Patent Grant
11029312
The present invention relates to the field of the in vitro detection of bacterial proteins in biological samples that may contain these proteins. In particular, the invention relates to the stabilization of glutamate dehydrogenase from a bacterium of the Clostridium genus so that this protein retains the antigenic properties thereof when it is in an aqueous solution.
Bacteria of the Clostridium genus are Gram-positive, sporulated, anaerobic bacteria which are incapable of reducing sulfates to sulfites. Some species are very pathogenic, such as Clostridium difficile, Clostridium botulinum and Clostridium perfringens, these being the most well-known.
The Clostridium difficile bacterium is the main agent responsible for diarrhea following the administration of antibiotics. It is formidable because of its very high contagion potential. Although approximately 5% of the population are asymptomatic carriers of the bacterium, its pathological manifestations are closely linked to time spent in hospital. This bacterium develops in an intestinal flora weakened by treatment with antibiotics and can secrete two toxins, A and B. Only the toxin-producing strains are pathogenic. Toxin A, an enterotoxin, causes modification of intestinal epithelium permeability; toxin B, a cytotoxin, directly attacks the cells of the epithelium. The combined effect of the two toxins is a decrease in intestinal transit time and in intestinal absorption, thereby resulting in diarrhea. More rarely, the Clostridium difficile bacterium can cause a severe inflammation of the colon (pseudomembraneus colitis).
The Clostridium botulinum bacterium, for its part, is responsible for botulism. It produces spores which represent the resistance form of the bacterium. These spores can withstand weak heat treatments, such as pasteurization, which can pose food safety problems, and can then give a metabolically active bacterial cell capable of multiplying. This bacterium secretes one of the most powerful toxins of the living world, the botulinum toxin. Active by ingestion, this toxin then diffuses in the organism and acts by blocking neuromuscular transmission: it inhibits the motor neurons of muscle contraction. This infection can cause death by paralysis of the respiratory muscles if no treatment is put in place.
The Clostridium perfringens bacterium is a bacterium which develops in wombs, sometimes very deeply. This bacterium will produce necrotoxins, thus causing necrotizing enteritis. The most common major toxin is the alpha toxin, essentially produced by Clostridium perfringens type A. This toxin is involved in a very large number of cases of gangrene in humans and animals. Alone or in combination with other toxins, it also causes abrupt mortalities in pigs and ruminants.
The detection of the presence of these bacteria and of the secretion of their toxin is therefore a major public health problem which requires laboratories to have detection tests that are reliable, both in terms of sensitivity/specificity, and in terms of reproducibility of the results obtained with these tests. To do this, laboratories in particular need tests which include reagents that do not degrade over time and therefore remain stable.
The detection of the presence of bacteria in samples can be carried out by various techniques, such as the use of culture media or the technique of immunoassays, which are widely known to those skilled in the art. The immunoassay technique is a technique consisting broadly in detecting the presence of proteins using binding partners of these proteins. In the context of the detection of bacteria of the Clostridium genus, one of the detectable proteins, representative of the presence of this bacterium, is glutamate dehydrogenase (GDH). Other detectable proteins are the toxins secreted when the bacteria are toxigenic. The detection or quantification of GDH by immunoassay is a technique which makes it possible to have a greater diagnostic sensitivity than the detection or quantification of toxins by immunoassay. It is used as a screening means on populations at risk. In the event of a positive result, a search for toxins is then recommended since this technique has, for its part, a greater specificity.
Several diagnostic companies propose kits for detecting GDH in Clostridium difficile. Mention may, for example, be made of the VIDAS® GDH kit from the applicant.
In addition to the binding partners, the immunoassay technique also requires the use of reagents for calibrating and/or controlling the test. Thus, in the context of a GDH immunoassay, such reagents comprise GDH as such, which must retain its antigenic properties for as long as required from the viewpoint of the shelf life of the diagnostic kit in which it is contained.
The properties of a protein can be disrupted by any structural modification, both from a chemical point of view and from a physical point of view. The chemical modifications of a protein are based on changes at the level of the covalent bonds, due for example to oxidation, hydrolysis, etc., reactions, while the physical modifications, also called denaturation, cause a disorganization of the tertiary structure or three-dimensional conformation of the protein, without breaking of the covalent bonds. Protein denaturation can be induced by many chemical or physical factors, such as, inter alia, temperature, pH modification or a chemical agent. The consequence of such modifications is a disruption of the protein's actual activity. Thus, in the context of an enzyme such as GDH, such modifications can modify its enzymatic activity, something which the various authors have tried to overcome.
