Combined saliva test

Abstract

Disclosed is an integrated or combined device for a saliva test for at least two analytes comprising a sampling member, an intermediate section and an immunolabeling section comprising a plurality of antibodies for the specific binding of the analytes and a plurality of second antibodies for detecting the analytes, which analytes are the histidine-rich protein (HRP) and the lactate dehydrogenase (LDH) protein of Plasmodium sp..

Description

[0001] COMBINED SALIVA TEST

[0002] technical field

[0003] The present invention relates to a saliva collection device coupled to an immunological test of at least two analytes of Plasmodium sp.

[0004] Previous art

[0005] Antigen detection from blood samples is known.

[0006] For example, the Optimal-IT test allows the detection of Plasmodium in a patient's blood with a detection limit of up to 100 parasites / µL of blood, although sensitivity drops for loads below 500 parasites / µL (Valéa et al., Tropical Medicine and International Health, 2009, pp. 338-340). The antigen is LDH.

[0007] The CN 1 10 343 161 B document describes a nitrocellulose membrane for the detection of two Plasmodium falciparum analytes, LDL and HRP; if present, these analytes are fixed to the membrane via proteins selected by phage display.

[0008] On the other hand, the antigenic detection of a salivary analyte is also known. For this purpose, document EP 4 201 343 describes a saliva collection device comprising a rigid head decorated with multiple orifices, a longitudinal handle with fluidic connection, and elements for measuring a saliva characteristic. Means for active saliva absorption or displacement can be incorporated. For example, saliva tests have been attempted for infectious diseases, such as Covid-19.

[0009] However, antigenic detection of a salivary analyte is very challenging due to low concentrations or variable levels, as well as the composition and abundance of saliva, which can vary between patients (age, pathological condition) and may contain interfering molecules. Furthermore, such tests are rarely integrated from saliva collection to detection, even though such integration represents a significant advantage.

[0010] The present invention aims to provide a salivary immunological testing device for the detection of the presence of one or more analyte(s) which overcomes the aforementioned difficulties and is simple to use while being fast.

[0011] Brief summary of the invention

[0012] A first aspect of the present invention relates to an integrated device 1 for a saliva test of at least two analytes comprising a sampling member 2, an intermediate section 3 and an immunological labeling section 4, said sampling member comprising a collection channel 21 and being arranged for the collection of a sufficient quantity of salivary fluid via the application of a vacuum force or suction, said intermediate section 3 comprising an arrangement of polymers in the form of fibers and said immunological labeling section comprising several antibodies for the specific binding of said analytes and several second antibodies for the detection of said analytes, said analytes being the Histidine Rich protein (HRP) and the Lactate dehydrogenase protein (LDH) of Plasmodium sp.

[0013] Preferably, in this device, the collection channel 21 includes a non-return system.

[0014] Preferably, the depression in the harvesting limb 2 is caused by a suction mechanism.

[0015] An associated aspect of the present invention relates to a combined device 1 1 for a saliva test of at least two analytes comprising on the one hand a sampling member 2 coupled to an intermediate member 3, said sampling member 2 comprising a collection channel 21 and being arranged for the collection and retention of a sufficient quantity of salivary fluid, said retained salivary fluid being transferable to said intermediate section by means of a trigger, said intermediate section 3 comprising an arrangement of polymers in the form of fibers and being arrangeable to an immunolabeling section, and on the other hand, an immunolabeling section 4 comprising several antibodies for the specific binding of said analytes and several second antibodies for the detection of said analytes, said analytes being the "Histidine Rich" Protein (HRP) and the Lactate dehydrogenase (LDH) protein of Plasmodium sp.Preferably, in this combined device, the action for transferring the sampling limb to the immunological labeling section is the application of pressure so as to eject a determined quantity of salivary fluid towards the labeling section 4 or a movement allowing the sampling salivary fluid to come into contact with said labeling section 4.

[0016] Advantageously, the sampling member 2 of this integrated device 1 or combined device 1 has a geometry suitable for stimulating salivary secretion.

[0017] Preferably, the sampling member 2 of this integrated or combined device 1 comprises a salivary stimulation molecule, preferably selected from an acid, a sugar, a pungent agent, or olfactory stimulants.

[0018] Advantageously, the intermediate section 3 of this integrated or combined device 1 contains fibers organized according to a gradient of hydrophilicity and / or pore size, so as to cause (reinforce) a movement of salivary fluid towards the immunolabeling section 4.

