BE1024123B1 - Mainly peptide composition for the detection of asbestos - Google Patents

Abstract

The present invention relates to a mainly peptide composition for detecting asbestos in a sample comprising a first peptide fragment P1 arranged to recognize and bind to asbestos fibers of the amphibole family and having a percentage identity of at least 70% with a sequence X3X1X2X1X4X2X4, in which X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid, a second peptide fragment P2 arranged to recognize and bind at least to asbestos fibers of the serpentine family together with said first peptide fragment P1, and at least one detection means arranged to detect the recognition and binding of said first and second peptide fragments P1 and P2.

Description

Mainly peptide composition for the detection of asbestos

The present invention relates to a predominantly peptide composition for the detection of asbestos in a sample comprising: a first peptide fragment Pi specific for asbestos fibers, arranged to recognize and bind asbestos fibers of the family of amphiboles a second peptide fragment P2 specific for asbestos fibers, arranged to recognize and bind at least asbestos fibers of the family of serpentines together with said first peptide fragment Pi, and at least one detection means arranged to detect recognition and binding said first peptide fragment Pi to said asbestos fibers and recognizing and binding said second peptide moiety P2 to said asbestos fibers. Asbestos is a general term covering different types of natural minerals based on fibrous silicate. To date, there are six types of asbestos fibers that are classified into two families: the chrysotile type that is part of the serpentine family and the amosite, crocidolite, tremolite, actinolite and anthophyllite types that form the amphibole family. . These six types of asbestos fibers differ in composition and shape. Asbestos is known to resist mechanical forces, to be chemically and thermally stable and more specifically to be fire resistant. Due to its specific properties, asbestos has many applications ranging from car brake pads to fire blankets, building materials, window sills, tiles, ceiling panels, plaster or materials. insulators.

Given the health hazard of asbestos fibers, the use of asbestos has been banned in many countries. In fact, asbestos fibers are fine (some diameters are 2000 times smaller than that of a hair), of variable length and are easily found in the air. Once inhaled, they are deposited in the lungs and can cause inflammation, benign diseases or cancers such as mesothelioma and lung cancer. Indeed, it is recognized that asbestos fibers generate chromosome abnormalities leading to a cancerous transformation of cells.

However, despite their prohibition, many buildings still contain asbestos-containing materials, which poses a risk to residents and construction professionals, especially in the case of renovation work on old buildings. In the case where the presence of asbestos is suspected, the site is shut down until the receipt of the results of analyzes of samples taken to detect the presence or absence of asbestos fibers. Once the results are obtained, a decision can be made on how to proceed to reduce the risk of exposure to asbestos fibers.

There is therefore a real need to develop reliable and efficient detection means that are able to provide results quickly to confirm the presence or absence of asbestos fibers.

Various means for detecting asbestos are known from the state of the art. Indeed, there are currently tests that are based on light microscopy and electron microscopy. However, such tests must be performed in the laboratory, which results in long delays before obtaining a result.

In addition, a detection device (PhazIR ™, Polychromix, USA) using infrared spectroscopy has recently been developed to allow in situ detection of the presence or absence of asbestos fibers. Nevertheless, the use of this device requires a staff specifically trained to hope the realization of a relevant test. In addition, studies show a lack of sensitivity of such devices on some materials with a risk of false negatives. These erroneous answers were mainly found for low asbestos levels and for some materials such as tile joints and glues. False positives have also been reported in the case of analyzed materials containing silicate minerals or carbonates which have a spectrum similar to that of asbestos, which reflects a lack of specificity of such a detection device according to the state of the technique.

There is therefore a growing interest in developing detection means that are easy to implement and that make it possible to obtain a fast and reliable result.

In this regard, the document US2014 / 0170773 discloses an asbestos detection method comprising a first step of contacting, with the sample to be tested, an asbestos-binding protein coupled to a fluorescent molecule. Then, a detection step is performed using a fluorescence microscope: detection of the asbestos-binding protein (labeled with a fluorescent molecule) which is effectively bound to asbestos fibers. This prior document further discloses that the asbestos-binding proteins are, on the one hand, the H-NS protein which recognizes asbestos fibers of the amosite type of the amphibole family and, on the other hand, the Dksa protein. which recognizes, as for it, chrysotile asbestos fibers of the serpentine family. In order to distinguish these two types of asbestos fibers, fluorescent molecules that emit at different wavelengths are used to label the H-NS and Dksa proteins. Unfortunately, it appears that such a means of asbestos detection greatly limits the type of asbestos fibers detected and may lead to erroneous conclusions, which may prove to be dangerous to health.

The inventors of the present patent application have also developed a method for detecting the presence of asbestos fibers, this method being the subject of a patent application PCT / EP2016 / 069126 still unpublished at this time. day. The object of the invention is to overcome the drawbacks of the state of the art by providing a predominantly peptide composition which makes it possible to detect directly and rapidly, in a sample, the presence or absence of the six types of asbestos fibers of the family. amphiboles and the serpentine family more reliably (more sensitive), thereby reducing the risk of being exposed to asbestos fibers on the one hand and of false negative results, ultimately minimizing the risk of adverse health effects. Indeed, the present invention, by providing a composition for immediately detecting the presence of the six types of asbestos fibers at a site (in situ), makes it possible to quickly isolate people from these fibers in the case of positive detection. without having to wait for long periods to obtain the results of the test for detecting the presence or absence of asbestos fibers.

To solve this problem, it is provided according to the invention a mainly peptide composition for the detection of asbestos as indicated at the beginning, characterized in that said first peptide fragment PI has an identity percentage of at least 70. %, preferably at least 85%, with a sequence X3X1X2X1X4X2X4, wherein X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid. Preferably, X is T or S; X2 is Lou M; X3 is E; X4est R.

For the purposes of the present invention, the term "polar amino acid" includes the following amino acids: S (Ser), T (Thr), Y (Tyr), C (Cys), Q (Gin) or N (Asn), the terms "apolar amino acid" include the following amino acids: W (Trp), G (Gly), A (Ala), P (Pro), F (Phe), L (Leu), I (Ile), M ( Met) or V (Val), the term "acid amino acid" of the present invention includes the following amino acids: D (Asp) or E (Glu), while the terms "basic amino acid" include the following amino acids: H (His), R (Arg) or K (Lys).

In the context of the present invention, contrary to the document US2014 / 0170773, it has been demonstrated that the mainly peptide composition according to the invention makes it possible to detect, in a sample, the presence of not only amosite and chrysotile fibers but also four other types of asbestos fibers, the detection according to the invention being simple, fast and effective but also reliable and sensitive.

Indeed, the detection method according to the document US2014 / 0170773 makes it possible to highlight the asbestos fibers belonging to the family of serpentines (the chrysotile type) but makes it possible to detect a single type of fibers of the family of amphiboles namely amosite. Such a detection method thus lacks sensitivity to the fibers of the amphibole family and therefore has a real risk of obtaining a false negative result. In other words, the method according to this prior document would lead to a negative result whereas amphibole-type asbestos fibers, different from the amosite, are present in the sample. Such a method may therefore be responsible for unprotected exposure and consequently lead to health problems ranging from benign diseases to cancers of the lungs and pleura.

In the context of the present invention, it has surprisingly been found that the two peptide fragments Pi and P2 of the composition according to the invention are sufficient to detect the six types of asbestos fibers. The composition according to the invention therefore makes it possible to improve the detection sensitivity of asbestos fibers in a sample by highlighting asbestos fibers of chrysotile, amosite, actinolite, anthophyllite, tremolite and crocidolite type. In this way, the risk of obtaining a false negative result is reduced or even eliminated, this minimizing the exposure of people to all types of asbestos fibers and therefore the adverse effects of these on health.

Advantageously, said first peptide fragment Pi has the sequence SEQ ID NO: 1, as mentioned below: SEQ ID NO: 1: ETLSRMR (Glu Thr Leu Ser Arg Met Arg).

Preferably, said first peptide fragment Pi and said second peptide fragment P2 are fused, in any order, and form a hybrid peptide.