Thus, for example, the authors of patent application WO 2007/003936 have described the stabilization of the enzymatic activity of various proteins using one or more stabilizing compounds having the following characteristics:
Garcia-Galan C. et al., 2013, have for their part indicated that Escherichia coli GDH can be stabilized so that it maintains its enzymatic activity by coating the surface of the GDH with polyethyleneimine in the presence of lithium+.
However, no author has shown an interest in searching for how to stabilize GDH so that it retains its antigenic properties, although this is a problem encountered when this protein is used in an aqueous solution, in particular in a very dilute manner, for example at a concentration of about a few ng/ml.
Indeed, GDH is usually stored in lyophilized form since it is known that it does not retain its antigenic properties when it is placed in an aqueous solution. Thus, when GDH must be placed in an aqueous solution, for example in the context of a GDH immunoassay, the laboratory assistant takes up the lyophilized GDH in an aqueous solvent, and prepares aliquots that must then be frozen at −20° C. The expiration date of the aqueous solution is then quite short, on average two months stored between 2 and 8° C. Furthermore, taking up the GDH in an aqueous buffer has the drawbacks of leading not only to additional manipulations, but also to additional risks of error when it is taken up. Finally, this also requires the presence of a freezer.
The applicant has found, against all expectations, that it is possible to stabilize GDH in an aqueous solution so that its three-dimensional structure is at least partly preserved such that it keeps its antigenic properties. Such a stabilization is carried out by addition, as stabilizing compound, of a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or of a salt thereof. By virtue of the addition of this compound, the aqueous solution comprising the GDH can be stored between 2 and 8° C., for many months.
Thus, a first subject of the invention relates to a process for stabilizing glutamate dehydrogenase from a bacterium of the Clostridium genus in order to maintain the antigenic activity thereof, comprising the step of mixing, with said glutamate dehydrogenase in an aqueous solution, a stabilizing compound which is a carboxylic acid having a carbon-based chain of at least three carbon atoms and at least two —COOH groups, or a salt thereof.
Another subject of the invention relates to the stabilized aqueous compositions thus obtained, and also to the diagnostic kits comprising these compositions.
Yet another subject of the invention relates to the use of the compositions for establishing standard ranges in the context of a GDH immunological assay.
Yet another subject of the invention relates to the use of the compositions as a calibrator and/or control in the context of a GDH immunological assay.
Finally, a last subject of the invention relates to the processes for detecting the presence of a bacterium of the Clostridium genus using a composition of the invention, the bacterium being, where appropriate, toxigenic.
The applicant has therefore shown, against all expectations, that the use of specific compounds makes it possible to stabilize GDH from bacteria of the Clostridium genus in order to maintain the antigenic properties thereof when the GDH is in an aqueous solution, in particular in concentrations of about a few ng/ml, for example from 0.75 to 10 ng/ml, from 2 to 10 ng/ml or else from 3 to 6 ng/ml, which is particularly important in the context of tests for detecting these bacteria by immunoassay.
The expression “maintaining the antigenic properties of the GDH” is intended to mean that the GDH retains its property of binding to the binding partners used in the context of the immunoassay since its structure is preserved, at least in terms of the antigenic determinant involved in the binding of the binding partner.
Of course, the prefix “immuno” in the term “immunoassay”, for example, is not to be considered in the present application as strictly indicating that the binding partner is necessarily a partner of immunological origin, such as an antibody or an antibody fragment. Indeed, as is well known to those skilled in the art, this term is more widely used to denote tests and processes in which the binding partner is not a partner of immunological origin/nature, but consists, for example, of a receptor of the analyte that it is desired to detect and/or quantify. The requirement is that the binding partner concerned be capable of binding to the analyte being sought, preferably specifically. Thus, it is known practice to refer to the ELISA assay for assays which use binding partners that are not immunological in the strict sense, more widely known as “ligand binding assays”, whereas the term “immuno” is included in the title in extenso corresponding to the acronym ELISA. In the interest of clarity and uniformity, the term “immuno” is used in the present application to denote any biological analysis using at least one binding partner suitable for binding to the analyte being sought and detecting and/or quantifying the latter, preferably specifically, even when said binding partner is not of immunological nature or origin in the strict sense.
The term “GDH binding partner” is intended to mean any molecule capable of binding to GDH. By way of example of a GDH binding partner, mention may be made of antibodies, antibody fragments, nanofitins, GDH receptors, aptamers, DARPins or any other molecule which is known to have an interaction with GDH.
The binding partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies.
