Tear sample eluent, tear sample diluent, preparation method of tear sample eluent and preparation method of tear sample diluent, and tear collecting and processing device

Abstract

The invention discloses a method for preparing a tear diluent, and provides a tear sample eluent and a tear sample collecting and processing device. A small-volume tear sample can be stably collected by using an absorptive material, and a to-be-detected substance of the sample can be stably released after the sample eluent provided by the invention is used for elution.

Description

Tear sample eluent, diluent and preparation method thereof, and tear collection and processing device

[0001] The present invention claims:

[0002] Priority to the prior application, patent application number 2022112199260, filed with the State Intellectual Property Office of China on September 30, 2022, entitled “Tear Sample Collection and Processing Device”;

[0003] Priority to the prior application, patent application number 2022112198906, filed with the State Intellectual Property Office of China on September 30, 2022, entitled “Tear sample eluate and its use”;

[0004] Priority to the prior application, patent application number 2022112198893, filed with the State Intellectual Property Office of China on September 30, 2022, entitled “Method for Preparing Tear Diluent”;

[0005] The entire contents of said prior application are incorporated herein by reference. Technical Field

[0006] The present invention relates to the field of biological sample collection, and in particular provides a tear sample eluent, a diluent and a preparation method thereof, and a tear sample collection and processing device. Background Art

[0007] Tears are a weakly alkaline transparent liquid that mainly contains water, and also contains inorganic salts and bioactive molecules such as proteins, lipids, lysozyme, complement system and other substances. It is a very complex system. Tears are evenly distributed in the conjunctival sac, thereby forming a thin liquid film called the tear film. The tear film located in front of the cornea is usually called the precorneal tear film (PCTF). The precorneal tear film has a thickness of only 6 to 10 nm, but it can keep the eyeball moist and improve the refraction of glasses by filling the tiny bumps on the cornea.

[0008] The secretion of the precorneal tear film is not only influenced by the metabolic state of the ocular surface, but analysis of its composition can also detect the presence of ocular surface diseases and the balance of the human internal environment. Therefore, biochemical analysis of PCTF components has the potential to detect the presence or progression of diseases and be applied to clinical treatment. In addition, because tears are present on the body surface (ocular surface), the process of sampling them is non-invasive, providing subjects with a more comfortable, hygienic, and simpler option than collecting blood, urine, and fecal samples.

[0009] However, compared to routine clinical samples such as blood, urine, and feces, the amount of tear samples that can be collected is too small, resulting in poor detection rate, accuracy, reproducibility, and reliability of test results, or requiring great effort to ensure reliable results.

[0010] In addition, the current tear sample collection methods mainly include stimulated tear collection and capillary tear collection. The former requires stimulating tear secretion, which is usually achieved by applying irritating substances such as cooling oil to the lower eyelid or using a needle to stimulate the Jingming acupoint. It can easily cause discomfort to the subject, and the sample amount cannot be collected quantitatively, so the test results fluctuate greatly; the latter requires the use of capillaries to absorb tears on the ocular surface after the appearance of reflex tears in the eyes. It is extremely demanding on the operator, otherwise it is easy to hurt the subject's eyes, and is limited by the capacity of the capillary tube. The amount of sample collected is too small, which is not conducive to subsequent testing. In addition, since the stimulated tear collection method uses irritating substances, there is a risk of contaminating the sample and it cannot truly reflect the condition of the tears.

[0011] At the same time, current immunological detection technologies, including fluorescent immunochromatography, colloidal gold immunochromatography, magnetic particle immunochemiluminescence, latex microsphere immunochromatography, and enzyme-linked immunosorbent assay (ELISA), require large sample volumes and multiple separate instruments to obtain more reliable test results. These methods are cumbersome and lengthy, place high demands on operators, and are time-consuming, thus limiting their application in testing small sample volumes. Furthermore, if small sample volumes are used in these methods, the results are unreliable, making it difficult to obtain clinically meaningful evidence.

[0012] Therefore, there is an urgent need in the art for means to reliably collect and process small-volume tear samples.