Preferably, said first peptide fragment Pi and / or said second peptide fragment P2 and / or said at least one detection means are fused, possibly in the form of repetitions, via at least one binding domain selected from the poly-linker domains. G or G-rich, the length of which may vary, including GGG, GGS, GGGS and GGSSG. According to the invention, said at least one detection means is added to the N-terminus or C-terminus of said hybrid peptide formed by said first peptide fragment Pi and said second peptide fragment P2 merged.

Advantageously, said first peptide fragment Pi and said second peptide fragment P2 are each independent of each other, in the form of a mixture. Said at least one detection means is added at the N-terminus or C-terminus of said first peptide fragment Pi and said second peptide fragment P2 and may be the same or different. If the detection means coupled to said first peptide fragment Pi and to said second peptide fragment P2 are different from each other, it is then possible to visually distinguish the asbestos fibers detected by said first peptide fragment Pi, the d asbestos detected by said P2 peptide fragment.

In a particular embodiment, said second peptide fragment P2 has an identity percentage of at least 50%, preferably at least 65%, preferably at least 80%, with a sequence selected from the group consisting of of X1X2X3X4X2X4X1 and X2X1X1X4X1X2X2X1X4X1X4, wherein X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid. Preferably, Xi is C, Q, N, Y, S or T; X2 is F, P, G, W, A or L; X3 is D; X4 is H or R.

Advantageously, said second peptide fragment P2a for sequence SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, as illustrated below: SEQ ID NO: 2: FYSHSALYRTH (Ser Phe Ser Ser Ser Ala Leu Tyr Arg

Thr His), SEQ ID NO: 3: QFDHWRN (Gin Phe Asp His Trp Arg Asn), SEQ ID NO: 4: GHCSHPT (Gly His Cys Ser His Pro Thr).

In a particular embodiment, said first peptide fragment P1 is arranged to recognize and bind asbestos fibers of amosite, tremolite, actinolite and crocidolite types and said second peptide fragment P2 is arranged to recognize and bind to the fibers of asbestos of chrysotile, anthophyllite, amosite and tremolite types, together with said first peptide fragment Pi.

Thus, the composition according to the invention comprising SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 makes it possible to detect, in said sample, the presence or absence of the six types of asbestos namely types amosite, tremolite, actinolite, crocidolite, anthophyllite and chrysotile, this in contrast to the detection technique according to the previous document US2014 / 0170773. Indeed, as indicated above, the proteins used in this prior document to detect the presence or absence of asbestos fibers are proteins that recognize only the types chrysotile and amosite. Thus, with a detection method according to this document US2014 / 0170773, the types crocidolite, tremolite, actinolite and anthophyllite are not highlighted, which generates an asbestos detection result which can be falsely negative. In other words, according to this prior document, crocidolite, tremolite, actinolite and anthophyllite asbestos fibers are not detected, which consequently entails a significant risk of exposure to these carcinogenic fibers.

In contrast to this document US2014 / 0170773, the composition according to the invention detects all the asbestos fibers of the family of serpentines but also all the asbestos fibers of the amphibole family. Such a composition, according to the invention, thus improves the detection sensitivity of asbestos, thus making it possible to obtain results with greater reliability by reducing the number of false-negative results. As a result, the risk of unprotected exposure to asbestos is lower and the risk of serious health consequences is lower.

Preferably, the mainly peptide composition according to the invention further comprises a third peptide fragment P3 arranged to recognize and bind to said asbestos fibers of said family of amphiboles and / or to said asbestos fibers of said family of serpentines and having an identity percentage of at least 50%, preferably at least 65%, preferably at least 80%, with a sequence selected from the group consisting of X1X2X3X4X2X4X1 and X2X1X1X4X1X2X2X1X4X1X4, where X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid. Preferably, Xi is C, Q, N, Y, S or T; X2 is F, P, G, W, A or L; X3 is D; X4 is H or R.

Recognition and binding of said third peptide fragment P3 to said asbestos fibers of said family of amphiboles and / or said asbestos fibers of said family of serpentines is detected by said at least one detection means.

Advantageously, said third peptide fragment P3a for sequence SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.

In a particularly advantageous embodiment, said third peptide fragment P3 and said second peptide fragment P2 are different from each other.

According to the invention, said third peptide fragment P3 is a peptide specific for asbestos fibers of chrysotile, amosite, anthophyllite and tremolite types. It is therefore designed to recognize and bind asbestos fibers of chrysotile, amosite, anthophyllite and tremolite types.

Such a composition further comprising said third peptide fragment P3 has improved specificity for asbestos fibers. Indeed, it has been demonstrated that the composition according to the invention comprising said third peptide fragment P3 and said second peptide fragment P2 which have different sequences reduces the risk of detecting other fibers (for example glass fibers ) that asbestos fibers and therefore allows to minimize the risk of obtaining a false positive result.

Preferably, said third peptide fragment P3 and said second peptide fragment P2 are fused, in any order, and form a hybrid peptide, said first peptide fragment P3 being independent, said hybrid peptide and said first peptide fragment P3 being in the form of a mix. Said third and second peptide fragments P3 and P2 are fused optionally in the form of repeats, via at least one binding domain chosen from the poly-G or G-rich binding domains, the length of which may vary, including GGG, GGS, GGGS and GGSSG. Said at least one detection means is added at the N-terminus or C-terminus of said hybrid peptide formed by said third peptide fragment P3 and said second peptide fragment P2 fused and at the N-terminus or C-terminus of said first peptide fragment Pi.

Advantageously, said third peptide fragment P3 and said first peptide fragment P3 are fused, in any order, and form a hybrid peptide, said second peptide fragment P2 being independent, said hybrid peptide and said second peptide fragment P2 being in the form of a mixed. Said third and first peptide fragments P3 and Pi are optionally fused as repeats via at least one binding domain selected from poly-G or G-rich binding domains, the length of which may vary, including GGG, GGS, GGGS and GGSSG. Said at least one detection means is added at the N-terminus or C-terminus of said hybrid peptide formed by said third peptide fragment P3 and said first peptide fragment Pi fused and at the N-terminus or C-terminus of said second peptide fragment P2.

Preferably, said first peptide fragment Pi and said second peptide fragment P2 are fused, in any order, and form a hybrid peptide, said third peptide fragment P3 being independent, said hybrid peptide and said third peptide fragment P3 being in the form of a mix. Said first and second peptide fragments P1 and P2 are fused optionally in the form of repeats, via at least one binding domain chosen from the poly-G or G-rich binding domains, the length of which may vary, including GGG, GGS, GGGS and GGSSG. Said at least one detection means is added at the N-terminus or C-terminus of said hybrid peptide formed by said first peptide fragment Pi and said second peptide fragment P2 fused and at the N-terminus or C-terminus of said third peptide fragment P3.

In a particular embodiment, said second peptide fragment P2, said third peptide fragment P3 and said first peptide fragment Pi are each independent of each other in the form of a mixture. Said at least one detection means is added to the N-terminus or C-terminus of said first peptide fragment Pi, said second peptide fragment P2 and said third peptide fragment P3.

Preferably, said third peptide fragment P3 <said second peptide fragment P2 and said first peptide fragment Pi are fused, in any order, and form a hybrid peptide. Said third, second and first peptide fragments P3, P2 and P1 are optionally fused in the form of repeats, via at least one binding domain chosen from the poly-G or G-rich binding domains, the length of which may vary. , including GGG, GGS, GGGS and GGSSG. Said at least one detection means is added at the N-terminus or C-terminus of said first peptide fragment Pi, said second peptide fragment P2 and said third peptide fragment P3 fused.

Advantageously, said first peptide fragment Px and / or said second peptide fragment P2 and / or said third peptide fragment P3 and / or said at least one detection means are fused, possibly in the form of repetitions, via at least one domain of bond selected from the poly-G or G-rich binding domains, the length of which may vary, including GGG, GGS, GGGS and GGSSG.

Advantageously, said third peptide fragment P3, said second peptide fragment P2 and said first peptide fragment Pi are fused and form a hybrid peptide having the sequence X3XiX2XiX4X2X4GGGX2XiXiX4XiX2X2GGGXiX4XiX4XiX2X3X4X2X4Xi, where X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents a acidic amino acid and X4 represents a basic amino acid. Preferably, Xi is Q, N, T, S, C or Y; X2 is F, W, L, M, A, P or G; X3 is D or E; X4 is H or R.