The polyclonal antibodies can be obtained by immunization of an animal with the target GDH as immunogen, followed by recovery of the desired antibodies in purified form, by taking the serum of said animal, and separation of said antibodies from the other serum constituents, for example by affinity chromatography on a column to which is attached an antigen specifically recognized by the antibodies, in particular the immunogen, or by means of a protein A or G.
The monoclonal antibodies can be obtained by the hybridoma technique widely known to those skilled in the art. The monoclonal antibodies can also be recombinant antibodies obtained by genetic engineering, using techniques well known to those skilled in the art.
By way of example of antibody fragments, mention may be made of Fab, Fab′, and F(ab′)2 fragments and also scFvs (single chain variable fragments) and dsFvs (double-stranded variable fragments). These functional fragments can in particular be obtained by genetic engineering.
Nanofitins (commercial name) are small proteins which, like antibodies, are capable of binding to a biological target, thus making it possible to detect it, to capture it or quite simply to target it within an organism.
Aptamers are oligonucleotides, generally RNA or DNA, identified in libraries containing up to 1015 different sequences, by means of an in vitro combinatorial selection method known as SELEX for “Systematic Evolution of Ligands by Exponential Enrichment” (Ellington A D and Szostak J W., 1990). Most aptamers are composed of RNA, owing to the capacity of RNA to adopt varied and complex structures, thereby making it possible to create at its surface cavities of varied geometries, making it possible to bind various ligands. These are biochemical tools of interest which can be used in biotechnological, diagnostic or therapeutic applications. Their selectivity and their ligand-binding properties are comparable to those of antibodies.
“DARPins” for Designed Ankyrin Repeat ProteINS (Boersma Y L and Plackthun A, 2011) are another class of proteins which make it possible to mimic antibodies and to be able to bind to target proteins with high affinity and high selectivity. They derive from the family of ankyrin proteins, which are adapter proteins which enable the attachment of integral membrane proteins to the spectrin/actin network which constitutes the “backbone” of the cell plasma membrane. The structure of ankyrins is based on the repetition of a unit of approximately 33 amino acids and the same is true of DARPins. Each unit has a secondary structure of helix-turn-helix type. DARPins contain at least three, preferably four to five, repeat units and are obtained by screening combinatorial libraries.
The binding partners used may or may not be specific for GDH. They are termed specific when they are capable of binding exclusively or virtually exclusively to GDH. They are termed nonspecific when the GDH-binding selectivity is lower and they are then capable of binding to other ligands, such as other proteins or antibodies. According to one preferred embodiment, the specific binding partners are preferred.
The glutamate dehydrogenase that needs to be stabilized is any glutamate dehydrogenase from Clostridium of which it is desired to detect the presence, for example that of Clostridium difficile, of Clostridium botulinum or of Clostridium perfringens. It includes all the possible variants. Such proteins are known and their sequences are described for example in the Uniprot database (www.uniprot.org).
Thus, the GDH from Clostridium difficile (Uniprot accession no. P27346) is a protein of 421 amino acids, the reference amino acid sequence of which is the following SEQ ID NO 1:
And the variants of which are:
Clostridium difficile (strain 630)
Clostridium difficile (strain CD196)
Clostridium difficile (strain R20291)
Clostridium difficile 002-P50-2011
Clostridium difficile 050-P50-2011
Clostridium difficile NAP07
Clostridium difficile 70-100-2010
Clostridium difficile NAP08
The GDH from Clostridium perfringens is a protein which does not yet have a reference sequence in the Uniprot base. The first protein given in the Uniprot base (Uniprot accession No. Q8XK85) is the protein of strain 13/type A, of 448 amino acids, the amino acid sequence of which is the following SEQ ID NO 2:
And the variants of which are:
Clostridium perfringens (strain 13/Type A)
Clostridium perfringens (strain SM101/Type A)
Clostridium perfringens (strain ATCC
Clostridium perfringens B str. ATCC 3626
Clostridium perfringens C str. JGS1495
Clostridium perfringens E str. JGS1987
Clostridium perfringens CPE str. F4969
Clostridium perfringens D str. JGS1721
Clostridium perfringens NCTC 8239
Clostridium perfringens F262
Clostridium perfringens WAL-14572
The GDH from Clostridium botulinum is a protein which does not yet have a reference sequence in the Uniprot base. The first protein given in the Uniprot base (Uniprot accession No. A5I2T3) is the protein of strain Hall/type A (ATCC 3502, NCTC 13319), of 421 amino acids, the amino acid sequence of which is the following SEQ ID NO 3:
And the variants of which, of 421, 447 or 450 amino acids according to the strains, are:
Clostridium botulinum (strain Okra/Type B1)421
Clostridium botulinum (strain Loch Maree/Type A3)
Clostridium botulinum (strain ATCC 19397/Type A)
Clostridium botulinum (strain H04402 065/Type A5)
Clostridium botulinum (strain Eklund 17B/Type B)
Clostridium botulinum (strain Kyoto/Type A2)
Clostridium botulinum (strain Alaska E43/Type E3)
Clostridium botulinum (strain 657/Type Ba4)
Clostridium botulinum (strain 230613/Type F)
Clostridium botulinum BKT015925
Clostridium botulinum (strain Langeland/NCTC
Clostridium botulinum C str. Eklund
Clostridium botulinum D str. 1873
Clostridium botulinum NCTC 2916
Clostridium botulinum Bf
Clostridium botulinum CFSAN001627
Clostridium botulinum CFSAN001628
Clostridium botulinum E1 str. ‘BoNT E Beluga’
According to one particular embodiment, the glutamate dehydrogenase is an enzyme from the bacterium of the species Clostridium difficile.
