic reticulum. The Ca2 + is then released in the cytosol and binds to troponin C, which leads to a strengthening of the interaction between troponin I and troponin C and, consequently, a change in conformation of the troponin 1, T, C complex. release of actin - myosin interaction sites, allowing movement of

is muscle contraction.

  When the muscle is damaged, irreversibly, it is the heart muscle, during myocardial necrosis following a myocardial infarction, or it is the skeletal muscle during prolonged physical exertion, the troponins then released appear (more or less

  quickly) into the bloodstream.

  Thus, the dosage of troponin has recently been advocated for the early diagnosis of myocardial infarction, whether that of troponin T in Circulation (1991,) 83, pp. 902-912, or troponin I in Am. Heart J.

  (1987), 110, pp. 1333-1344, and Molecular Immunology (1992), 29 (2), pp. 271-278.

  Similarly, the assay of cardiac troponin T has been proposed to measure the success of thrombolytic therapy following a myocardial infarction in Br. Heart J., (1994) ,, pp. 242-248, as well as the assay of skeletal troponin I for the measurement of damage to skeletal muscles (D. Rama et al, Clinical Chemistry (1996), 42 no 12 p. 2033). It should be noted that the dosage of -2- different cardiac and skeletal troponins is today a very

  useful for the diagnosis of various human and animal pathologies.

  To evaluate the different cardiac troponins, we use

  usually blood samples (serum, plasma or whole blood).

  However, this choice may be limited by the method implemented, since it is known that the serum is an inappropriate biological sample for certain methods for evaluating troponin, for example with rapid methods for evaluating troponin T, and that whole blood does not always allow a quantitative assay to be made. In the case of immunoassays carried out with heparin-containing plasma, there are often unreliable results, even when the methods used are very effective. This problem is generally encountered in the case where the concentration of troponins in the plasma is low (P. O. Collison et al., Ann. Clin. Biochem., (1995), 32, pp. 1t 454-458). Indeed, it is known that the presence of heparin in the blood sample can interfere with the various immunoassays and thus considerably modify the final result and, therefore, the practitioner's clinical diagnosis. However, the dosage of troponins is carried out for the diagnosis or monitoring of serious diseases which requires obtaining very precise results. Therefore, the use of material containing heparin (heparinized tubes etc.) must often be avoided both for biological sampling and for

perform immunoassays.

  In addition, before angioplasty or after a myocardial infarction or during certain treatments for cardiovascular conditions, patients are administered significant amounts of heparin and this treatment is often extended beyond 24 hours. Depending on the dose administered, the concentration of heparin in the plasma one hour after administration can vary between 0.1 to 2 IU / ml. Presence of heparin in blood samples due to patient treatment is a problem

  important for the dosage of troponins.

  A number of products are known which are capable of either degrading or capturing heparin such as heparinase 1, L-histidine, protamine sulphate, cation exchange resins, etc. These products are commonly used in biology as heparin inhibitors. However, their use during the implementation of an immunoassay does not always make it possible to obtain the suppression of the interference due to heparin and often their

  presence during immunoassay may generate additional interference (E.

  W. Nielsen et al., J. Immunological Methods, (1994), 173, pp. 245-251).

  I () It has now been found that it is possible to avoid interference due to heparin during immunoassays of the various troponins. Thanks to the invention, we are now able to carry out the determination of troponins on heparinized biological samples or from patients treated with heparin with very high precision, without decreasing the actual values found in

  blood circulation regardless of the immunological process used.

  The present invention relates more specifically to an immunological assay of troponin I (cardiac or skeletal), T or C, and 2o of troponins 1, T or C associated with one another in the form of dimers (IT, IC or TC) or under form of trimer (ITC) in a biological sample containing heparin, characterized in that the immunoassay is carried out

  presence of hexadimethrin bromide (polybrene).

  The amount of polybrene used during the immunoassay of troponin I (cardiac or skeletal), T or C, and troponins 1, T or C associated with one another in the form of dimers (IT, IC or TC) or under Trimer form (ITC) may vary depending on the immunoassay procedure used. Advantageously, the ratio [molar concentration of the polybrene used / molar concentration of heparin present in the sample to be analyzed] can be from 1 to 1000. Depending on the dosage, it can be more especially -4- to 300, preferably from 60 to 180 or even only from 1 to 10, from

preferably 4 to 10.

