Patent Grant
6867011
The present invention relates to synthetic biepitope compounds which can be used as standards in immunoassays, for the assay of troponin I, method for preparing them, compositions and kits containing such compounds as well as immunoassay methods using such compounds.
It is known that troponin is a myofibrillar protein complex consisting of three proteins, troponins I, T and C. This protein complex makes it possible to contribute to the regulation of muscle contraction by the Ca2+ ion by interacting with myosin and actin. More precisely, it is known that when a nerve impulse arrives at the level of the motor endplate of a muscle, there is generation of an action potential which is transmitted to the sarcoplasmic reticulum. Ca2+ is then released into the cytosol and binds to troponin C, which brings about a strengthening of the interaction between troponin I and troponin C and, consequently, a change in conformation of the troponin I, T, C complex. There is then liberation of the sites of actin-myosin interaction, which allows the muscle contraction movement.
When the muscle is damaged, whether it is the cardiac muscle, during myocardial necrosis following a myocardial infarction, or whether it is the skeletal muscle during sustained physical effort, the troponins then released appear more or less rapidly in the blood stream.
Thus, the assay of troponin has recently been recommended for the early diagnosis of myocardial infarction, whether that of troponin T in Circulation (1991,) 8, pp. 902-912, or of troponin I in Am. Heart J. (1987), 110, pp. 1333-1344, and Molecular Immunology (1992), 29 f 2), pp. 271-278. Likewise, the assay of cardiac troponin T for measuring the success of thrombolytic therapy following a myocardial infarction has been proposed in Br. Heart J., (1994), 7, pp. 242-248, as well as the assay of skeletal troponin I for the measurement of damage to the skeletal muscles (abstract No. 35 of the American Association for Clinical Chemistry, 46th National Meeting, New Orleans, Jul. 17-21, 1994). It should be noted that the assay of the various cardiac and skeletal troponins is nowadays a very useful means for the diagnosis of human and animal pathologies.
It is well known that the immunoassays performed in biological analysis laboratories require the supply by the manufacturer, besides the reagents necessary for the assay (that is to say antibodies, labelled or otherwise, revealing agents and diluting solutions), of a standard for the compound to be determined which, used under conditions similar to those of the sample to be studied, will serve as reference for calculating the results and/or as positive control.
To obtain the standard and/or the control for the compound to be determined, it is possible to use the said compound purified in freeze-dried form (accompanied by a solvent in which the compound will be dissolved by the user before use) or ready for use.
Because biological reagents are unstable, the standard or control solutions prepared from a freeze-dried product are frozen in unit doses and stored at −80° C. It has been observed, moreover, that these solutions were not stable for more than a few hours at +4° C., even if protease inhibitors or antibacterial agents were added to them. This therefore requires the users to prepare their standard solutions immediately before use.
The patent application published under the number FR-A-2,701,954 discloses a stabilized solution of troponin I or T for immunoassay, characterized in that it consists of an aqueous solution containing troponin I or troponin T, mixed with troponin C and particularly in proportions of 1 to 10 molar equivalents of troponin C per equivalent of troponin I or T, and calcium chloride. This technique allows preservation for several days at +4° C. of standard solutions, diluted to a greater or lesser extent, of troponin I or T.
The patent application published under the number FR 2,734,267 describes standard solutions of troponin composed of a ternary complex formed by troponin I, troponin T and troponin C.
The raw materials used to obtain these standards are of human or animal origin, and the standards or controls thus obtained are stable for about one month at +4° C.
Application WO 94/15217 describes some synthetic peptides useful as immunogens for the preparation of antibodies recognizing the N-terminal peptide of troponin I. Some of these peptides can be used as standards in immunoassays of troponin I, using the antibodies which are the subject of the invention covered by application WO 94/15217.
Likewise, patent application WO 94/27156 relates to a method of assaying cardiac troponin I, using antibodies specific for cardiac troponin I. These antibodies can be prepared from peptide fragments having a sequence absent from skeletal muscle troponin I and therefore specific for cardiac troponin I. However, this application neither discloses nor suggests the possibility of using certain peptide fragments as standards in immunoassays of troponin I.
It is also known that application WO 96/27661 describes aqueous solutions for stabilizing proteins and peptides. These solutions find application in particular in diagnostic tests for proteins or peptides.
According to WO 97/27661, these aqueous solutions even make it possible to increase the stability of the fragments of troponin I which are known to be less stable than whole troponin I.