[0013] Summary of the Invention

[0014] In light of the problems existing in the prior art, the inventors discovered that by using absorbent materials, it is possible to stably collect small tear samples and, after elution with a sample eluent of a specific composition, stably release the test substance from the sample. This allows for further qualitative, semi-quantitative, and quantitative analysis of the test substance. In additional steps, the analysis results of different test substances can be used to analyze various physiological parameters of the subject. This has led to the completion of the present invention.

[0015] Therefore, in a first aspect, provided herein is a tear sample eluate comprising water as a solvent, a buffer, a surfactant, a stabilizer, a preservative, and an osmotic pressure regulator, and having a pH value of 7.0 to 9.0.

[0016] In one embodiment, a pH adjuster is optionally used to adjust the sample eluate to a pH value of 7.0 to 9.0. Preferably, the pH adjuster comprises an organic or inorganic acid, such as hydrochloric acid, sulfuric acid, phosphoric acid; or an organic or inorganic base, such as sodium hydroxide, potassium hydroxide. The content of the pH adjuster is not particularly limited, and those skilled in the art can select its concentration and dosage based on the final pH value of the sample eluate so that the sample eluate reaches the final desired pH value. In one embodiment, the sample eluate is weakly alkaline, for example, with a pH value of about 7.1 to 8.5, preferably 7.5 to 8.2, more preferably 7.8 to 8.1, for example, about 7.4, 7.6 or 8.0. Preferably, the final pH of the sample eluate is adjusted by an HCl solution.

[0017] In one embodiment, the buffer is selected from an organic amine buffer, preferably an organic amine buffer with a hydroxyl group. As an example, the buffer can be selected from tris(hydroxymethylaminopropane), i.e., tromethamine (Tris) and / or tris(hydroxymethylaminopropane)-hydrochloric acid (Tris-HCl). The buffer content in the sample eluent can be between 10 mM and 100 mM, preferably between 20 mM and 80 mM, more preferably between 35 mM and 60 mM, and especially about 50 mM.

[0018] In one embodiment, a combination of Tris as a buffer and HCl as a pH adjuster is preferred.

[0019] In one embodiment, the surfactant may include a first surfactant and a second surfactant, which may be the same or different. Preferably, the surfactant is selected from a nonionic surfactant. In one embodiment, the first surfactant is different from the second surfactant. Preferably, the first surfactant is selected from a polysorbate surfactant, more preferably a surfactant of the Tween (TWEEN) series, such as Tween 20, Tween 80, etc. Preferably, the second surfactant is selected from a block polyether surfactant, such as a polyethylene glycol surfactant with a terminal hydroxyl group, more preferably a polyethylene glycol phenyl ether surfactant. As an example, the second surfactant can be polyethylene glycol mono-4-octylphenyl ether, which can for example be known under the name Triton (Triton) TM) or, for example, a polymer surfactant of methyl oxirane, 1,2-ethylenediamine, and ethylene oxide. As an example, it can be obtained under the name Tetronic 1307 (S9). In the sample eluent, the content of the surfactant can be between 0.1% by weight and 5% by weight, preferably between 0.5% by weight and 4% by weight, and more preferably between 1% by weight and 3.5% by weight. The weight ratio between the first surfactant and the second surfactant can be between 10:1 and 1:10, preferably between 1:1 and 1:10, more preferably between 1:1 and 1:8, for example, about 1:5. In one embodiment, the first surfactant is different from the second surfactant, and the first surfactant is selected from the Tween series of surfactants and the second surfactant is selected from the Triton series of surfactants; a combination of Tween 20 and Triton X-100 is preferred, and their concentration ratio is preferably about 1:1, 1:2, 1:5 or 1:10; in the most preferred embodiment, the sample eluate contains about 0.5% by weight of Tween 20 and about 2.5% by weight of Triton X-100.

[0020] In one embodiment, the stabilizer is selected from albumin, casein, gelatin, and the like. Preferably, the stabilizer is selected from casein. The content of the stabilizer in the sample eluate may be between 0.1% and 5% by weight, preferably between 0.5% and 4% by weight, more preferably between 0.8% and 2% by weight, for example, about 1% by weight.