  The polybrene can be added directly to the biological sample containing or likely to contain heparin or it can be further added to one of the immunoassay reagents such as buffer solutions, solution of the conjugate reagent, etc. It is preferred to add the polybrene in buffer solutions used during the immunoassay. The term buffer solution includes any solution used during the first immunoassay steps present I) together with the plasma sample. The washing solutions are

excluded from this term.

  The present invention can be implemented with any immunological method allowing the evaluation of troponin I (cardiac or In skeletal), T or C, and troponins 1, T or C associated with each other in the form

  of dimers (IT, IC or TC) or in the form of a trimer (ITC).

  According to the invention, immunoenzymatic methods are preferred which allow the determination of troponin I (cardiac or skeletal), T or C or of dimers or trimers thereof in a biological sample, which use polybrene to suppress interference due to Heparin. Among these methods, the sandwich type methods are preferred. The sandwich type processes can be carried out in one or more stages (two stages, three stages

  etc.) and use two or more monoclonal or polyclonal antibodies.

  2S5 Immunoenzymatic methods are already known allowing the determination of troponin I. C. Larue et ai in Mol. Immunol. (1992), 29 (2), pp. 271-278 and Clin. Chem. (1993), 39/6, pp. 972-979 describe a sandwich enzyme immunoassay method which uses the chromogenic substrate tetramethylbenzydine (TMB) - H202 for enzymatic development. After the revelation, the absorbance is read at 450 nm and the detection limit, -5- according to the authors, is 0.2 pg / l. A dosing kit implementing this method is commercially available (Sanofi Diagnostics Pasteur, Marnes-la Coquette, France). The detection limit of this kit is of the order of 0.03 to 0.04 pg / l (J. Mair et al. Clin. Chim. Acta. (1996), 245, pp. 19-38) Another immunoenzymometric method of troponin I is described by Bodor et al. This method makes it possible to detect the latter at concentrations of the order of 115 to 3.1 pg / l (Bodor et al, C / lin. Chem. (1992), 38/11, p. 2203-2214; Adams J . et ai, Circulation (1993), 88 (1), pp. 101-106). This assay I (uses for the enzymatic revelation a chromogenic substrate of phosphatase

  alkaline, p-nitrophenylphosphate, and the measurement is carried out at 405 nm.

  Patent application WO 96/22535 also describes an immunoenzymatic assay method of the sandwich type allowing the quantitative assay of cardiac troponin I. This process uses for the enzymatic revelation a chemiluminescent substrate, chosen from derivatives of a diacylhydrazine, either a derivative of 1,2-dioxetane and can be carried out either by a manual system (diagnostic assay kit), or by a system automated. Depending on the embodiment, it is possible to carry out either a metering in a time, or a metering

in two times.

  The immunoenzymatic method used for the automated quantitative determination of troponin I and marketed by Dade Diagnostics, Munich Germany (L.P. Kuhr et al, Eur. J. Chem. Clin.

Biochem. (1997), 35 (5), p. 399-404).

  A kit allowing the immunoenzymatic assay of troponin T is also commercially available. This is the "Troponin T / ES 300 Analyzer" kit sold by Boehringer Mannheim, Mannheim Germany (N. Genseret al, Clin. Chem. Acta (1997), 265, pp. 207-217;

  3) H. Baum et ai, C / lin. Chem. (1997), 43/10, p. 1877-1977).

  Patent application WO 96/33415 also describes sandwich-type immunoassays making it possible to evaluate the amounts of troponins I and T or of complexes of troponins 1, C and / or T in biological media. A method of immunoenzymatic assay of troponin I

  skeletal is described by Rama et al in Clin. Chem. (1996), 42 no 2, p. 2033.

  The immunoenzymatic methods for the evaluation of troponin 11, cardiac or skeletal, T or C in individual proteins, dimeric or tridimeric, mentioned above are given by way of example and do not constitute an exhaustive list of the immunoenzymatic methods known troponins

by the skilled person.

  The method according to the invention can be applied as indicated below.

  above to any type of immunological process of troponins.

  For example, the method according to the invention can be applied during immunoassays (for example as described by C. Larue et al in L'information

  2 cardiology (1991) vol. XV n 1, pp. 17-21) and fluoroimmunoassays (L.

  Bellanger et al., Clin. Chem. (1997), vol 43 no 6, p. S159, no 240).

  The use of polybrene during immunoassay of troponin I (cardiac or skeletal), T or C, and troponins 1, T or C associated together in the form of dimers (IT, IC or TC) or in the form of trimer (ITC), to avoid interference from heparin is part of the

present invention.