[0019] Preferably, the intermediate section 3 comprises a chemical treatment mechanism or a physical treatment mechanism for saliva, or both; preferably, the chemical treatment of saliva comprising the presence of disulfide bond-reducing molecules and / or a protease inhibitor; preferably, the physical treatment of saliva comprising sieving (filtering) said saliva to reduce the air bubble content. Preferably, the labeling section of this integrated or combined device 1 comprises chromatography, preferably lateral flow chromatography 41.

[0020] Advantageously, the integrated device 1 or combined device 1 (and / or its labeling section) is suitable when the analytes are from Plasmodium falciparum, Plasmodium vivax and / or Plasmodium malariae (and / or Plasmodium ovale).

[0021] Advantageously, the antibodies are selected and matched to allow sensitive detection of analytes.

[0022] Preferably, this integrated device 1 or combined device 1, or its labeling section 4, comprises a binding antibody and a detection antibody for HRP2 and HRP3 of P. falciparum, and two binding antibodies for the LDH proteins of P. falciparum and P. vivax (respectively) and at least one detection antibody for these LDH proteins of P. falciparum and P. vivax.

[0023] Other features, details and advantages of the invention will become apparent from the description given below, by way of non-limitation and with reference to the drawings and examples.

[0024] Brief description of the drawings

[0025] Figure 1 is a schematic view of the integrated device according to the invention.

[0026] Figure 2 is a schematic view of the combined device according to the invention.

[0027] CORRECTED SHEET (RULE 91) ISA / EP Detailed description of an embodiment of the invention

[0028] The inventors had the intuition that malaria diagnosis is not carried out satisfactorily: either the tests are too complicated or too expensive, or they are invasive (depending on the cultures or sanitary facilities available locally, a blood test may be considered invasive), or their performance (sensitivity, specificity) leaves something to be desired.

[0029] As a result, in case of doubt, many patients receive antimalarial treatments when it is not necessary, and other patients who need them do not receive them.

[0030] Besides the problem at the individual patient level, overuse of antimalarial drugs leads to protozoan resistance mechanisms, and failure to treat patients increases the protozoan reservoir, which increases the risk of contagious bites.

[0031] In seeking to solve this problem, the inventors developed the integrated device 1, or combined device 1 1, which will be described in more detail below.

[0032] A first aspect of the present invention therefore relates to an integrated device 1 (or a combined device 1 1; see below), for a saliva test of at least one analyte comprising a sampling member 2, an intermediate section 3 and an immunological labeling section 4, said sampling member comprising a collection channel 21 and being arranged for the collection of a sufficient quantity of salivary fluid via the application of a vacuum and / or suction force, said intermediate section comprising an arrangement of polymers in the form of fibers and said immunological labeling section comprising one or more antibodies for the specific binding of said analyte(s) and one or more second antibodies for the detection of said analyte(s).

[0033] The inventors noticed that it was possible to create a complete device, from saliva collection to the immunolabeling detection system. This avoids several manipulations, potentially affecting the result, and also allows for simplified distribution and therefore widespread use, even in hard-to-reach areas.

[0034] The sampling method involves applying a vacuum force to the sampling limb 2 (preferably to the sampling duct 21). This can be achieved via a specific mechanism, such as a bulb connected to the sampling duct 21. Alternatively, the device can be designed so that the patient applies suction to the sampling device 2, which draws saliva into the sampling duct 21; this can be done using a teat placed in the mouth, whereby suction induces a vacuum, drawing up the saliva.

[0035] Furthermore, the presence of a depression at the sampling point eliminates the need for gravity flow, thus allowing, for example, the use of the device even in bedridden individuals. Advantageously, the sampling member 2 has a geometry designed to stimulate salivary secretion, advantageously resembling a teat.

[0036] Indeed, the inventors measured that a grooved structure at the sampling device 2 stimulates the patient's saliva production.

[0037] On the other hand, the inventors believe that a mouth device of almost universal shape is instinctively associated with the stimulation of the salivary glands. Such a device is therefore advantageous, for example, for sick or elderly patients who would otherwise have more difficulty producing sufficient saliva.

[0038] One of the advantages of this saliva collection device is that it allows sufficient saliva to be collected in less than a minute, for example in about 30 seconds (between 15 and 50, preferably between 20 and 40 seconds). This is more comfortable for the patient.