Application EP-A-752 426 published on 8 Jan. 1997 also describes troponin I standards composed of one or more peptides bound to a carrier molecule such as high-molecular weight proteins (>100 KD) or polymers.
Application EP-A-650 053 describes synthetic standards containing active sites for one or more receptors, linked to each other with an arborescent structure. This application describes more particularly synthetic standards for troponin T which are stable in solution for only 3 weeks.
It has now been found that it is possible to obtain synthetic standards which can be used for the evaluation of troponin I, which are stable for several months.
Indeed, the specific structure of the compounds of the invention confer an excellent stability on them.
The compounds of the present invention therefore make it possible to obtain highly standardized standards, which are stable in solution for several months, and to avoid the purification steps and the complex extraction procedures which are necessary for the preparation of troponin I standards from animal organs.
The compounds of the invention comprise two epitopes of troponin I, linked to each other by a linker and correspond to the general formula I:
Ξ-E1-Z-E2-Ψ (I)
in which:
It is considered that -E1-Z-E2 - is not a portion of the troponin I sequence when -E1-Z-E2 - differs from a given fragment of the troponin I sequence by a nonconservative substitution, deletion or insertion of at least one amino acid, preferably of 2 amino acids, more particularly of 5 amino acids.
Among the preferred compounds of the invention are the compounds in which Ξ represents either a chain of formula IIa, or a mixed construct IIIa to Va, or an N-α-acetylated chain of formulae IIa to Va or a chain of formulae IIa to Va carrying a biotinyl, biocytinyl or cysteinyl residue.
Among the preferred compounds of the invention are also the compounds in which Ψ represents either a chain of formula IIb, or a mixed construct of formulae IIIb to Vb, or a chain of formulae IIb to Vb carrying a hydroxyl radical or an amino radical.
The compounds of the invention in which Z represents either a chain of formula II or a mixed construct of formulae III to V are preferred in particular.
The peptide sequences comprising a minimum epitope of troponin I have been determined with the aid of anti-troponin I monoclonal antibodies. Such antibodies are described by Larue et al. (Molec. Immunology, (1992), vol. 20 No. 29, pp. 271-278), Bodor et al. (Clin. Chem. (1992), vol 38 No. 11, pp. 2203-2214), Granier et al (Protein Science, (1997), vol. 6 suppl. 1. p. 61) and in application WO 94/15217. The majority of these antibodies are commercially available (Hytest LTD-Turku, Finland).
To determine the peptide sequence comprising a minimum epitope of troponin I, the simultaneous synthesis of peptides of 10 amino acids, in which the sequence overlaps with the sequence of the preceding peptide by 7 amino acids, was carried out according to the following scheme:
As a whole, the peptides synthesized and tested describe the entire sequence of the protein (Vallins J. et al., FEBS Letters (1990), vol. 270 No. 1-2, pp. 57-61).
The peptides which react with each of the monoclonal antibodies used were then identified by an immunoenzymatic test for binding of each peptide with each antibody.
Indicated for example below, for some anti-troponin I monoclonal antibodies, are the peptide sequence comprising a minimum epitope immunologically reactive for each antibody and the position of this peptide sequence in the troponin I sequence, as described in FEBS Letters (1990), vol. 270 No. 1-2, pp. 57-61.
The peptide sequences derived from the said peptide sequences by substitution, deletion or insertion of an amino acid also belong to the field of the invention since they exhibit equivalent affinity for binding to the antibody.
The following linear sequences E1 and/or E2 No. 1 to 5 are preferred.
The above sequences are given by way of a non-limiting example.
When the linker Z comprises a peptide sequence, the latter may form with the sequences E1 and/or E2 an extended epitope of troponin I. This extended epitope may in particular have a sequence (SEQ ID NOS 48-55 respectively, in order of appearance); -TEPHAKK- or -QALLGAR- or -PTLRRVRISA- or -GKFKRPTLRRVR- or -LGFAELQD- or -NYRAYATEPH- or -AYATEPH- or -LGFAELQ-, etc.
These extended epitopes, which amplify the immunological response for antibodies were determined by adding amino acids to the peptide sequence comprising a minimum epitope determined as described above.
As has been shown above, the peptide sequence of the linker Z may comprise from 1 to 40 amino acids. It is preferable that the number of amino acids which form the peptide sequence of Z is less than 30, more particularly less than 20.