The glutamate dehydrogenase placed in an aqueous solution is either of natural origin, or of recombinant origin. The natural, or otherwise termed native, glutamate dehydrogenase can be obtained after culturing the Clostridium bacterium and purifying the protein from the bacterial lysate. The recombinant glutamate dehydrogenase can be obtained by genetic engineering, using techniques well known to those skilled in the art. Such obtaining is described, for example, by Anderson B M et al., 1993. The recombinant glutamate dehydrogenase can be obtained from companies such as Holzel Diagnostika GmbH (Germany).
The term “aqueous composition or solution” is intended to mean a clear liquid solution obtained by complete dissolution of one or more compounds and the major solvent of which is water, representing at least 50% by volume, generally at least 60%, 70%, 80% or 90%, relative to the total volume of the solution.
In the context of the present invention, the aqueous composition or solution is obtained by diluting the GDH in a solvent comprising predominantly water and a stabilizing compound as defined hereinafter. The stabilizing compound to be added to the aqueous solution containing the GDH to be stabilized is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or a salt thereof.
The expression “carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups” is intended to mean a molecule consisting of:
For example, the —CX— groups are chosen independently from —CH2—, ═CH—, —C(H)OH—, —C(H)NH2— and —C(O)—. The —CX groups are for their part chosen independently from —CH3, —COOH (if there are more than two —COOH groups in the molecule) and —C(O)NH2.
Thus, for example, the stabilizing compound may be succinic acid, of formula OH(O)C—(CH2)2—C(O)OH, which is a molecule having a contiguous linear carbon-based chain of four carbon atoms, two —COOH groups at the chain end and two —CH2— groups.
Another example consists of fumaric acid of formula OH(O)C—(CH═CH)—C(O)OH, which is a molecule having a contiguous linear carbon-based chain of four carbon atoms, two —COOH groups at the chain end and two —CH— groups.
Yet another example consists of N-(2-acetamido)iminodiacetic acid, of formula H2NC(O)—CH2—N(CH2—COOH)2, which is a molecule having a branched carbon-based chain of six carbon atoms, interrupted with a nitrogen atom, consisting of two —COOH groups at the chain end, of three —CH2— groups and of one —C(O)NH2 group.
According to one particular embodiment, the stabilizing compound is chosen from: fumaric acid, succinic acid, malic acid, glutaric acid, citric acid, tartaric acid, N-(2-acetamido)iminodiacetic acid, glutamic acid, adipic acid, aspartic acid, pimelic acid, malonic acid, and salts thereof, the formulae of which are given in
The term “carboxylic acid salt” is intended to mean a salt of a monovalent cation. By way of monovalent cation, mention may be made of ammonium (NH4+), silver (Ag+), diamine silver (Ag(NH3)2+), cesium (Cs+), copper (I) (Cu+), mercury (Hg+), methanium (CH5+), methylium (CH3+) and nitrosium (NO2+) ions and ions of the alkali metals sodium (Na+), potassium (K+) and lithium (Li+).
When the stabilizing compound is in salt form, at least one proton H+ of the —COOH groups is replaced with a monovalent cation described above.
According to one preferred embodiment, the stabilizing compound is chosen from succinic acid, fumaric acid, and salts thereof, in particular alkali metal salts, as defined above.