  The invention also relates to immunoassay kits for troponin 1 (cardiac or skeletal), T or C and troponins 1, T or C associated with each other in the form of dimers (IT, IC or TC) or in the form of trimer (ITC),

  containing among the polybrene immunoassay reagents.

  The following examples are given without implied limitation to illustrate the invention.

EXAMPLE I

  I (Determination of cardiac troponin I using serums and Piasmas overloaded with heparin - treatment of samples with different

heparin inhibitors.

  Human sera possibly containing troponin I were overloaded with heparin, part of the sera without heparin was kept as a control and assayed at the same time. Different heparin inhibitors (heparin lithium) have been tested, in particular heparinase I (heparinase I from Flavobacterium heparinum Sigma ref. H 2519), L-histidine (Sigma ref. H 8000), 20. protamine sulfate ( protamine sulfate grade X from salmon Sigma ref. P 4020), antithrombin III (antithrombin III from human plasma Sigma ref. A 7388) and cation exchange resins QSFF (Q Sepharose Fast Flow, Pharmacia Biotech ref. 17051001) and DEAE (DEAE Sepharose Fast Flow, Pharmacia Biotech

ref. 17070901).

  The automated system used for the enzyme immunoassay is the Access immunoassay system, system marketed by Beckman. The kit used is the Access-Troponin I kit, marketed by the same company. The assay is carried out as follows: into the assay cup are introduced 50 μl of the sample to be assayed, 25 μl of a solution of mouse immunoglobulins at 4 mg / ml, 10 μl of a 0.1 M succinic acid solution containing the heparin inhibitor and 50 pI of the anti-troponin I monoclonal antibody conjugate 8El mouse cardiac alkaline phosphatase

(concentration: 5 pg / ml).

  After incubation for 5 minutes at 37 ° C., 50 μl of ferric latex beads are introduced (Rhône Poulenc ref. MI-070/60) to which are fixed by covalent bonds cardiac anti-troponin I monoclonal antibodies

mouse 1 1 E12.

  The mouse cardiac anti-troponin I antibodies 8E1 WIc and 11E12 are part of the Troponin I kit. The mixture is incubated for 36 seconds at 37 ° C., then the ferric latex beads are separated using a magnetic field. The washing is carried out using a Tris pH 8 buffer solution, then 200 μl of

  Lumi-Phos 530 substrate. This substrate is also part of the Access-

  1 Troponin I. The revelation is carried out at 37 ° C. and the luminescence is measured

  generated by the reaction with a luminometer.

  The total analysis time is 15 minutes.

  Troponin I solutions are used for the calibration range

human heart.

  A calibration range corresponding to

  concentrations between 0 and 50 pg / l.

  The results of this study are given in Tables I to VI below.

after.

  -9- TABLE I: Heparinase I test Without Heparinase I | Heparinase I heparinase I 2 IU / mi 7 IU / ml Sample C ** (ng / ml) C ** (ng / mi) C ** (ng / ml) Tnl negative serum * 0.005 0.000 0.008 TnI negative serum + heparin 0.005 0.000 0.011 IU / ml TnI extract + diluent 7.929 8,646 9,148 TnI extract + heparin 30IU / ml 2,225 2,506 '. 742 TnI extract + heparin 60UI / ml 1,913 2,052., 2 05 Serum positive in TnI + diluent 1,002 1.03 5 0.975 Serum positive in TnI + heparin 0.231 0.252 0.331 IU / ml] _ Serum positive in TnI + heparin 0.175 0.216 0.286 IU / ml 'I * TnI = Troponin I ** C = troponin concentration I TABLE II: Test with L-histidine I () Without L-histidine L- histidine L-histidine x 1000 '** x 3000 *** Sample C ** (ng / ml) IC ** (ng / mi) C ** (ng / ml) TnI negative serum * 0.014 0.011 0.016 TnI negative serum + heparin 20UI / ml 0.012 0.011 0.010 Tnl negative serum + heparin 40UI / mli 0.015 0.013 0.014 TnI + extract diluent 5,440 5,312 '4,794 TnI extract + hepari ne 20UI / ml 1,723 1,721 1,617 TnI extract + heparin 40UI / ml 1,497 1,403 1,498 TnI positive serum + diluent 0.861 0.872_ 0.925 TnI positive serum + heparin 20UI / ml 0.256 0.264 0.317 TnI positive serum + heparin 0.2U 0.249 0.290 * TnI = Troponin I * C = troponin I concentration ** mol L- histidine / mol heparin -10-