Advantageously, said hybrid peptide has the sequence SEQ ID NO: 5 consisting of SEQ ID NO: 3 - GGG - SEQ ID NO: 1 - GGG - SEQ ID NO: 2 or SEQ ID NO: 6 formed of SEQ ID NO: 3 - GGG - SEQ ID NO: 1 - GGG - SEQ ID NO: 4

Preferably, said first peptide fragment Pi, said second peptide fragment P2 and said third peptide fragment P3, alone, in a mixture or merged, are GEPIs. GEPIs (Genetically engineered peptide or polypeptide for inorganics) are small peptides that have a high affinity for inorganic materials. They are defined as having an amino acid sequence that specifically and selectively binds an inorganic surface. The molecular mechanisms relating to this peptide recognition and this binding to solid materials are little known to date, but several studies seem to show that this bond is largely governed by the combination of electrostatic interactions and hydrogen and hydrophobic bonds.

In a particularly advantageous embodiment, said at least one detection means is selected from luciferase, alkaline phosphatase, fluorescent proteins, beta-galactosidase, beta-lactamase, diphorase, peroxidase, maltose, laccase, lipase, glycosyl hydrolase, bovine serum albumin, colloidal gold, colored beads, preferably colored latex beads, or biotin and mixtures thereof. Thanks to colorimetric techniques, the detection of the presence or absence of asbestos fibers in a sample using the peptide composition according to the invention can be implemented directly on site and therefore makes it possible to obtain the results of the presence or absence of asbestos fibers more quickly, this reducing the exposure time and therefore the risk of adverse health effects.

In the case of the detection of the said at least one detection means using a fluorescence technique, an analysis device may also be used to study the images obtained using a microscope. fluorescence.

Preferably, the mainly peptide composition according to the invention is in the form of a solution, in a solvent such as an aqueous solution, a polar organic solvent, and preferably in water.

Advantageously, said sample is a sample of building material, pure asbestos fibers, an air sample and / or a sample of biological material. For example, said sample may be a sample of car brake pads, fire blankets, building materials, window sills, tiles, ceiling panels, plaster or an insulating material. Said sample may also be pure asbestos fibers. In another alternative, said sample may be air that has been removed from where air contaminated with asbestos fibers is suspected, for example, locations near sites under construction or renovation. In another aspect, said sample is biological material, such as a biopsy or a post-mortem body sample. In the case of the present invention, said sample is preferably in the form of a suspension in a liquid medium, for example a blocking buffer. Other embodiments of a composition according to the invention are indicated in the appended claims.

The present invention also relates to a method for detecting asbestos in a sample comprising: preparing the sample by suspending a test sample for the presence of asbestos, contacting said sample with at least one mainly peptide composition according to the invention, for a time interval sufficient to allow the peptides of the predominantly peptidic composition according to the invention to bind to the sample, a revelation of the presence of peptide fragments of the composition according to the invention. related to asbestos fibers of the serpentine family and / or the amphibole family.

This method according to the invention is advantageous since it makes it possible to improve the detection sensitivity of asbestos fibers in a sample by highlighting asbestos fibers of chrysotile, amosite, actinolite, anthophyllite, tremolite and crocidolite type. In this way, the risk of obtaining a false negative result is reduced or even eliminated, this minimizing the exposure of people to all types of asbestos fibers and therefore the adverse effects of these on health.

Preferably, the method according to the invention further comprises at least one step of separating said bound peptide fragments from peptide fragments not bound to asbestos fibers, such as a step of removing said unbound peptide fragments, for example by percolation through a filter wall or for example, by a washing separation step. The separation step according to the invention can be carried out using a separation means, such as a filter or a sinter, to retain the asbestos fibers but to pass through it. the composition according to the invention which would not have been bound to the fibers of the sample to be tested, once the withdrawal thereof has been carried out. This withdrawal can be achieved by suction, by addition of a washing solution or by any other equivalent means, such as for example by making an opening allowing the fluid to flow under the effect of gravity or by capillarity. Other embodiments of the asbestos detection method according to the invention are indicated in the appended claims.

The present invention also relates to an asbestos detection kit comprising at least one support provided with at least a first zone for receiving a sample to be tested, and at least one peptide composition according to the present invention, in which at least said peptide fragments P1, P2 and optionally additional peptide fragments such as P3 are packaged together or each separately, preferably in a syringe or in a cartridge comprising a plurality of compartments.

It is in fact provided according to the present invention to provide an asbestos detection kit comprising a first reception zone in which a sample to be tested for the presence of asbestos is placed.

The kit therefore comprises a mainly peptide composition according to the present invention, comprising: a first peptide fragment Pi specific for asbestos fibers, arranged to recognize and bind asbestos fibers of the family of amphiboles and having a percentage of identity at least 70%, preferably at least 85%, with a sequence X3X1X2X1X4X2X4, where Xx represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid, a second P2 peptide fragment specific for asbestos fibers, arranged to recognize and bind at least asbestos fibers of the family of serpentines together with said first peptide fragment Pi, and at least one detection means arranged to detect the recognition and the bonding said first Px peptide fragment to said asbestos fibers and recognizing and binding said second peptidic fragment P2 to said asbestos fibers.

The composition according to the invention is preferably packaged in a syringe or in a cartridge comprising several compartments. In this way, when the sample is placed in the receiving zone, the mainly peptide asbestos detection composition is added to the sample for which it is desired to detect if it contains asbestos for a period of time. sufficient to allow the binding of the peptides of the composition according to the invention to the sample. Once the time interval has elapsed, the excess of mainly peptide composition according to the present invention is eliminated and the analysis is carried out.

Advantageously, the detection kit according to the present invention comprises at least one revelation means, preferably packaged in a syringe or in a cartridge comprising a plurality of compartments, arranged to display said at least one means for detecting the peptide composition, said at least one a revealing means being a means of revealing in the visible, such as, for example, a means of revealing by colorimetry, for example nitrocele or any other compound allowing a revelation by colorimetry.

In a preferred embodiment of the present invention, said at least one detection means is selected from the group consisting of luciferase, beta-galactosidase, beta-lactamase, laccase, lipase, glycosyl hydrolase, phosphatase alkaline, diphorase, peroxidase and their mixtures and is more particularly beta-lactamase. In these cases, said at least one detection means is a protein having an enzymatic activity. When the detection means is present, and therefore has been bound to the sample to be tested, the enzymatic activity is visibly revealed, as for example when the detection means is a beta-lactamase of which the enzymatic activity on a chromogenic substrate, such as nitrocine, UW154, UW57, UW58 (Ghavami et al., Assay for drug discovery: synthesis and testing of nitrocefin analogues for use as beta-lactamase substrates, Anal Biochem October 2015, vol 486, pages 7-75), Chromacef®, Cefesone (SI), HMRZ-86 (Hanaki et al., Characterization of HMRZ-86: a novel chromogenic cephalosporin for the detection of extended-spectrum beta-lactamases, J Antimicrob Chemother. May 2004, vol 53 (5), pages 9-888), CLS405 (Makena et al., Chromophore-linked substrate (CLS405): probing metallo-beta-lactamase activity and inhibition, ChemMedChem, December 2013, Vol 8 (12) , pages 9-1923), CENTA® or PADAC (Jorgensen et al., Pyridinium-2-azo-p-dimethylaniline chromopho A new chromogenic cephalosporin for rapid beta-lactamase testing, Antimicrobial Agents and Chemotherapy 1982, Vol 22 (1), pages 4-162) (revealing means) allows for visible revelation by color change.

In another preferred embodiment of the present invention, the detection kit according to the present invention further comprises at least one revelation means, preferably packaged in a syringe or in a cartridge comprising a plurality of compartments, arranged to display the said at least one means for detecting the peptide composition, said at least one revelation means being a fluorescence revelation means. For example, said at least one detection means is beta-lactamase, peroxidase or alkaline phosphatase and said at least one means of disclosure is Fluorocillin Green 495-525, Ampliflu ™ Red or 4-methylumbelliferyl phosphate disodium , respectively.

According to yet another preferred embodiment of the present invention, said at least one detection means is a fluorescent molecule, revealed by excitation by means of a wave.