The carbon-based chain may comprise one or more of the following characteristics:
In particular, the carboxylic acid may have one or more of the following characteristics:
The amount of GDH present in the aqueous solution depends on the final use of the aqueous composition which contains it. In the context of a conventional immunoassay, the GDH may be present in a proportion of from 0.75 to 10 ng/ml, or from 2 to 10 ng/ml, preferably from 3 to 6 ng/ml. In the context of an “ultrasensitive” immunoassay, the GDH will be present in a much lower amount, for example less than one pg/ml, or even about one fg/ml.
The amount of stabilizing compound to be added to the aqueous solution containing the GDH is in large excess relative to the amount of GDH. It depends on whether the stabilizing compound is used only as a stabilizing compound, another molecule then being added as a buffer, or else whether it is used both as a stabilizing compound and as a buffer. Thus, for example, when the stabilizing compound is only used as a stabilizing compound, from 20 to 100 molecules of stabilizing compound are added per GDH monomer, preferably from 30 to 80 molecules of stabilizing compound per GDH monomer, more preferably from 40 to 60 molecules of stabilizing compound per GDH monomer. In this case, the compound added as a buffer is any compound known to those skilled in the art having a pH of between 4.5 and 7, preferably between 5.5 and 6.5, a pH of 5.8 being preferred. By way of example of a compound added as a buffer, mention may be made of phosphate and acetate. When the stabilizing compound is used both as a stabilizing compound and as a buffer, from 108 to 1010 molecules of stabilizing compound are added per GDH monomer, preferably from 1×109 to 5×109 molecules of stabilizing compound per GDH monomer, more preferably from 1×109 to 2×109 molecules of stabilizing compound per GDH monomer.
Other compounds may also be added to the aqueous composition in the process of the invention. Thus, for example, a polyol may be added. The addition of polyol makes it possible to promote the heat stability of the proteins (resistance to heat denaturation) and also to prevent aggregation. Generally, polyols have a co-solvent effect and contribute to maintaining the native conformation of proteins in an aqueous solution.
By way of examples of polyol, mention may be made of monosaccharide polyols such as triols, for example glycerol, tetraols, for example erythritol, pentols, for example xylitol, arabitol and ribitol, hexols, for example sorbitol, dulcitol and mannitol, heptols, for example volemitol, and also disaccharide polyols such as maltitol, isomaltitol and lactitol.
According to one embodiment of the invention, the polyol added to the aqueous composition of the process of the invention is sorbitol.
The polyol is added to the aqueous composition in a proportion of at least 1%, preferably at least 5% and more preferably at least 10%, with at most 50%.
The composition may also comprise another macro molecule, generically referred to as a “filler protein” even though macromolecules other than proteins are appropriate, which has nothing to do with the GDH, but is present in large excess relative to the GDH, further improving the stabilization of the GDH. This “filler protein” has a shield effect in the sense that it will partially undergo physicochemical modifications in the aqueous solution, thus making it possible to protect the molecule of interest, in this case GDH. This added macromolecule may for example be a protein such as BSA (bovine serum albumin), or else synthetic polymers of dextran or polyethylene glycol type. BSA, for example, is added in a proportion of 50 g/l compared with 3 mg/l of GDH.
The aqueous composition is buffered so as to have a pH of between 4.5 and 7, preferably between 5.5 and 6.5, a pH of 5.8 being preferred. As previously indicated, the stabilizing compound of the invention may also be used as a buffer, or else another buffer compound may be added.
The aqueous compositions comprising the glutamate dehydrogenase from a bacterium of the Clostridium genus and a stabilizing compound which is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or a salt thereof, it being understood that the stabilizing compound is neither glutamate, nor alpha-ketoglutarate, produced from hydrolysis of the glutamate by the GDH enzyme and the NAD+ coenzyme, nor citrate, nor succinate, nor glutarate, are novel and constitute another subject of the invention.
According to another embodiment, the aqueous compositions of the invention also exclude glutamic acid, alpha-ketoglutaric acid, glutaric acid, succinic acid and/or citric acid.
When the bacterium of the Clostridium genus is chosen from the species Clostridium difficile, Clostridium botulinum and Clostridium perfringens, then the aqueous compositions comprising the glutamate dehydrogenase from one of these bacteria and a stabilizing compound which is a carboxylic acid having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups, or a salt thereof, it being understood that the stabilizing compound is neither glutamate, nor alpha-ketoglutarate, nor citrate, are novel and constitute another subject of the invention.
According to another embodiment, the aqueous compositions of the invention also exclude glutamic acid, alpha-ketoglutaric acid and/or citric acid.
The same characteristics and preferences described previously, in particular with regard to the choice of the GDH, of the stabilizing compound, of the compounds to be added and of the relative amount thereof, given in relation to the process, also apply to the aqueous compositions according to the invention.