[0039] Preferably, the collection channel 21 includes a non-return device, for example a ball, or a valve which is moved to the open position, due to the depression and which blocks the collection channel 21 in the absence of depression.

[0040] This prevents saliva from flowing back into the mouth, for example saliva that may have come into contact with internal elements of the integrated device 1. This is advantageous in itself, since it reinforces the one-way flow of saliva towards the detection device 4. This is particularly advantageous when chemical agents are present in the integrated device 1.

[0041] In addition, it is preferably provided that the integrated device 1 according to the invention, in particular the sampling member 2, comprises a salivary stimulation molecule, preferably selected from taste stimulants (acids such as citric acid, malic acid, tantric acid; sweets such as sucrose, glucose, xylitol, fructose; pungents such as capsaicin, ginger, cinnamon, menthol or bitters) or olfactory stimulants (such as mint, citrus, vanilla aromas for example).

[0042] Advantageously, as described above, the vacuum in the sampling limb is created by a suction mechanism (an alternative to a bulb system that would be activated to generate a vacuum). This alternative is advantageous because it avoids additional manual action, and therefore the risk of errors during sampling.

[0043] Preferably, the intermediate section 3 includes a chemical treatment mechanism or a physical treatment mechanism for saliva, or both a chemical treatment mechanism and a physical treatment mechanism for saliva.

[0044] A preferred chemical treatment of saliva includes the presence of disulfide bond reducing molecules and / or a protease inhibitor.

[0045] Indeed, the inventors noticed that reducing the disulfide bonds of the proteins present in saliva reduces its viscosity.

[0046] Beneficial molecules include cysteine ​​or its derivatives, such as acetylcysteine, or glutathione.

[0047] Alternatively, or in addition, the physical treatment of saliva involves passing it through a porous structure to reduce the content of air bubbles and unwanted compounds (e.g., exogenous particles from food). The porosity enabling this filtration is advantageously achieved via the fibers present in intermediate section 3, for example, due to the three-dimensional organization of these fibers. This is complementary and even synergistic with mucolytic treatment, for example, by reducing disulfide bonds, resulting in more fluid saliva. This enhances the power of downstream detection, especially in the case of viscous saliva or saliva obtained under conditions that result in excessive air content.

[0048] A simplified alternative to the integrated device 1 consists of a combined device 1 1, or a "kit of parts" 1 1, where elements 2 and 3 are not always in direct contact with the marking section 4.

[0049] This may involve two parts that will be brought into contact or assembled as soon as the saliva has been collected, or two elements that are already associated externally, but without internal fluidic continuity, this being permitted only via an action, such as a rotational force to allow this fluidic continuity by removing an obturation element or by arranging two parts of the canal to make one.

[0050] Another way of bringing the saliva into contact with the marking section 4 is to apply pressure to the element 2, so as to extract the saliva, preferably a determined quantity, and expel it into the marking section 4.

[0051] As with the integrated device 1, the elements 2 or 3 of the combined device 11 advantageously also include a fiber system which may also preferably include filtration systems (e.g., a fibrous, porous structure), bubble management systems, and saliva viscosity management systems (e.g., reducing agents based on -SH groups) as described above, or even include a protease inhibitor. Advantageously, the labeling section 4 of the integrated device 1, or of the combined device 11 of the invention, includes chromatography, preferably lateral flow chromatography 41.

[0052] Thus, advantageously, saliva will migrate on the chromatography, potentially present analytes will associate with the detection antibody, and the analyte-detection antibody complex will continue its migration until it is fixed (immobilized) by fixation antibodies localized to the detection zone assigned to that analyte.

[0053] Since several analytes need to be detected, several pairs of detection and binding antibodies are present in the chromatography, with the detection antibodies for the second analyte being placed in a different area from those for the first analyte, unless a different detection system, compatible with the first, is used.

[0054] The detection systems can advantageously be the same; it can be a colored substrate, or an enzymatic activity emitting a color or fluorescence in situ.

[0055] In some cases (see an example below), identical detection antibodies can be used for two homologous analytes (the antibody recognizing a common epitope, for example, HRP2 and HRP3, or the HRP proteins of several Plasmodium species, or the LDH proteins of several Plasmodium species), with specificity then ensured by the binding antibody, even though, theoretically, a binding antibody can also specifically recognize several homologous HRP or LDH proteins of Plasmodium. Advantageously, HRP antibodies, for both binding and detection, recognize both HRP2 and HRP3 from several Plasmodium species. This simplifies kit design while ensuring good specificity and sensitivity, and covering at least Piasmodium falciparum and Piasmodium vivax, and preferably also Piasmodium malariae and Piasmodium ovale.