TABLE III

  Protamine sulphate test (protamine) Without Protamine Protamine protamine x 1 ** x 5 *** Sample C ** (ng / ml) C ** (ng / ml) C (ng / mi) Serum negative in TnI * 0.010 0.000 [0.000 TnI negative serum + heparin 40IU / ml 0.020 0.000 0.000 TnI extract + diluent 0.310 02 000 0.050 TnI extract + heparin 40UI / ml 0.070 0.00 0.000 TnI positive serum + diluent 0.570 0.340 0.100 Serum positive in TnI + heparin 40IU / ml 0.120 0.05 d 0.000 * TnI = Troponin I * C = concentration in troponin I *** mol protamine sulfate / mol heparin

TABLE IV

  Antithrombin III test Without Antithrombin Antithrombin antithrombin III 11 11I 2UI / ml 7UI / ml Sample C ** (ng / ml) C ** (ng / ml) C ** (ng / ml) Negative serum in TnI * 0,000 0.000 0.000 TnI negative serum + heparin 0.000 0.000 0.000 U1 / ml TnI extract + diluent 11,600 12,130 11,370 TnI extract + heparin 30UI / ml 2,800 3,130 2,830 TnI positive serum + diluent 0.800 0.830 0.830 Positive TnI serum 0.200 200 0.2 IU / ml * TnI = Troponin I ** C = troponin I concentration

- 11 -

TABLE V

  Test with the cation exchange resin QSFF Sample C ** (ng / ml) Negative serum in TnI * r 0, 000 Negative serum in TnI + heparin 40UI / ml 0.010 Negative serum in TnI + heparin 40UI / ml + resin 50mg 0,000 Serum TnI negative + SOmg resin 0.000 TnI positive serum + diluent 0.950 TnI positive serum + heparin 40UI / ml 0, 180 TnI positive serum + heparin 40UI / ml + 50mg resin 0.110 TnI positive serum + resin 50mg 0.960 TnI extract + diluent 5,270 TnI extract + heparin 40IU / ml 1,580 TnI extract + heparin 40UIml + resin 50mg 1,240 TnI extract + resin 50mg 5,160 * TnI = Troponin I ** C = troponin I concentration

TABLE Vl

  DEAE cation exchange resin test Sample C ** (ng / ml) TnI negative serum * 0.010 TnI negative serum + heparin 40IU / ml 0.010 TnI negative serum + heparin 40UI / ml + mg resin 0.018 Negative serum TnI + resin 1 mg 0.014 TnI positive serum + diluent 0.910 TnI positive serum + heparin 40UI / mlI 0.200 TnI positive serum + heparin 40UI / ml + resin I mg 0.310 TnI positive serum + Img resin 0.410 TnI extract + diluent 6.080 TnI extract + heparin 40UI / ml 1.540 TnI extract + heparin 40U / ml + resin l mg 2.610 TnI extract + resin lmg 3.600 I () * TnI = Troponin I ** C = concentration of troponin I -12 - The results of this study demonstrates that neither the heparin inhibitors used nor the cation exchange resins make it possible to inhibit interference due to heparin. In addition, it has been observed that protamine sulphate I causes a significant increase in background noise (results not

mentioned in Table III).

EXAMPLE II

  Assay of cardiac troponin I with an enzyme immunoassay using polybrene to avoid interference due to heparin For the immunoassay, the Troponine I Pasteur Ji kit was used (Sanofi Diagnostics Pasteur, Marnes-la Coquette, France) . The assay is carried out as follows:

  In polystyrene tubes coated with anti-monoclonal antibodies

  cardiac troponin I of mouse 8E1, 150 μl of a succinate buffer solution comprising polybrene and containing 0.2% Tween 20, 2 of non-specific mouse immunoglobulins and 0.1% of Kathon, 50 μl, are introduced of

  monoclonal antibody anti-troponin I mouse heart 1 1E12-

  peroxidase, and 200 μl of sample to be assayed or of a standard solution used for the

calibration range.

  For the calibration range, human serum containing

  0 to 1.74 pg / I of purified human cardiac troponin I.