Advantageously, the asbestos detection kit according to the present invention further comprises a washing solution, preferably packaged in a syringe or in a cartridge comprising a plurality of compartments.

Preferably, in the asbestos detection kit according to the present invention, said support further comprises a second reception zone arranged to receive a negative control or a positive control.

More particularly, in the detection kit according to the present invention, said support further comprises a second and a third reception zone arranged respectively to receive a negative control and a positive control or vice versa.

In a variant of the asbestos detection kit according to the present invention, said kit further comprises at least one means of revealing and said support comprising said first reception zone of a suspended sample to be tested is a tab on which the first reception zone of said test sample is positioned at one end thereof, said tab also comprising at least two stop strips in the direction opposite to said first reception zone in which primary binding proteins are immobilized or secondary of said at least one detection means or said developing means, for example antibodies, streptavidin, or the like. Such tags without immobilized molecules are commercially available. Then, primary or secondary binding proteins of said at least one detection means or said at least one developing means are immobilized in the stop bands. The sample is prepared by suspending a test sample for the presence of asbestos. The sample is then brought into contact with the mainly peptide composition according to the present invention for a period of time sufficient to allow the peptides of the composition according to the invention to bind to the sample. The amount of composition according to the invention is relatively small relative to the amount of sample to be tested to ensure that if there are asbestos fibers in the sample, the entirety of the peptides are related to asbestos fibers.

Once the time interval has elapsed, the suspended sample thus prepared is brought into contact with the receiving zone, for example by soaking said rod in the sample in suspension. If there are asbestos fibers in the suspended sample, the peptides bound to them do not migrate along the tab and their presence is not detected on the tab. The band farthest from the receiving zone of the sample to be tested comprises a control (migration control) to show that the test has worked correctly. If the bands, located between the control band and the receiving zone of the sample to be tested and the control band are colored, this means that the test has worked and that the peptides of the composition according to the invention have migrated to the bands. . They are therefore not related to asbestos fibers. We can therefore conclude that there is no asbestos fiber. If the farthest band is not colored, in this case, the test has not worked and it should be reproduced.

In a preferred embodiment of the asbestos detection kit, said at least one detection means comprises at least one fluorescent molecule, for example fluorescein isothiocyanate, or at least one carrier protein such as bovine serum albumin, maltose binding protein, biotin, an enzyme such as beta-galactosidase, beta-lactamase, laccase, lipase, glycosyl hydrolase, alkaline phosphatase, diphorase, peroxidase and more particularly beta-lactamase, lactamase, said at least one fluorescent molecule or said at least one carrier protein being coupled to said at least one developing means such as colloidal gold or to colored beads, preferably colored latex beads and wherein the immobilized binding in the stopping bands are primary binding proteins selected from antibodies directed against said at least one carrier protein, or antibodies directed against said at least one fluorescent molecule, or avidin (or the like), or streptavidin.

In a also preferred variant of the asbestos detection kit according to the invention, said at least one detection means comprises at least one carrier protein or at least one fluorescent molecule, such as a fluorescein isothiocyanate, recognized by at least one revealing means such as an antibody directed against said at least one fluorescent molecule, for example an antibody directed against fluorescein isothiocyanate, or an antibody directed against said at least one carrier protein, said antibodies being coupled to colloidal gold, colored beads, such as colored latex beads and wherein the binding proteins immobilized in the stop bands are secondary binding proteins chosen from secondary antibodies directed against said antibodies directed against said at least one molecule fluorescent and said at least one carrier protein.

The detection kit according to the invention is particularly easy to use, even by unskilled personnel, and provides results in a short time. It has been established that the results can be obtained in approximately 1 hour, preferably in less than 30 minutes, preferentially in less than 15 minutes, more preferably in approximately 10 minutes.

Preferably, in the kit according to the invention, said first peptide fragment Pi is fused to a first detection means and said second peptide fragment P2 or said third peptide fragment P3 is fused to a second detection means, different from said first means of detection. detection, said first, second or third peptide fragments Pi, P2 or P3 being in the form of a mixture. Other embodiments of a kit according to the invention are indicated in the appended claims. The invention also relates to a use of a mainly peptide composition according to the invention for the detection of asbestos in a sample. The invention also relates to a use of a method according to the invention for the detection of asbestos in a sample.

Finally, the invention also relates to a use of a kit according to the invention for the detection of asbestos in a sample. Other forms of use of a composition, process or kit according to the invention are indicated in the appended claims. Other features, details and advantages of the invention will emerge from the description, examples and figures given below, without limitation.

FIG. 1 represents the results of a comparative example of detection of asbestos by the peptide SEQ ID NO: 1 fused to the detection means, namely TEM-1.

FIG. 2 represents the results of a comparative example of detection of asbestos by peptide SEQ ID NO: 4 fused to the detection means, namely TEM-1.

FIG. 3 represents the results of an example according to the invention for the detection of asbestos by the peptide SEQ ID NO: 1 fused to the detection means, namely TEM-1 and the peptide SEQ ID NO: 4 fused by means of detection, namely TEM-1, in the form of a mixture.

FIG. 4 represents the results of an example according to the invention for detecting asbestos by a hybrid peptide containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 fused to the detection means, know TEM-1.

FIG. 5 represents the results of an example according to the invention for the detection of asbestos by a hybrid peptide containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 fused to the detection means, namely fluorescein isothiocyanate.

FIG. 6 represents the results of an example according to the invention for detecting asbestos by a hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 3 fused to the detection means, namely fluorescein isothiocyanate. .

FIG. 7 represents the results of an example according to the invention for the detection of asbestos by a hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 2 fused to the detection means, namely fluorescein isothiocyanate. .

FIG. 8 represents an asbestos detection kit according to the invention. Figure 8A shows a first embodiment and Figure 8B a second embodiment of an asbestos detection kit according to the invention.

Figure 9 shows a variant of the asbestos detection kit according to the invention. FIG. 9A shows a first embodiment and FIG. 9B a second embodiment of a variant of the asbestos detection kit according to the invention.

Examples Selection of peptides by the method of "Phase Display" The approach used to isolate peptides recognizing asbestos is the "Phage display" method. Various libraries expressing random peptides have been selected: Ph.D. ™ -7 phage display peptide library (NEB), Ph.D. ™ -12 phage display peptide library (NEB); Ph.D. ™ -C7C phage display peptide library (NEB). Sequencing of amino acids

Sequencing of phages (bacteriophage M13) (after three selection steps) was performed with the primer CCC TC A TAG TTA G CG T AA CG. Sequences were reviewed with the Chromas Lite Software (Technelysium Pty Ltd) and the Expasy tools Translate program available online. Sequence alignment was achieved via the ClustalW program available online.

Following the realization of this protocol and from these libraries, the following peptides were generated: ETLSRMR (SEQ ID NO: 1), FYSHALYRTHT (SEQ ID NO: 2), QFDHWRN (SEQ ID NO: 3), GHCSHPT ( SEQ ID NO: 4).

Comparative Example 1.-Detection of Asbestos by Peptide SEQ ID NO: 1 Fused with TEM-1:

Peptide SEQ ID NO: 1 was cloned into 6 repeats upstream of a gene encoding TEM-1 β-lactamase (P62593-BLAT_ECOLX). A sequence encoding the GGG binding domain was introduced between the sequence of interest SEQ ID NO: 1 and TEM-1. The NcoI and BamHI restriction sites were used to clone the TEM-1 fused peptide into the pET28a vector (to obtain the pET28a / peptide-TEM-1 vector).

The E. coli bacterium expressing the pET28a / peptide-TEM-1 vector was cultured at 37 ° C overnight (time: 16 h) with stirring in 50 ml of LB medium enriched with 50 μg / ml kanamycin. The bacterial suspension was diluted 100-fold in a total volume of 1 L of fresh LB medium enriched with kanamycin (50 μg / ml) and the expression of peptide-TEM-1 was induced with isopropyl-thiogalactopyranoside. (final concentration of ImM) as soon as the culture had reached an absorbance at 600 nm of 0.6. The induced culture was then incubated at 18 ° C for 16 h with shaking. Peptide-TEM-1 was purified by zinc affinity chromatography (HiTrap Chelating HP 2 x 5 ml (GE Healthcare, Machelen, Belgium)). 50 μl of the solution containing the peptide (4 mg / ml) was added to 1 mg of pure asbestos fibers. The resulting solution was incubated for 10 min at room temperature and gentle stirring.