[0056] Indeed, the inventors noticed that some promising antibodies do not accurately recognize certain species, or even certain mutants. However, combining at least two labels combines the specificity and sensitivity of the best identified antibodies, while compensating for the risk of false negatives for species or mutants poorly recognized by one antibody: the other label still provides a signal. Thus, the risk of false negatives is significantly reduced, thanks to both the sensitivity of the antibodies and the labeling of at least two analytes.

[0057] The device according to the invention is particularly well suited for the detection of one or more analyte(s) of Plasmodium sp., preferably Piasmodium falciparum, Plasmodium vivax, Plasmodium malariae and / or Piasmodium ovale, preferably at least Plasmodium falciparum and Plasmodium vivax, advantageously all 4 species.

[0058] To this end, the binding antibody(ies) and the detection antibody(ies) are advantageously chosen for the HRP protein (preferably the antibody recognizes HRP 2 and 3) and / or LDH of Plasmodium sp. (pan-Plasmodium), preferably one binding antibody and one detection antibody for HRP2 of Plasmodium sp., and two binding antibodies and at least one detection antibody for the LDH proteins of P. falciparum and / or P. vivax. The inventors have identified a detection antibody recognizing an epitope common to P. falciparum and P. vivax, and which does not interfere with the selected binding antibody.

[0059] In the context of the present invention, HRP preferably refers to one of the "histidine-rich" proteins of Plasmodium sp. It is known in the literature to search for the presence of such molecules in a blood sample.

[0060] Conversely, in the context of the present invention, LDH preferably refers to a "Lactate Dehydrogenase" protein. It is known in the literature to test for the presence of such molecules in a blood sample.

[0061] However, the combination of these markers in a saliva test is not known, especially via the device described above.

[0062] This combination ensures good specificity and sensitivity. It allows, if necessary, for the discrimination of the Plasmodium species that may be present.

[0063] The antibodies can advantageously be pan-Plasmodium antibodies, or species-specific antibodies, for example P. falciparum, or a mixture of both. This combination is preferred because it maximizes the chance of detecting the presence of Plasmodium and provides an indication of the species that might be present.

[0064] Other features and advantages of the present invention will be derived from the following non-limiting description, and with reference to the drawings and examples.

[0065] Examples

[0066] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made to it without departing from the scope of the attached claims; among the modifications, other binding or detection antibodies, even at high dilution, of the HRP2 protein and / or the HRP3 protein, of Plasmodium sp, preferably of Plasmodium falciparum or a pan-Plasmodium or other antibodies allowing the binding or detection of the LDH protein, of Piasmodium sp, preferably at least Piasmodium falciparum and Piasmodium malariae and, advantageously in addition, Piasmodium vivax and Piasmodium ovale are identifiable or developable without undue difficulty in light of the present invention.

[0067] Example 1:

[0068] The inventors have developed an integrated device 1 comprising 3 elements including a collection element 2 which the patient inserts into their mouth and sucks, an intermediate section 3 for pre-treatment of saliva (viscosity, proteolytic activities, bubbles) which includes a network of polymeric fibers, and a detection section 4 based on fixation antibodies and labeling antibodies.

[0069] The inventors noted that detecting malaria (Piasmodium) in a saliva sample faces three major difficulties: low antigen content, a difficult sample type (proteases, viscosity, bubbles), and that saliva properties vary greatly from patient to patient.

[0070] The first area of ​​improvement focused on optimizing detection, using a collection of blood samples from infected patients: the inventors sought conditions that allowed detection even in high dilutions, compared to commercial tests.

[0071] For the HRP protein (pan-Piasmodium; HRP2 and 3), the inventors selected, rather than the commercially available antibodies for this purpose, a binding antibody, 10-P90B (Fitzgerald), and two detection antibodies, HM086 or HM246 (both from Medix). Another test was performed using HM086 for binding and HM246 for detection. Compared to the standard commercial test (blood test, here used on model samples), the inventors obtained a detection limit 16 to 40 times better. The 10-P90B / HM246 combination was the best tested and was therefore selected.