The aqueous compositions of the invention, in particular those obtained according to the process of the invention, are particularly useful for detecting the presence of a bacterium of the Clostridium genus in a biological sample that may contain such a bacterium. Thus, the kits containing such compositions constitute another subject of the invention.
The biological samples that may contain a bacterium of the Clostridium genus may be samples from the clinical field or from the field of testing innocuity, or even sterility of industrial products. In the clinical field, the sample is an animal, preferably human, biological specimen, such as stools or derivatives, for example an extract of fecal proteins, urine, blood or derivatives, for example serum or plasma, pus, etc. In the industrial field, the sample comes from a food or cosmetic product.
The kits according to the invention may also contain the compounds required for carrying out a process for detecting, for example by immunoassay, the presence of a bacterium of the Clostridium genus, and in particular Clostridium difficile. Thus, for example, the kits may contain one or more GDH-binding partners as previously described, and all the compounds required for demonstrating the reaction between the binding partner(s) and the GDH.
The qualitative or quantitative GDH immunological assay will preferably be a sandwich assay, which is an assay widely known to those skilled in the art using two GDH-binding partners. One of the two partners may be coupled to a label so as to form a conjugate or a tracer. The other binding partner may be captured on a solid support. The term “capture partner” is then used for the latter and detection partner is then used for the former.
The measured signal emitted by the conjugate is then proportional to the amount of GDH in the biological sample.
The term “label” is intended to mean in particular any molecule containing a group that reacts with a group of the binding partner, directly without chemical modification, or after chemical modification so as to include such a group, which molecule is capable of directly or indirectly generating a detectable signal. A nonlimiting list of these direct detection labels consists of:
Indirect detection systems may also be used, for instance ligands capable of reacting with an anti-ligand. The ligand then corresponds to the label so as to constitute, with the binding partner, the conjugate.
Ligand/anti-ligand pairs are well known to those skilled in the art, which is the case, for example, of the following pairs: biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin, polynucleotide/polynucleotide complementary to said polynucleotide.
The anti-ligand may then be directly detectable by the direct detection labels previously described or be itself detectable by another ligand/anti-ligand pair, and so on.
These indirect detection systems may result, under certain conditions, in an amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference may be made to the applicant's prior patent applications FR 2781802 or WO 95/08000.
Depending on the type of labeling used, those skilled in the art will add reagents which allow the visualization of the labeling or the emission of a signal detectable by any type of appropriate measuring apparatus, for instance a spectrophotometer, a spectrofluorimeter, a densitometer or else a high-definition camera.
In the GDH assay processes, aqueous compositions of the invention, where appropriate obtained according to the process of the invention, are particularly useful for establishing a standard range, this constituting another subject of the invention. The establishing of the standard range, a step that is necessary in order to be able to quantify the GDH, is a step widely known to those skilled in the art. Briefly, it consists in measuring the signal generated by increasing and known amounts or concentrations of the GDH analyte, in plotting the curve giving the signal as a function of the amount or the concentration and in finding a mathematical model which represents this relationship in the most faithful way possible. To do this, several aqueous compositions of the invention are used, each containing a different GDH concentration. The mathematical model will be used to determine by extrapolation the unknown amounts or concentrations of GDH contained in the biological sample to be tested.
The aqueous compositions of the invention, where appropriate obtained according to the process of the invention, are also particularly useful as a calibrator, which constitutes another subject of the invention. In this case, the GDH concentration of the composition is fixed and known. The signal generated during the use of the immunoassay kit by the calibrator is also known. The calibrator is used to verify that the measurement (signal) produced during the use of the immunoassay kit indeed corresponds to the expected value. If this is not the case, the calibrator is used to measure the shift which may, where appropriate, be corrected mathematically or by a physical intervention on the measuring instrument (adjustment).
The aqueous compositions of the invention, where appropriate obtained according to the process of the invention, are also particularly useful as a control, which constitutes another subject of the invention. In this respect, they are used, for example, to verify that the immunoassay kit operates according to expectations (also called positive control) and that the detection of the GDH in the biological sample is not falsely negative in so far as the detection method has operated correctly with the aqueous composition of the invention.
The detection of GDH in a biological sample makes it possible to conclude that the bacterium is present.
Thus, another subject of the invention relates to a process for detecting the presence of a bacterium of the Clostridium genus in a biological sample that may contain such a bacterium, characterized in that it comprises the steps of (i) carrying out a process for detecting the presence of a bacterium of the Clostridium genus by detecting or quantifying glutamate dehydrogenase in said sample using an aqueous composition of the invention, where appropriate as obtained according to the process of the invention, or else a kit as previously defined, and
(ii) if the process of step (i) is positive, concluding that the bacterium is present.