[0021] In one embodiment, the sample eluate does not contain particles with a size greater than 0.50 μm, preferably does not contain particles with a size greater than 0.45 μm, and more preferably does not contain particles with a size greater than 0.40 μm.

[0022] In one embodiment, the preservative is selected from Proclin-300, sodium azide, thimerosal, etc. In the sample eluate, the content of the preservative may be between 0.01 and 0.5% by weight, preferably between 0.05 and 0.4% by weight, more preferably between 0.1 and 0.3% by weight, for example, 0.1%, 0.2% or 0.3% by weight.

[0023] In one embodiment, the osmotic pressure regulating agent is an alkali metal salt, such as an inorganic salt or an organic salt; preferably a sodium salt or a potassium salt; more preferably NaCl.

[0024] In one embodiment, the sample eluent may or may not contain an organic solvent. Preferably, the sample eluent does not contain an organic solvent. In a preferred embodiment, the sample eluent contains only water as a solvent.

[0025] In one embodiment, the sample eluent may comprise about 10 mM to 100 mM of a buffer, about 0.1% to 5% by weight of a surfactant, about 0.1% to 5% by weight of a stabilizer, and optionally about 0.01% to 0.5% by weight of a preservative and about 0.1% to 5% by weight of an osmotic pressure regulator; preferably, the eluent also has a pH value of about 7.0 to 9.0.

[0026] In one embodiment, the sample eluent may comprise about 35 mM to 60 mM of a buffer, about 1% to 3.5% by weight of a surfactant, about 0.8% to 2% by weight of a stabilizer, and optionally about 0.1% to 0.3% by weight of a preservative and about 0.5% to 1.2% by weight of an osmotic pressure regulator; the surfactant comprises a first surfactant and a second surfactant, and the weight ratio between the first surfactant and the second surfactant may be between 10:1 and 1:10, preferably between 1:1 and 1:10, and more preferably between 1:1 and 1:8; preferably, the eluent also has a pH value of about 7.4 to 8.1.

[0027] In one embodiment, the sample eluent may comprise about 50 mM of a buffer, about 0.5% of a first surfactant, about 2.5% by weight of a second surfactant, about 1% by weight of a stabilizer, and optionally about 0.2% by weight of a preservative and about 0.9% by weight of NaCl, preferably, the eluent also has a pH of about 7.4.

[0028] As an example, the sample eluent may comprise about 50 mM Tris buffer, about 0.5% Tween 20, about 2.5% by weight Triton X-100, about 1% by weight casein, and optionally about 0.2% by weight Proclin-300 and about 0.9% by weight NaCl, preferably, the eluent also having a pH of about 7.4.

[0029] As an example, the sample eluent contains about 50 mM Tris buffer, about 0.5% Tween 20, about 2.5% by weight of Triton X-100, about 1% by weight of casein, about 0.2% by weight of Proclin-300 and about 0.9% by weight of NaCl. Preferably, the eluent has a pH of about 7.4.

[0030] In one embodiment, the sample elution liquid is used to elute the tear fluid absorbed by the first absorbent sheet. In another embodiment, the first absorbent sheet and the sample elution liquid constitute a tear fluid sample collection and processing device.

[0031] In one embodiment, the first absorbent sheet may have one or more dyed marking line layers. The dyed marking line layer may contain a fluorescent substance such as sodium fluorescein. Preferably, the first absorbent sheet is a filter paper strip. As an example, the first absorbent sheet is, for example, a filter paper strip for Schirmer's tear secretion test. In one embodiment, the first absorbent sheet also has one, two or more readable scales starting from the end. The end is preferably a sampling end. There is no particular limitation on the matrix material of the first absorbent sheet. In one embodiment, the first absorbent sheet is a hydrophilic inert sheet, the matrix material of which may contain cellulose; a polymer such as polyurethane.