[0072] Similarly, for LDH, the T3 (BioMapper) and 4C8 (Vista) binding antibodies were tested, and only one detection antibody, DH60 (Vista), was tested. Again, and depending on the Plasmodium species, the results were 8 to 64 times better (with the sensitivity limit reduced accordingly) than the commercial test (again, a blood test, here used on model samples in increasing dilutions), except for Plasmodium ovale, where the improvement was only 2 to 4 times, and for the second condition, which did not detect Plasmodium vivax, which, a priori, represents a problem. The inventors therefore combined the two pairs for the test, one detecting all Plasmodium species (falciparum, malariae, ovale, and vivax), and the other only 3, but which is more sensitive for falciparum and malariae.The inventors believe that a combination of HRP and LDH already sufficiently compensates for the risk of "false negatives", but that the combination of HRP with dual LDH detection offers even greater security against "false negatives".

[0073] For both LDH and HRP, sensitivity and specificity were 99.5% or higher.

[0074] These three pairs of binding and detection antibodies were placed on a lateral flow chromatography system (Coris), and the device was successfully tested on patients with malaria.

Claims

DEMANDS 1. An integrated device (1) for a saliva test of at least two analytes comprising a collection member (2), an intermediate section (3) and an immunolabeling section (4), said collection member comprising a collection channel (21) and being arranged for the collection of a sufficient quantity of salivary fluid via the application of a vacuum force or suction, said intermediate section (3) comprising an arrangement of polymers in the form of fibers and said immunolabeling section comprising several antibodies for the specific binding of said analytes and several second antibodies for the detection of said analytes, said analytes being the Histidine Rich (HRP) protein and the Lactate dehydrogenase (LDH) protein of Plasmodium sp.

2. The device according to claim 1 in which the collection channel (21) includes a non-return system.

3. The device according to claim 1 or 2, wherein the depression in the sampling limb (2) is caused by a suction mechanism.

4. A combined device (11) for a saliva test of at least two analytes comprising, on the one hand, a sampling member (2) coupled to an intermediate member (3), said sampling member comprising a collection channel (21) and being arranged for the collection and retention of a sufficient quantity of salivary fluid, said retained salivary fluid being transferable to said intermediate section by means of a trigger, said intermediate section (3) comprising an arrangement of polymers in the form of fibers and being able to be arranged to an immunological labeling section, and on the other hand, an immunological labeling section (4) comprising several antibodies for the specific binding of said analytes and several second antibodies for the detection of said analytes, said analytes being the Histidine Rich Protein (HRP) and the Lactate dehydrogenase (LDH) protein of Plasmodium sp.

5. The device according to claim 4, wherein the action for transferring the sampling limb to the immunological labeling section is the application of pressure so as to eject a determined quantity of salivary fluid towards the labeling section (4) or a movement enabling the sampling salivary fluid to come into contact with said labeling section (4).

6. The device according to any one of the preceding claims wherein the sampling member (2) has a geometry suitable for stimulating salivary secretion, preferably a teat.

7. The device according to any one of the preceding claims wherein the sampling member (2) comprises a salivary stimulation molecule, preferably selected from an acid, a sugar, a pungent agent or olfactory stimulants.

8. The device according to any one of the preceding claims wherein the fibers of said intermediate section (3) are arranged according to a gradient of hydrophobicity and / or pore size, so as to cause movement of salivary fluid towards the immunological labeling section (4).

9. The device according to any one of the preceding claims wherein the intermediate section (3) comprises a chemical treatment mechanism or a physical treatment mechanism for saliva or both a chemical treatment mechanism and a physical treatment mechanism for saliva.

10. The device according to claim 9 in which the chemical treatment of saliva comprises the presence of disulfide bridge reducing molecules and / or a protease inhibitor. 1 1. The device according to claim 9 or 10 wherein the physical treatment of saliva comprises a sieving (a filtration) of said saliva so as to reduce the content of air bubbles therein.

12. The device according to any one of the preceding claims wherein the marking section (4) comprises a chromatography, preferably a lateral flow chromatography (41).

13. The device according to any of the preceding claims in the analytes are of Plasmodium falciparum, Plasmodium vivax and / or Plasmodium malariae.

14. The device according to any of the preceding claims in which antibodies are selected and matched to enable sensitive detection of analytes.

15. The device according to any one of the preceding claims comprising a binding antibody and a detection antibody for HRP2 and HRP3 of P. falciparum, and two binding antibodies for LDH proteins of P. falciparum and P. vivax and at least one detection antibody for these LDH proteins of P. falciparum and P. vivax.

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