  The incubation is carried out for 15 minutes precisely with horizontal stirring at ambient temperature, then the tubes are washed as follows: - the contents of the tubes are inverted in a container and the tubes are sponged on absorbent paper,

- 13 -

  washing is carried out by adding 1 ml of 0.1 M phosphate buffer, pH 6.8 containing 0.1% Tween 20 and 0.3% Kathon, keeping the temperature of the washing solution at most at 8 C. This step is repeated 4 times. After washing and removing all traces of the solution used for the latter, the enzymatic development is carried out using 600 μl of a mixture consisting of 1 part of tetramethylbenzydine and 100 parts of

  citrate buffer containing 4% DMSO and 0.03% hydrogen peroxide.

  tff It is left at room temperature and in the dark for 15 minutes

  and the absorbance is measured at X = 450 nm.

  The results of this study are shown in Table VII.

TABLE VII

  Without Polybrene polybrene x10 ** Sample C ** (ng / ml) C ** (ng / ml) TnI negative serum * 0.000 0.020 TnI negative serum + heparin 40UI / ml 0.000 0.000 TnI extract + diluent 0.950 0.990 TnI + heparin 40IU / ml 0.410 1.040 Positive serum in TnI + diluent 1.160 1.240 Positive serum in TnI + heparin 40UIml 0.480 1.170 * TnI = Troponin I ** C = concentration in Troponin I *** mol polybrene / mol heparin The results in table VII demonstrate that polybrene avoids interference due to heparin. A molar concentration 10 times higher than that of the heparin present in the sample is necessary when the troponin I assay is carried out with the Troponine I Pasteur kit (Sanofi

  Diagnostics Pasteur, Marnes-la Coquette, France).

EXAMPLE III

  Assay of cardiac troponin I with an automated system o The automated system used for the enzyme immunoassay is the Access immuoassay system, system marketed by Beckman. The immunoassay method is that described in Example I. Plasmas from five patients containing heparin were analyzed. The results of this study are given in Table VIII. From this study it emerges that when the automated system described above for the determination of Troponin I is used, it is necessary to use from 60 to 120 mol of

  polybrene per mol of heparin present in the sample.

TABLE VIII

  Polybrene free Polybrene Polybrene Polybrene mols / mol 90 mols / mol 120 months / mol heparin heparin heparin Sample C * (ng / ml) C * (ng / ml) C * (ng / ml) C * ( ng / ml)

1 0.139 0.649 0.800 0.868

2 0.066 0.439 0.520 0.595

3 0.044 0.441 0.541 0.585

4 0.046 0.486 I 0.660 0.736

0.085 0.367 0.471 0.570

  * C = Troponin I concentration -15 -

Claims (1)

CLAIMS> 1. Immunological assay method for troponin 1, cardiac or skeletal, T or C and troponins 1, T or C associated together in the form of IT, IC or TC dimers or in the form of ITC trimer in a biological sample containing heparin, characterized in that the immunoassay is carried out in the presence of polybrene. I) 2. Method for the immunological assay of troponin 1, cardiac or skeletal, T or C and of troponins 1, T or C associated together in the form of IT, IC or TC dimers or in the form of ITC trimer according to claim 1 characterized in that the ratio [molar concentration of the polybrene used 1In / molar concentration of heparin present in the sample to be analyzed] is from 1 to 1000. 3. Immunological assay method according to claims 1 or 2 characterized in what the ratio [molar concentration of the polybrene used / molar concentration of heparin present in the sample to be analyzed] is   1 to 10, preferably 4 to 10.   4. Immunological assay method according to claims 1 or 2   characterized in that the ratio [molar concentration of the polybrene used / molar concentration of heparin present in the sample to be analyzed] is   to 300, especially from 60 to 180.   5. Method according to any one of claims 1 to 4   characterized in that the polybrene is added in solutions used during   of the first steps in immunoassay. - 16-   6. Method according to any one of claims 1 to 5   characterized in that the determination of troponin 1, cardiac or skeletal, T or C and of troponins 1, T or C associated together in the form of dimers IT, IC   or TC or as ITC trimer is an enzyme immunoassay.   7. Method according to any one of claims 1 to 6,   characterized in that the immunoenzymatic process is of the sandwich type.   o 8. Use of the polybrene during an immunological assay of troponin 1, cardiac or skeletal, T or C and of troponins 1, T or C associated together in the form of IT, IC or TC dimers or in the form of trimer   ITC to avoid interference from heparin.   Ji 9. Immunoassay kits for troponin 1, cardiac or skeletal, T or C and troponins 1, T or C combined together in the form of IT, IC or TC dimers or in the form of ITC trimer containing among the reagents polybrene immunoassay.