A particularly preferred peptide sequence of Z includes a sequence selected from the following amino acid sequences(SEQ ID NOS 6, 8, 17, 22, 56, & 21, respectively, in order of appearance):
This particularly preferred peptide sequence of Z may contain a repeated stretch of amino acids and has a number of amino acids less than 30.
By way of example, there are given the Z peptide sequences of the following formulae in which n represents an integer from 1 to 5:
Z may also represent a mixed construct of formulae III to V, in which m is an integer from 1 to 10.
By way of a non-limiting example, Z may thus correspond to the following sequences:
Preferred compounds of the present invention are the biepitope compounds of formula I, in which Z includes a sequence selected from the sequences No. 6 to 26, as defined above.
When Ξ and/or Ψ contain a peptide sequence, this sequence may form an extended epitope with E1 and/or E2 with which it is linked.
Advantageously, the chemical structure of Ξ is such that it allows the binding of the biepitope compounds of the invention to a natural carrier protein, to a peptide construct or to a solid phase.
The synthetic biepitope peptides of formula Ia and Ib form part of the peptides of formula I and are preferred peptides: (These formula peptides are encompassed by various peptide sequences listed throughout the application, including those peptides disclosed in the tables shown on page 14.);
Ξ-Thr Glu Pro His-Z-Leu Leu Gly Ala Arg-Ψ (Ia)
Ξ-Pro Ala Pro IIe Arg Arg Arg-Z-Thr Glu Pro His-Ψ (Ib)
In the formulae Ia and Ib:
Any peptide including one of the sequences 27 to 33, which are the following, is preferred:
The synthetic biepitope peptides of formula Ia and Ib below are particularly preferred peptides. In the two tables which follow, the peptide sequences are given using the single-letter code.
In the formulae Ia and Ib, the underlined amino acid sequences, corresponding to E1 and E2, represent a minimum epitope of troponin I. The underlined amino acid sequences of Z form with E1 or E2, to which they are linked, an extended epitope of troponin I.
Among the compounds indicated above, compounds 5, 6, 10, 11, 12, 13, 14 and 15 are particularly preferred compounds.
These peptides have the following sequences:
The synthetic biepitope compounds of formula I, which are the subject of the present invention, are obtained by solid phase synthesis according to conventional methods: R. B. Merrifield, J. Amer. Chem. Soc. (1963), 85, pp. 2149-2154; R. C. Sheppard, in “Peptides 1971”, Nesvadba H (ed.) North Holland, Amsterdam, pp. 111; E. Atherton and R. L. Sheppard, in “Solid phase peptide synthesis, a practical approach”, IRL PRESS, (1989), Oxford University Press, pp. 25-34. As automatic synthesizer, the Millipore 9050 Plus Pep Synthesizer or an equivalent synthesizer can be used.
The solid support used for the syntheses should be compatible with the technique and the chemistry used. For example, for synthesis on the 9050 Plus Pep Synthesizer, it is recommended to use a resin suited to the so-called “continuous flow” technique; the PEG PS resins meet these criteria. These supports consist of a spacer based on polyethylene glycol (PEG) situated between the functional group of the polystyrene of the beads and the point of attachment of the first amino acid. The nature of this point of anchorage may vary according to the C-terminal functional group selected. For example, for a peptide in the form of an amide, a PAL PEG PS type resin may be taken.
The starting resin and the amino acids used as raw material are products which are commercially available (PerSeptive-Biosystem).
The following side chain protecting groups were used:
The primary amine functional group was temporarily protected at the α position of the amino acids with the aid of the 9-fluorenylmethyloxycarbonyl (Fmoc) group. The deprotection is carried out with a 20% piperidine solution in dimethylformamide.
For the coupling, an excess of diisopropylcarbodiimide (DIPCDI) and of 1-hydroxybenzotriazole (HOBt) is preferably used.
After synthesis, the resin is washed with organic solvents (dimethylformamide and then dichloromethane), dried under vacuum and then treated with a trifluoroacetic acid-based solution cooled to 0° C. and containing appropriate scavengers. It will be possible to use, for example, the K reagent containing 82% trifluoroacetic acid, 5% phenol, 5% water, 5% thioanisole and 3% ethanedithiol.
The synthetic biepitope peptides thus isolated are then precipitated and rinsed with ether.