The fibers were then precipitated by centrifugation (10000 g, 3 min) and the supernatant was removed. The fibers were then suspended in 1 ml of 10 mM phosphate buffer at pH 8.0 and collected by further centrifugation (10000 g, 3 min).

The collected fibers were finally suspended in 100 μl of revealing agent (UW154, 200 mM) and incubated at room temperature for 5 min.

A dark red color is induced under conditions where asbestos fibers are detected, while the coloring remains light yellow in case of negative detection.

Different samples of asbestos were tested according to the detection protocol mentioned above and according to Table 1. The results resulting from this analysis are shown in FIG.

Table 1.-

As can be seen in FIG. 1, the peptide SEQ ID NO: 1 recognizes and binds to crocidolite asbestos fibers. Peptide SEQ ID NO: 1 also recognizes, but in a less sensitive manner, the type actinolite, amosite, anthophyllite, tremolite and chrysotile. Negative controls were validated on fiberglass (G) but also on viscose, polyester, Cerawool® fibers, SAFIL ™, Nicalon ™, silicon carbide, Kevlar® and still on acrylic (data not presented). Control (H) was also performed by incubating the revealing solution (UW154) with the asbestos fibers without adding the peptide SEQ ID NO: 1 fused to TEM-1.

Comparative Example 2, Detection of Asbestos by Peptide SEQ ID NO: 4 Fused with TEM-1:

The production protocol of peptide SEQ ID NO: 4 fused to TEM-1 is identical to that described in Comparative Example 1, with the difference that the peptide SEQ ID NO: 4 was cloned in five repetitions.

The protocol for detecting asbestos fibers is identical to that described in Comparative Example 1.

Various samples of asbestos were tested according to the detection protocol mentioned above and according to Table 2. The results resulting from this analysis are shown in FIG.

Table 2, -

Peptide SEQ ID NO: 4 recognizes and binds to asbestos fibers of anthophyllite, chrysotile (Zimbabwe and Canada) and crocidolite type. The peptide SEQ ID NO: 4 also recognizes, but in a less sensitive manner, the actinolite type. This peptide does not, however, bind to the amosite and tremolite type. Negative controls were validated on fiberglass (9) but also on viscose, polyester, Cerawool® fibers, SAFIL ™, Nicalon ™, silicon carbide, Kevlar®, and acrylic (data not represented). A control (1) was also performed by incubating the revealing solution (nitrocine) with the asbestos fibers without adding the peptide SEQ ID NO: 1 fused to the TEM-1.

EXAMPLE 1 Detection of Asbestos with Peptide SEQ ID NO: 1 Fused with TEM-1 and Peptide SEQ ID NO: 4 Fused with TEM-1, in the Form of a Mixture:

The production protocol of peptide SEQ ID NO: 4 fused to TEM-1 and peptide SEQ ID NO: 1 fused to TEM-1 is identical to that described in Comparative Examples 1 and 2.

The protocol for detecting asbestos fibers is identical to that described in Comparative Example 1.

Various samples of asbestos were tested according to the detection protocol mentioned above and according to Table 3. The results resulting from this analysis are shown in FIG.

Table 3.-

The mixture comprising the peptide SEQ ID NO: 4 and the peptide SEQ ID NO: 1 recognizes and binds to the asbestos fibers of the family of serpentines and the family of amphiboles. In fact, following this analysis, it has been demonstrated that the mixture of these two peptides binds asbestos fibers of the crocidolite, tremolite, anthophyllite, actinolite, amosite and chrysotile type. Control (G) was performed by incubating the revealing solution (UW154) with the asbestos fibers without adding the mixture comprising SEQ ID NO: 1 and SEQ ID NO: 4 fused to TEM-1.

Example 2. Detection of Asbestos by a Hybrid Peptide Containing SEQ ID NO: 1 SEQ ID NO: 2 and SEQ ID NO: 3 fused to TEM-li

The production protocol of the hybrid peptide containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 fused to TEM-1 is identical to that described in Comparative Example 1, with the difference that The sequence encoding the GGG binding domain was further introduced between the sequences of interest SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and that the peptide SEQ ID NO: 1 was cloned into a sequence. repetition.

The protocol for detecting asbestos fibers is identical to that described in Comparative Example 1.

Various asbestos samples were tested according to the detection protocol mentioned above and according to Table 4. The results resulting from this analysis are shown in FIG.

Table 4.-

The hybrid peptide containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 fused to TEM-1 recognizes and binds to asbestos fibers of the family of serpentines and the family of amphiboles. In fact, following this analysis, it has been demonstrated that this hybrid peptide binds asbestos fibers of the crocidolite, tremolite, anthophyllite, actinolite, amosite and chrysotile type. Control (A) was performed by incubating the revealing solution (nitrocine) with the asbestos fibers without adding the mixture comprising SEQ ID NO: 1 and SEQ ID NO: 4 peptide fused to TEM-1.

Example 3 - Detection of Asbestos by a Hybrid Peptide Containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 Fused with Fluorescein Isothiocvanate

The asbestos-recognizing hybrid peptide containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 fused to fluorescein was synthesized by Biomatik (Cambridge, Ontario, Canada) by adding fluorescein to the N-terminal and amidation at the C-terminal portion of the peptide.

The fluorescent hybrid peptide synthesized was then solubilized in water at a concentration of 10 mg / ml and the solution was stored at -20 ° C.

A buffer was used for the detection of asbestos fibers in fluorescence. This buffer is a 50 mM pH 7.0 phosphate buffer, 150 mM NaCl, 2% Tween-20. 500 μ! of a suspension solution (40 μg / ml, asbestos fibers in buffer) were centrifuged (13000 rpm, 10 min, 4 ° C.). After removing the supernatant, 1 ml of a buffer solution was added to the pellet and the whole was then vigorously mixed for 20 seconds. The supernatant was removed after centrifugation (13000 rpm, 10 min, 4 ° C.) and 1 ml of a buffer solution containing the asbestos-recognizing fluorescent peptide (50 μg / ml) was then added. After 10 min incubation at room temperature and protected from light, the solution was then centrifuged (13000 rpm, 10 min, 4 ° C) and the supernatant was removed. The pellet was then subjected to two washing steps before 30 μΙ of mounting buffer was added to finally be spread on a glass slide. Fiber analysis was performed by fluorescence microscopy (Olympus BX60). Fluorescein was detected using an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

Various samples of asbestos were tested according to the detection protocol mentioned above and according to Table 5. The results resulting from this analysis are shown in FIG.

Table 5.-

The hybrid peptide containing SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 fused to fluorescein recognizes and binds asbestos fibers of the serpentine family and the amphibole family. In fact, following this analysis, it has been demonstrated that this hybrid peptide binds asbestos fibers of the crocidolite, tremolite, anthophyllite, actinolite, amosite and chrysotile type. A negative control has been validated on the fiberglass (G).

Example 4. Detection of Asbestos by a Hybrid Peptide Containing SEQ ID NO: 1 and SEQ ID NO: 3 Fused with Fluorescein

The production protocol of the hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 3 fused to fluorescein is identical to that described in Example 3.

The protocol for detecting asbestos fibers with the hybrid containing SEQ ID NO: 1 and SEQ ID NO: 3 fused to fluorescein is identical to that described in Example 3.

Various asbestos samples were tested according to the detection protocol mentioned above and according to Table 6. The results resulting from this analysis are shown in FIG.

Table 6.-

The hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 3 fused to fluorescein recognizes and binds asbestos fibers of the serpentine family and the amphibole family. In fact, following this analysis, it has been demonstrated that this hybrid peptide binds asbestos fibers of the crocidolite, tremolite, anthophyllite, actinolite, amosite and chrysotile type.

Example 5 - Detection of asbestos by a hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 2 fused to fluorescein:

The production protocol of the hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 2 fused to fluorescein is identical to that described in Example 3.