In other words, for step (ii), a positive result in step (i) makes it possible to conclude that the bacterium is present.
On the other hand, this provides no information as to whether or not this bacterium produces at least one toxin, which is particularly important as an aid to diagnosis when a patient presents symptoms that might be caused by the presence of a bacterium which is toxigenic and which expresses at least one toxin.
Thus, another subject of the invention relates to a process for detecting the presence of a toxigenic bacterium of the Clostridium genus which produces at least one toxin, in a biological sample that may contain such a bacterium and at least one such toxin, characterized in that it comprises the steps of:
(i) carrying out a process for detecting the presence of a bacterium of the Clostridium genus by detecting or quantifying glutamate dehydrogenase in said sample using an aqueous composition of the invention, where appropriate as obtained according to the process of the invention, or else a kit as previously defined, and
(ii) if the process of step (i) is negative, concluding that the bacterium is absent, or
(ii′) if the process of step (i) is positive, carrying out a process for detecting or quantifying at least one toxin that may be released by said bacterium of the Clostridium genus, in the same biological sample, or in a new biological sample from the same individual, and concluding that the bacterium is toxigenic and produces said at least one toxin if said at least one toxin is present.
In other words, for step (ii), a negative result in step (i) makes it possible to conclude that the bacterium is absent and a positive result in step (i) makes it possible to conclude that the bacterium is present.
Step (i) above consisting in detecting the presence of a bacterium of the Clostridium genus by detecting or quantifying glutamate dehydrogenase in the biological sample has been described previously. The aqueous composition of the invention, where appropriate obtained by means of the process of the invention, can then be used to produce the standard range and/or as a calibrator and/or as a positive control.
Step (ii′) above consisting in detecting or quantifying at least one toxin that may be released by said bacterium of the Clostridium genus is a step widely known to those skilled in the art. Such detecting or quantifying may be carried out, for example, by immunoassay using partners for binding to the toxins being sought. The toxin immunoassay is carried out in a manner similar to the GDH immunoassay as described previously. Kits for immunoassaying toxin from bacteria of the Clostridium genus are commercially available, for instance the VIDAS® Clostridium difficile A&B kit which makes it possible to detect Clostridium difficile toxins A and B.
Mass spectrometry may also be used to carry out the step of detecting/quantifying the toxin. This technique is an analytical technique which makes it possible to determine the molar mass of the compounds analyzed, and also to identify their molecular structure, or even to quantify them. Applied to a complex mixture such as a biological fluid or stools, it needs to be coupled to a separative technique which makes it possible to reduce the complexity thereof. This is usually gas chromatography (GC) or liquid chromatography (LC). Tandem mass spectrometry (MS/MS) combines two analyzers and may be used for the purposes of detection/quantification. The ionic compounds selected in the first analyzer and then fragmented are analyzed more finely in the second. This double analysis makes it possible to significantly increase the specificity of the method. For this technology, reference may in particular be made to Van den Broek et al., 2013.
The detecting and/or quantifying of the toxins may also be carried out by means of a test for immunotoxicity in the stools (CTA) which makes it possible to demonstrate the biological effects of toxins in the stools (Eckert C. et al., 2011).
The detecting or quantifying of the toxin is carried out in the same biological sample as that used for detecting or quantifying the GDH, a part of which has been kept in this respect, or else in a new biological sample from the same origin, i.e. from the individual from whom the first biological sample tested with respect to the presence of GDH came if the biological sample is a clinical sample or from the same source if the biological sample is an industrial sample. The second biological sample is either of the same nature, or of different nature, the first case being preferred.
According to one particular embodiment, the toxigenic bacterium of which it is desired to detect the presence is Clostridium difficile and said at least one toxin comprises toxin A, toxin B or both.
Other toxigenic bacteria of the Clostridium genus have been described previously.
The invention will be understood more clearly by means of the following examples which are given by way of nonlimiting illustration, and also by means of
The gluD gene encoding the GDH from Clostridium difficile (Genbank accession No. M65250), with a sequencing encoding a HIS tag added thereto, is cloned into the pMR78 vector (bioMérieux, France). The expression plasmid thus constructed is introduced into E. coli BL21 bacteria and derivatives (Stratagene, Agilent Technologies). The cultures are carried out in 2×YT medium (Difco), in the presence of ampicillin, at 37° C. with shaking. The expression of the protein is induced by adding 1 mM of IPTG (isopropyl beta-D-1-thiogalactopyranoside). The bacteria are collected by centrifugation at the end of culturing.