The synthetic biepitope compounds are then purified by reversed-phase liquid chromatography and their purity is determined by mass spectrometry. The Bondapak C18 phase can be used, for example, as phase. The peptides are eluted by forming a linear gradient between two buffer solutions, the first of which is essentially aqueous (for example water-TFA 0.1%) and the second of which is rather organic (for example a mixture containing 60% acetonitrile, 40% water and 0.08% TFA). The pure fractions collected are combined, concentrated under vacuum and freeze-dried.
The biepitope character of the compounds of formula I was determined using a troponin I assay kit. It is possible to use, for example, a kit allowing a sandwich-type immunoassay to be used and containing monoclonal antibodies which react with the peptide sequences E1 and E2.
To determine the biepitope character, the compounds of formula I are diluted in the diluent of the kit, which may be for example normal human serum or a buffer solution, and they are assayed like a patient's sample.
The use of compounds of formula I as standard for the immunological assay of troponin I also forms part of the invention.
The present invention also relates to compositions containing the compounds of formula I. They are preferably aqueous solutions or compositions consisting of compounds of formula I in a buffer solution. As buffer solution, it is possible to use, for example, a phosphate buffer solution (KH2PO4/K2HPO4 pH=6.5-7.5) containing Kathon and BSA or Kathon, Régilait and EDTA, or Kathon, Plasmion and optionally EDTA, or Kathon, casein and EDTA.
It is also possible to use succinate buffer (pH=5-6) or Tris-HCl buffer (pH=7.5-8.5) solutions containing Kathon and BSA or Kathon, Régilait and EDTA or Kathon, Plasmion and optionally EDTA, or Kathon, casein and EDTA.
Buffer solutions containing glycine, Kathon, Régilait and EDTA can also be used.
Succinate or phosphate buffer solutions containing Kathon, Régilait and EDTA are preferred.
Kathon®, an antibacterial agent marketed by the company Rhom and Haas, consists of 5-chloro-2-methyl4-isothiazolin-3-one and 2-methyl4-isothiazolin-3-one (1.5%).
Régilait® is marketed by the company Régilait.
Plasmion® is marketed by Bellon Laboratories and consists of modified fluid gelatin (30 g/l), NaCl (5.382 g/l), MgCl (143 mg/l), KCl (373 mg/l), sodium lactate (3.360 g/l), in water.
The following buffer solutions are particularly preferred:
Compositions containing plasma and a compound of formula I also form part of the present invention.
Immunoassay procedures using, as standards or controls, the compounds of formula I also form part of the invention.
The invention also relates to kits for carrying out immunoassays which include a compound of formula I or a composition which contains a biepitope peptide of formula I.
The following examples illustrate the invention and are given with no limitation being implied.
This peptide was synthesized on a solid phase. The technique developed in 1963 by Merrifield (J. Am. Chem. Soc. (1963), pp. 2149-2154) consists in attaching the first amino acid onto a polymeric solid support (resin) by its acid functional group and in extending the peptide sequence from this first amino acid, the peptide being synthesized remaining anchored onto the resin.
For the synthesis of compound 10, the 9050 Plus Pep Synthesizer was used as synthesizer and the PEG PS resin described above, as resin.
The various steps of the synthesis are summarized in Table I:
At the end of the synthesis, the resin was washed with dimethylformamide, then with dichloromethane and dried under vacuum.
Next, the resin was treated with K reagent (82% trifluoroacetic acid; 5% phenol; 5% water; 5% thioanisole; 3% ethanedithiol). Compound 10 thus isolated by precipitation was then rinsed with diethyl ether. 0.529 g of compound 10 was thus obtained.
In a similar manner, and using the appropriate amino acids, the other compounds of the invention were synthesized.
The molecular weight, evaluated by mass spectrometry, of some compounds of formula Ia and Ib are given below.
The biepitope character of the compounds mentioned above was determined using a troponin I assay kit. It is the kit ERIA Troponin I Pasteur Code 79691.
Its principle is based on a sandwich type immunoenzymatic method which allows a quantitative assay of cardiac troponin I in human serum.
The solid phase consists of polystyrene tubes coated with an anti-cardiac troponin I monoclonal antibody (8E1).
The revealing is performed with the aid of a second anti-cardiac troponin I monoclonal antibody (11E12) coupled to peroxidase.
The carrying out of the test comprises the following steps:
The samples and the standards (troponin I standards of animal origin) as well as the peroxidase-coupled monoclonal antibody are incubated in the presence of the anti-cardiac troponin I monoclonal antibody immobilized on the solid phase.