The protocol for detecting asbestos fibers with the hybrid containing SEQ ID NO: 1 and SEQ ID NO: 2 fused to fluorescein is identical to that described in Example 3.

Different samples of asbestos were tested according to the detection protocol mentioned above and according to Table 7. The results resulting from this analysis are shown in FIG.

Table 7, -

The hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO: 2 fused to fluorescein recognizes and binds asbestos fibers of the family of serpentines and the family of amphiboles. In fact, following this analysis, it has been demonstrated that this hybrid peptide binds asbestos fibers of the crocidolite, tremolite, anthophyllite, actinolite, amosite and chrysotile type.

Conclusion

These various analyzes have made it possible to demonstrate that contacting asbestos fibers with the peptides SEQ ID NO: 1 and SEQ ID NO: 2 and / or SEQ ID NO: 3 makes it possible to detect the presence or absence of all of them. the types of asbestos fibers of the serpentine family and the amphibole family. This detection is observed both with a mixture comprising the peptide SEQ ID NO: 1 and SEQ ID NO: 2 and / or SEQ ID NO: 3, with a hybrid peptide containing SEQ ID NO: 1 and SEQ ID NO : 2 and / or SEQ ID NO: 3. In addition, this detection is valid not only in colorimetry but also in fluorescence. Indeed, the colorimetric and fluorescence analyzes are complementary and lead to a similar result. This makes it possible to conclude that the results obtained in fluorescence can be transposed to colorimetric techniques and vice versa.

As can be seen in FIG. 8A and FIG. 8B, the present invention also relates to an asbestos detection kit which comprises, in the preferred embodiment illustrated, a support comprising a first reception zone B of a sample to be tested, a second reception zone C arranged to receive a negative control and a third reception zone A arranged to receive a positive control. The first, second and third receiving zones A, B and C form three columns terminating, on one side, in a container D for collecting waste and are extended opposite each other by a tube E, F, G which connects the column to an H input for a fluid.

The second reception zone C arranged to receive a negative control is ideally pre-filled by a negative control, for example virgin glass fibers, containing no asbestos. The third reception zone A arranged to receive a positive control A is ideally pre-filled by a positive control, for example a non-asbestiform analogue of asbestos. The sample to be tested, for example pure asbestos fibers or a material (<1 cm3), which is preferably suspended in a volume of blocking buffer, is deposited in the receiving zone B for the sample to be tested. in the appropriate column.

Once the test sample has been introduced into the first reception zone A, a first syringe, in which the composition according to the invention is packaged, is connected by means of a quick connector (LUER LOCK ®) to the inlet H to introduce a fluid of the detection kit according to the invention.

The composition according to the invention comprises, in this preferred embodiment, said hybrid peptide having for sequence SEQ ID NO: 3 -GGG-SEQ ID NO: 1 -GGG SEQ ID NO: 2 coupled to beta-lactamase TEM-1. The composition according to the invention is therefore introduced in the same way into the three receiving zones A, B and C (first, second and third reception zone). Then, the composition according to the invention of the syringe 1 is incubated with the sample to be tested as well as with both controls (positive and negative) for a predetermined time interval, such as for example 10 minutes. During this time interval, the composition according to the invention remains in contact with the sample to be tested, the positive control and the negative control.

Each receiving zone comprises at its end facing the container D a separation means, such as a filter, or a sinter for retaining the asbestos fibers, the positive control and the negative control in the column, but to leave passing through the composition according to the invention which would not have been bound to the fibers of the sample to be tested, to the fibers of the positive control and to the fibers of the negative control once the withdrawal thereof has been carried out. This withdrawal can be achieved by suction, by addition of a washing solution or by any other equivalent means, such as for example by making an opening allowing the fluid to flow under the effect of gravity or by capillarity.

After the incubation of the composition according to the present invention, a washing solution which is packaged in the syringe 2 is injected in the same manner into the three columns containing the first, second and third reception zones. The washing solution is, for example, water and its injection makes it possible to eliminate the composition according to the invention which is not bound to the asbestos fibers, to the fibers of the positive control and to the fibers of the negative control.

After the washing is complete (after addition of 6 ml of washing solution), a yellow-based nitrocrefin-based developer solution, or the like, contained in a third syringe 3 (200 μΜ concentration, 500 μΙ applied volume) is contacting the test sample and the two controls. If the hybrid peptide has been bound to the fibers, the beta-lactamase hydrolyzes the nitrocele and forms its derivative which then becomes red in color. The red color in the column containing the receiving zone B for the sample to be tested means that the test is positive for the presence of asbestos fibers, whereas a yellow color means that there are no fibers. asbestos present.

As can be seen in FIG. 9A, the present invention also relates to an asbestos detection kit which comprises a first zone for receiving a test sample 1 which is positioned on a tab 9 at one end of that -this. The tag 9 also comprises two stop bands 2 (Ami) and 4 (T) in which are immobilized primary or secondary binding proteins of the detection means coupled to the peptide fragments of the composition according to the present invention. The sample is prepared by suspending a test sample for the presence of asbestos in a blocking buffer (<1 cm3 of sample for a volume of 1 ml). The sample is then brought into contact, in a separate container, with the mainly peptide composition according to the present invention for a period of time sufficient to allow the peptides of the composition according to the invention to bind to the sample.

The amount of composition according to the invention comprises, in a preferred embodiment, said hybrid peptide having for sequence SEQ ID NO: 3 - GGG - SEQ ID NO: 1 - GGG SEQ ID NO: 2 coupled to TEM beta-lactamase -1, and is put in relatively small amount relative to the amount of test sample to ensure that if there are asbestos fibers in the sample, the entirety of the peptide or peptides are related to asbestos fibers.

In another preferred embodiment, the kit according to the invention comprises two strips 9 and two compositions according to the invention. The first tab 9 according to the invention for the recognition of chrysotile asbestos fibers is soaked in a first solution comprising said sample in suspension and a composition according to the invention comprising SEQ ID NO: 2 (or SEQ ID NO: 3 ) is coupled to a carrier protein (eg TEM-1) conjugated to colloidal gold. Band 2 (Ami) has antibodies directed against said carrier protein. The second strip 9 according to the invention for the recognition of amphibole-type asbestos fibers is soaked in a second solution comprising said sample in suspension and a composition according to the invention comprising SEQ ID NO: 1 coupled to a carrier protein (for example example TEM-1) conjugated to colloidal gold, band 2 (Ami) having antibodies directed against said carrier protein.

In another embodiment, the kit according to the invention comprises a tab 9 and a composition according to the invention. The tab 9 according to the invention for the recognition of chrysotile and amphibole type asbestos fibers is soaked in a solution comprising said sample in suspension and a composition according to the invention comprising SEQ ID NO: 3 -GGG - SEQ ID NO: 1 - GGG - SEQ ID NO: 2 coupled to fluorescein isothiocyanate and an antibody directed against isothiocyanate conjugated to colloidal gold, band 2 (Ami) having antibodies directed the antibody directed against the fluorescein isothiocyanate.

In another embodiment, the kit according to the invention comprises a tab 9 and a composition according to the invention. The tab 9 according to the invention for the recognition of asbestos fibers of the chrysotile and amphibole type is soaked in a solution comprising said suspended sample and a composition according to the invention comprising SEQ ID NO: 1 and SEQ ID NO: 2 (or SEQ ID NO: 3) coupled to fluorescein isothiocyanate and an antibody directed against isothiocyanate conjugated to colloidal gold, band 2 (Ami) having antibodies directed the antibody directed against isothiocyanate fluorescein. In another embodiment, the kit according to the invention may contain two tabs 9 and two compositions according to the invention for separately detecting chrysotile and amphibole type asbestos fibers.

Once the time interval has elapsed (at least 2 min), the suspended sample thus prepared is brought into contact with the receiving zone 1, for example by soaking the tab 9 in the sample in suspension. If there are asbestos fibers in the suspended sample, the peptides bound to them do not migrate along the tab 9 and their presence is therefore not detected (B).