The bacterial pellets are taken up in 2×PBS buffer (phosphate buffered saline) and lysed. The lysates are centrifuged at 3000 g for 30 min at 4° C. The supernatant contains the soluble proteins, including the recombinant GDH to be purified.
The purification of the protein is carried out by one-step metal chelate affinity chromatography. The supernatant obtained after centrifugation is loaded onto an Ni-NTA-Agarose resin (Qiagen). After a washing cycle, the protein is eluted in the presence of an imidazol gradient. The protein is dialyzed in a 20 mM phosphate buffer.
The recombinant GDH, prepared in example 1, is diluted to 3 ng/ml in the following formulations, according to the indications relating to the calibrator and control (S1/C1) given in the information sheet for the VIDAS® GDH reagent (Ref 30125, bioMérieux, France):
Each composition (comparative and ADA, succinate and fumarate) was prepared beforehand as follows: each stabilizing compound (1.18 g of succinic acid —Merck, 1.6 g of dibasic sodium fumarate—Sigma, 1.90 g of ADA—Sigma or 0.78 g of phosphate NaH2PO4.2H2O+1.79 g of Na2HPO4.12H2O) was mixed with demineralized water so as to obtain 50 ml. 10 N NaOH was then added so as to adjust the pH to 5.8. Five g of BSA (Millipore) were added. Finally, demineralized water was added to as to obtain a solution of 100 ml.
The four compositions containing the GDH are then aliquoted into fractions of 1 ml and then stored at 37+/−1° C. The impact of the formulation on the stability of the antigenic properties of the recombinant GDH protein is evaluated by carrying out several GDH assays over a period of 91 days with the VIDAS® GDH kit (Ref 30125) and the VIDAS® instrument according to the instructions of the manufacturer (bioMérieux, France).
The implementation of the VIDAS GDH test adheres to the protocol of the kit sold:
Each aliquot is used for only one monitoring timepoint, but is systematically used in duplicate. The VIDAS instrument measures a fluorescence signal and the results are expressed as “relative fluorescence value” or RFV.
The RFV results obtained for the two measurements in duplicate (1 and 2) and also the D/D0 ratio (RFV on day D relative to RFV on day 0) are given in table 4 below and are also reproduced on the graph of
The results demonstrate that, at 37° C., the use of stabilizing compounds consisting of carboxylic acids having a carbon-based chain of at least three carbon atoms and comprising at least two —COOH groups makes it possible to very substantially improve the storage time of an aqueous solution containing GDH since, for the comparative composition, there is no longer any signal at 28 days, whereas the D/D0 ratio at this date for the compositions according to the invention is at least equal to 0.5.
The procedure of example 2 was repeated, except for the fact that citric acid (100 mM) was used as stabilizing compound and that the aqueous solutions were stored for a longer period of time, at 2-8° C. and at 37° C.
The RFV results obtained for the two measurements in duplicate (1 and 2) and also the D/D0 ratio are given in table 5 below.
The above results demonstrate that the addition of citric acid allows very good stability associated with the preservation of the antigenic properties of the GDH, with a virtually optimal stabilization, even after 18 months, when the aqueous composition is stored between 2-8° C.
The procedure of example 2 was repeated, except for the fact that 10% of sorbitol was also added to a succinate composition and that the aqueous solutions were stored for a longer period of time, at 2-8° C. and at 37° C.
The RFV results obtained for the two measurements in duplicate (1 and 2) and also the D/D0 ratio are given in table 6 below.
The above results demonstrate that succinic acid also allows a lengthy stabilization of the antigenic properties of the GDH in an aqueous solution, the addition of sorbitol not modifying this stabilization, with a stabilization which is virtually optimal, even after approximately 30 months, when the aqueous solution is stored between 2-8° C.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1362354 | Dec 2013 | FR | national |
This is a Continuation of application Ser. No. 15/038,554 filed May 23, 2016, which is a National Stage Entry of PCT/FR2014/053213 filed Dec. 8, 2014, which claims priority to FR 1362354 filed Dec. 10, 2013. The disclosures of the prior applications are hereby incorporated by reference herein in their entireties.
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| 5965375 | Valkirs | Oct 1999 | A |
| 10533999 | Bettsworth | Jan 2020 | B2 |
| 20040086532 | Donovan | May 2004 | A1 |
| 20100028372 | Jezek | Feb 2010 | A1 |
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| 9508000 | Mar 1995 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20200132690 A1 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15038554 | US | |
| Child | 16733681 | US |