After a series of washes, the enzymatic revealing is carried out by adding the tetramethylbenzidine chromogen.
After stopping the revealing, the optical density is read at λ=450 nm. The absorbance obtained is directly correlated with the concentration of cardiac troponin I present in a tube.
Compounds 1, 2, 5, 6, 10, 11 and 12 were dissolved in water (C=1 mg/ml or 2 mg/ml depending on the case). These solutions were rediluted using a 0.1 M succinate buffer pH=6 containing 0.05% Régilait and 0.2% Kathon and 2 mM EDTA. The “final solutions” were then assayed as “samples” according to the protocol described above.
Moreover, compounds 1, 2, 10 and 11 were tested after dilution in human serum. In this case, the “final solutions” were obtained by diluting, in normal human serum, aqueous “stock” solutions containing compounds 1, 2, 10 and 11 at a concentration of 2 mg/ml.
All the compounds are reactive in the assay of troponin I.
A compound according to the invention (compound 10) was diluted in water (C=2 mg/ml). From this “stock” solution, four “working” solutions S1, S2, S3 and S4 are obtained.
To carry out the various dilutions, a 0.1 M sodium succinate buffer solution (pH=6) containing Kathon (0.2%), Re gilait (0.05%) and 2 mM EDTA was used for solutions S1 (C=0.5 ng/ml) and S2 (C=1 ng/ml).
Solutions S3 (C=0.5 ng/ml) and S4 (C=1 ng/ml) were obtained by diluting the “stock” solution with phosphate buffer (KH2PO4/K2HPO4 pH=7.5) containing Kathon (0.2%), Régilait (0.05%) and 2 mM EDTA.
Solutions S1, S2, S3 and S4 were assayed, according to the protocol described in Example 2, on D0 (day of preparation of the solutions). The values obtained served as reference for monitoring stability.
Next, solutions S1, S2, S3 and S4 were stored at +4° C. and assayed periodically. During each series of assays, three “control solutions” were used. They are the freeze-dried troponin I control sera distributed in the assay measuring range. For these sera, it was demonstrated beforehand that the storage life of a freeze-dried control serum is greater than 18 months. During each stability test, it was checked that each of the controls tested was obtained at its target concentration. This therefore made it possible to compare the concentrations obtained for the peptides during the various tests. In particular, it was possible to compare the concentration of the solution of the compound according to the invention at D0 (day on which the solution was made) with the concentration of the compound according to the invention during the stability trials over time. The fact that, for the controls, a constant value is always found during the stability trials makes it possible to validate the tests carried out.
The results obtained during the stability trials are indicated in Tables II and III.
The results indicated in Tables I and II demonstrate the excellent stability of the solutions of compound 10 according to the invention.
Trials carried out with other compounds of formula I demonstrated that their stability is comparable to that of compound 10. For example, the trials carried out with compound 6 confirmed that this compound had an excellent stability for at least 9 months, when stored at +4° C. in solution in a 0.1 M sodium succinate buffer solution (pH=6) containing Kathon (0.2%), Régilait (0.2%) and 2 mM EDTA. Compound 6 was dissolved in the buffer indicated above in order to obtain concentrations of 0.25, 4.8 and 18 ng/ml.
This stability study was validated with the aid of compound 6 stored at −20° C. and used as reference.
It was checked beforehand that compound 6 is stable when stored at −20° C. for at least 18 months. The results of this trial are given in Table IV.
| Number | Date | Country | Kind |
|---|---|---|---|
| 96 14959 | Dec 1996 | FR | national |
This application is a 371 of PCT/FR97/02209 filed Dec. 4, 1997.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCTFR97/02209 | 12/4/1997 | WO | 00 | 10/12/1999 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO9824816 | 6/11/1998 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5846738 | Seidel et al. | Dec 1998 | A |
| 6072040 | Dave et al. | Jun 2000 | A |
| 6077676 | Shi et al. | Jun 2000 | A |
| 6165981 | Flaa et al. | Dec 2000 | A |
| Number | Date | Country |
|---|---|---|
| 42 43 648 | Jul 1994 | DE |
| 0 650 053 | Apr 1995 | EP |
| WO 94 27156 | Nov 1994 | WO |
| WO 96 27661 | Sep 1995 | WO |
| WO 9739132 | Oct 1997 | WO |