On the other hand, the band farthest from the reception zone 1 of the suspended sample to be tested comprises a witness T for visualizing that the test has worked correctly. This control T may for example be bovine serum albumin coupled to colloidal gold and revealed by an antibody directed against bovine serum albumin. If this band is colored, and the first band 2 (Friend), located near the receiving zone 1 of the sample to be tested, is also colored, it means that there is no asbestos (A ). If the two bands are stained, it means that the test has worked and that the peptides of the composition according to the invention have migrated to the bands. They are therefore not related to asbestos fibers (A). We can therefore conclude that there is no asbestos fiber. If the farthest band is not colored, in this case, the test has not worked and it must be reproduced (C and D).

As can be seen in FIG. 9B, the present invention also relates to an asbestos detection kit which comprises a first zone for receiving a test sample 1 which is positioned on a tab 9 at one end of that and which also comprises three stop bands 2, 3 and 4 in which are immobilized primary or secondary binding proteins of the detection means coupled to the peptide fragments of the composition according to the present invention. The sample is prepared by suspending a test sample for the presence of asbestos in a blocking buffer (<1 cm3 of sample for a volume of 1 ml). The sample is then contacted, beforehand in a separate container, with the mainly peptide composition according to the present invention for a time interval (at least 2 min) sufficient to allow the binding of the peptides of the composition according to the invention. to the sample.

The amount of composition according to the invention comprises, in this preferred embodiment, a mixture comprising the peptide having the sequence SEQ ID NO: 1 coupled to colloidal gold-conjugated beta-lactamase TEM-1 and the peptide having for sequence SEQ ID NO: 2 coupled to the maltose binding protein (MBP) conjugated to colloidal gold. Band 2 (Am) has antibodies to TEM-1 beta-lactamase and Band 3 (Ch) to antibodies to maltose binding protein (MBP).

This mixture is placed in a relatively small amount relative to the amount of sample to be tested in order to ensure that if there are asbestos fibers in the sample, all or the peptides are bound to the fibers of the sample. 'asbestos. Once the time interval has elapsed (at least 2 min), the suspended sample thus prepared is brought into contact with the receiving zone 1, for example by soaking the tab 9 in the sample in suspension. If there are amphibole and serpentine asbestos fibers in the suspended sample, the peptides bound to them do not migrate along tab 9 and their presence is therefore not detected (B). If there are only amphibole fibers, the peptides bound to them do not migrate along tab 9 and their presence is therefore not detected (D). If there are only serpentine fibers, the peptides bound to them do not migrate along the tab 9 and their presence is therefore not detected (C).

The strip farthest from the reception zone 1 of the suspended sample to be tested comprises a witness T to show that the test has worked correctly. This control T may for example be bovine serum albumin coupled to colloidal gold and revealed by an antibody directed against bovine serum albumin.

If the control strip 4 is colored and the first strip 2 (Am), located near the receiving zone A of the sample to be tested, is also colored, it means that there is no asbestos of amphibole type (C).

If the control strip 4 is colored and the second strip 3 (Ch) is also colored, it means that there is no serpentine asbestos (D).

If the three bands 2 (Am), 3 (Ch) and 4 (T) are stained, it means that the test has worked and that the peptides of the composition according to the invention have migrated to the bands. They are therefore not related to asbestos fibers (A). We can therefore conclude that there is no asbestos fiber.

If the farthest band is not colored, in this case, the test has not worked and should be reproduced (E, F, G and H).

It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made without departing from the scope of the appended claims.

LJSTAGE.DE SEQUENCES <110> Symbiosis Biomaterials <120> Mainly peptidic composition for the detection of asbestos <130> PAT2524407BE00 / MAWL <160> 6 <170> Patent-ln version 3.5

<210> 1 <211> 7 <212> PRT <213> Bacteriophage M13 <221> Peptide <223> Peptide binding and recognizing asbestos <400> SEQ ID NO: 1

Glu Thr Leu Ser Arg Met Arg 1 5

<210> 2 <211> 11 <212> PRT <213> Bacteriophage M13 <221> Peptide <223> Peptide binding and recognizing asbestos <400> SEQ ID NO: 2

Phe Tyr Ser His Ser Ala Leu Tyr Arg Thr His 15 10

<210> 3 <211> 7 <212> PRT <213> Bacteriophage M13 <221> Peptide <223> Peptide binding and recognizing asbestos <400> SEQ ID NO: 3

Gin Phe Asp His Trp Arg Asn 1 5

<210> 4 <211> 7 <212> PRT <213> Bacteriophage M13 <221> Peptide <223> Peptide binding and recognizing asbestos <400> SEQ ID NO: 4

Gly His Cys Ser His Pro Thr 1 5

<210> 5 <211> 31 <212> PRT <213> Bacteriophage M13 <221> Peptide <223> Peptide binding and recognizing asbestos <400> SEQ ID NO: 5

Gin Phe Asp His Trp Arg Asn Gly Gly Gly Glu Thr Leu Ser Arg Met Arg Gly Gly Gly Phe Tyr Ser Ser Ser Ala Leu Tyr Arg Thr His 15 10 15 20 25 30

<210> 6 <211> 27 <212> PRT <213> Bacteriophage M13 <221> Peptide <223> Peptide binding and recognizing asbestos <400> SEQ ID NO: 6

Gin Phe Asp His Trp Arg Asn Gly Gly Gly Glu Thr Leu Ser Arg Arg Arg Gly Gly Gly His Cys Ser His Pro Thr 15 10 15 20 25

Claims (39)

1. A mainly peptide composition for the detection of asbestos in a sample comprising: a first peptide fragment Px specific for asbestos fibers, arranged to recognize and bind to the asbestos fibers of said family of amphiboles, a second peptide fragment P2 specific for asbestos fibers, arranged to recognize and bind at least asbestos fibers of said family of serpentines together with said first peptide fragment Pj, and at least one detection means arranged to detect recognition and binding of said first peptide fragment Pi to said asbestos fibers and the recognition and binding of said second peptide fragment P2 to said asbestos fibers, characterized in that said first peptide fragment P1 has a percentage of identity of at least 70%, preferably of at least 85%, with a sequence X3X1X2XiX4X2X4 <where X1 represents a polar amino acid, X2 represents an acid a A non-polar amino, X3 represents an acidic amino acid and X4 represents a basic amino acid. 2. Mainly peptide composition according to claim 1, characterized in that said first peptide fragment Pi has the sequence SEQ ID NO: 1. 3. The predominantly peptidic composition according to claim 1 or 2, characterized in that said first peptide fragment Pi and said second peptide fragment P2 are fused, in any order, and form a hybrid peptide. 4. The predominantly peptidic composition according to claim 1 or 2, characterized in that said first peptide fragment Pi and said second peptide fragment P2 are each independent of each other, in the form of a mixture. 5. Mainly peptide composition according to any one of claims 1 to 4, characterized in that said first peptide fragment Pi and / or said second peptide fragment P2 and / or said at least one detection means are fused, optionally in the form of repetitions, via at least one binding domain selected from poly-G or G-rich binding domains, the length of which may vary, including GGG, GGS, GGGS and GGSSG. 6. Mainly peptide composition according to any one of Claims 1 to 5, characterized in that the said second peptide fragment P2 has a percentage of identity of at least 50%, preferably at least 65%, preferably of at least 80%, with a sequence selected from the group consisting of XiX2X3X4X2X4Xi and X2X1X1X4X1X2X2X1X4X1X4, wherein X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid. 7. Mainly peptide composition according to any one of claims 1 to 6, characterized in that said second peptide fragment P2 has the sequence SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4. 8. Primarily peptide composition according to any one of claims 1 to 7, characterized in that it further comprises a third peptide fragment P3 arranged to recognize and bind to said asbestos fibers of said family of amphiboles and / or said asbestos fibers of said family of serpentines and having an identity percentage of at least 50%, preferably at least 65%, preferably at least 80%, with a sequence selected from the group consisting of X1X2X3X4X2X4X1 and X2X1X1X4X1X2X2X1X4X1X4, wherein X1 represents a polar amino acid, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid. 9. The predominantly peptide composition according to claim 8, characterized in that said third peptide fragment P3 has the sequence SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4. 10. Primarily peptide composition according to claim 8 or 9, characterized in that said third peptide fragment P3 and said second peptide fragment P2 are different from each other. 11. Primarily peptide composition according to any one of claims 8 to 10, characterized in that said third peptide fragment P3 and said second peptide fragment P2 are fused, in any order, and form a hybrid peptide, said first peptide fragment Pi being independent, said hybrid peptide and said first peptide fragment Pi being in the form of a mixture. 12. The mainly peptide composition according to claim 8, wherein said third peptide fragment P3 and said first peptide fragment Pi are fused, in any order, and form a hybrid peptide, said second peptide fragment P2. being independent, said hybrid peptide and said second peptide fragment P2 being in the form of a mixture. 13. Mainly peptide composition according to any one of claims 8 to 10, characterized in that said first peptide fragment Pi and said second peptide fragment P2 are fused, in any order, and form a hybrid peptide, said third peptide fragment P3 being independent, said hybrid peptide and said third peptide fragment P3 being in the form of a mixture. 14. Primarily peptide composition according to any one of claims 8 to 10, characterized in that said second peptide fragment P2, said third peptide fragment P3 and said first peptide fragment Px are each independent of each other, in form of a mixture. The predominantly peptide composition according to any of claims 8 to 10, characterized in that said third peptide fragment P3, said second peptide fragment P2 and said first peptide fragment Pi are fused, in any order, and form a hybrid peptide. . 16. The predominantly peptide composition according to claim 8, characterized in that said first peptide fragment Pi and / or said second peptide fragment P2 and / or said third peptide fragment P3 and / or said at least one means of detection are fused, optionally as repeats, via at least one binding domain selected from poly-G or G-rich binding domains, the length of which may vary, including GGG, GGS, GGGS and GGSSG. 17. The predominantly peptide composition according to claims 15 and 16, characterized in that said third peptide fragment P3, said second peptide fragment P2 and said first peptide fragment Pi are fused and form a hybrid peptide having the sequence X3XiX2XiX4X2X4GGGX2XiXiX4XiX2X2GGGXiX4XiX4XiX2X3X4X2X4Xi where Xi represents an amino acid polar, X2 represents an apolar amino acid, X3 represents an acidic amino acid and X4 represents a basic amino acid. 18. The predominantly peptide composition as claimed in claim 17, characterized in that said hybrid peptide has for sequence the sequence SEQ ID NO: 5 consisting of SEQ ID NO: 3 - GGG - SEQ ID NO: 1 - GGG -SEQ ID NO: 2 or SEQ ID NO: 6 consisting of SEQ ID NO: 3 - GGG - SEQ ID NO: 1 -GGG-SEQ ID NO: 4. 19. The predominantly peptide composition according to any one of claims 8 to 18, characterized in that said first peptide fragment Pi, said second peptide fragment P2 and said third peptide fragment P3, alone, in a mixture or merged are GEPIs. 20. Mainly peptide composition according to any one of claims 1 to 19, characterized in that said at least one detection means is selected from luciferase, alkaline phosphatase, fluorescent proteins, beta-galactosidase, beta-lactamase , diphorase, peroxidase, maltose binding protein, laccase, lipase, glycosyl hydrolase, bovine serum albumin, colloidal gold, colored beads, preferably colored latex beads, or biotin and their mixtures. 21. Primarily peptide composition according to any one of claims 1 to 20, characterized in that it is in the form of a solution, in a solvent such as an aqueous solution, a polar organic solvent, and preferably in some water. 22. Primarily peptide composition according to any one of claims 1 to 21, characterized in that said sample is a sample of building material, pure asbestos fibers, an air sample and / or a sample of biological material . 23. A method for detecting asbestos in a sample comprising: preparing the sample by suspending a test sample for the presence of asbestos, bringing said sample into contact with at least one predominantly peptide composition according to any one of claims 1 to 22, for a period of time sufficient to allow the binding of the peptides of the predominantly peptide composition according to any one of claims 1 to 22 to the sample, a revelation of the presence of fragments peptides of the composition according to any one of claims 1 to 22, related to asbestos fibers of the family of serpentines and / or the family of amphiboles. 24. The method of claim 23, characterized in that it further comprises at least one step of separating said peptide fragments bound peptide fragments not bound to asbestos fibers, such as a step of removing said peptide fragments not linked, for example by percolation through a filter wall or for example, by a washing separation step. 25. Asbestos detection kit comprising: at least one support provided with at least a first reception zone of a test sample, and at least one peptide composition according to any one of claims 1 to 22, in wherein at least said peptide fragments P1, P2 and optionally additional peptide fragments such as P3 are packaged together or each separately, preferably in a syringe or in a cartridge comprising a plurality of compartments. The detection kit according to claim 25, further comprising at least one developing means, preferably packaged in a syringe or in a cartridge comprising a plurality of compartments, arranged to visualize said at least one means for detecting the peptide composition, said at least one means of revelation being a means of revealing in visible form, such as, for example, a means of revealing by colorimetry, for example nitrocele or any other compound allowing revelation by colorimetry. The detection kit of claim 26, wherein said at least one detection means is selected from the group consisting of luciferase, beta-galactosidase, beta-lactamase, laccase, lipase, glycosyl hydrolase, alkaline phosphatase, diphorase, peroxidase and their mixtures and is more particularly beta-lactamase. 28. A detection kit according to claim 25, further comprising at least one developing means, preferably packaged in a syringe or in a cartridge comprising a plurality of compartments, arranged to display said at least one means for detecting the peptide composition, said at least one revelation means being a fluorescence revelation means. The asbestos detection kit of claim 28, wherein said at least one detection means is a fluorescent molecule, revealed by excitation by means of a wave. An asbestos detection kit according to any one of claims 25 to 29, further comprising a wash solution, preferably packaged in a syringe or in a cartridge comprising a plurality of compartments. 31. The asbestos detection kit according to any one of claims 25 to 30, wherein said support further comprises a second receiving zone arranged to receive a negative control or a positive control. An asbestos detection kit according to any one of claims 25 to 30, wherein said support further comprises a second and a third receiving zone arranged respectively to receive a negative control and a positive control or vice versa. An asbestos detection kit according to claim 25, further comprising at least one developing means and wherein said support comprising said first receiving zone of a suspended sample to be tested is a tigette on which the first zone of receiving said sample to be tested is positioned at one end thereof, said tab also comprising at least two stop strips in the direction opposite to said first reception area in which are immobilized primary or secondary binding proteins of said least detecting means or said developing means. 34. The asbestos detection kit according to claim 33, wherein said at least one detection means comprises at least one fluorescent molecule, for example fluorescein isothiocyanate, or at least one carrier protein such as bovine serum albumin. , the maltose binding protein, biotin, an enzyme such as beta-galactosidase, beta-lactamase, laccase, lipase, glycosyl hydrolase, alkaline phosphatase, diphorase, peroxidase and more particularly beta lactamase, said at least one fluorescent molecule and said at least one carrier protein being coupled to said at least one revelation means such as colloidal gold or to colored beads, preferably colored latex beads and in which the proteins binders immobilized in the stop bands are primary binding proteins selected from the antibodies directed against the at least one carrier protein, antibodies to said at least one fluorescent molecule, or avidin (or the like), or streptavidin. 35. The asbestos detection kit according to claim 33, wherein said at least one detection means comprises at least one carrier protein or at least one fluorescent molecule, such as a fluorescein isothiocyanate, recognized by at least one means. disclaimer such as an antibody directed against said at least one fluorescent protein, for example an antibody directed against fluorescein isothiocyanate, or an antibody directed against said at least one carrier protein, said antibodies being coupled to colloidal gold , colored beads, such as colored latex beads and wherein the binding proteins immobilized in the stop bands are secondary binding proteins selected from secondary antibodies directed against said antibodies directed against said at least one fluorescent molecule and said at least one carrier protein. 36. A detection kit according to any one of claims 25 to 35, characterized in that said first peptide fragment Pi is fused to a first detection means and said second peptide fragment P2 or said third peptide fragment P3 is fused to a second detection means, different from said first detection means, said first, second or third peptide fragments Pi, P2 or P3 being in the form of a mixture. 37. Use of a predominantly peptide composition according to any one of claims 1 to 22 for the detection of asbestos in a sample. 38. Use of a method according to claims 23 or 24 for the detection of asbestos in a sample. 39. Use of a kit according to any one of claims 25 to 36, for the detection of asbestos in a sample.