[0032] In one embodiment, the length of the first absorbent sheet is at least about 5 mm, at least about 6 mm, at least about 10 mm, at least about 15 mm, or even at least about 20 mm. In one embodiment, the width of the first absorbent sheet is at least about 5 mm, at least about 6 mm, or at least about 7 mm. In one embodiment, the predetermined length is at least about 5 mm, at least about 6 mm, or at least about 7 mm, or at least about 8 mm, or at least about 10 mm.

[0033] In one embodiment, the tear fluid contained in the fully saturated portion of the predetermined length is approximately 0.0001 to 0.0050 mL, preferably approximately 0.001 to 0.004 mL, more preferably approximately 0.0015 to 0.0025 mL, even more preferably approximately 0.0018 to 0.0020 mL, and most preferably approximately 0.0020 mL. In the present disclosure, the tear fluid contained in the fully saturated portion of the predetermined length may also be referred to as "the same tear sample" because the first absorbent sheet is disposable and the tear sample collected during each collection operation is only used for one testing process.

[0034] In one embodiment, the predetermined volume of sample eluent is at least about 0.100 mL, or at least about 0.150, or at least about 0.200 mL, or at least about 0.250 mL, or at least about 0.300 mL, or at least about 0.500 mL.

[0035] In a second aspect of the present invention, a method for preparing a tear diluent is provided, comprising the following steps:

[0036] a. contacting the first absorbent sheet with the ocular surface to collect tears, obtaining a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a fully saturated portion of a predetermined length or is completely saturated with tears; and

[0037] b. contacting a completely saturated portion of a predetermined length of the first absorbent sheet with a predetermined volume of the sample eluent of the first aspect for a predetermined time to obtain a tear diluent, or immersing the first absorbent sheet completely saturated with tears in a predetermined volume of the sample eluent to obtain a tear diluent.

[0038] In one embodiment, the predetermined contact time between the first absorbent sheet and the sample eluent is 1 second to 5 minutes, preferably 3 seconds to 3 minutes, more preferably 5 seconds to 1 minute. The first absorbent sheet can be contacted with the sample eluent by immersion and / or stirring.

[0039] In the third aspect of the present invention, an immunological detection kit is also provided, which includes an immunological detection card, wherein the detection card includes a support, a blotting membrane fixedly connected to the support, an absorbent pad located at a first end of the blotting membrane and a sample receiving pad located at a second end of the blotting membrane, the absorbent pad and the sample receiving pad are spatially separated from each other and there is a control line close to the absorbent pad side and a detection line close to the sample receiving pad side on the blotting membrane between the two.

[0040] In one embodiment, the support is made of at least one material selected from the group consisting of a polymer, glass, and polyvinyl chloride (PVC), polystyrene (PS), or a combination thereof.

[0041] In one embodiment, the blotting membrane comprises at least one material selected from the group consisting of cellulose, nitrocellulose, and / or combinations thereof.

[0042] In one embodiment, the sample receiving pad is a glass cellulose membrane, nylon cellulose, or polyester cellulose. In one embodiment, the sample receiving pad comprises any of the following: fluorescent latex microspheres or colloidal gold probes. Preferably, the fluorescent latex microspheres are polystyrene latex microspheres conjugated with fluorescent marker probes.

[0043] In one embodiment, the immunological test card further comprises a second absorbent sheet. The second absorbent sheet may be the same or different in size and structure from the first absorbent sheet, but contains a defined amount of the target substance. Preferably, the first absorbent sheet and the second absorbent sheet have the same matrix material and dimensions.

[0044] In one embodiment, the immunological test includes fluorescent immunochromatography, colloidal gold immunochromatography, latex microsphere immunochromatography, magnetic microparticle chemiluminescence and / or enzyme-linked immunosorbent assay, etc. In one embodiment, the immunological test card is a fluorescent immunochromatography test card or a colloidal gold immunochromatography test card.

[0045] In one embodiment, the target substances may include: IL-8, IL-1a, IL-1b, IL-12, IL-6, IL-8, tumor necrosis factor alpha (TNF-a) and interferon gamma (INF-g), lysozyme, lactoferrin, epidermal growth factor (EGF), lipid deposition protein-1, cystatin S100, alpha 1 antitrypsin, alpha enolase, alpha-1-acid glycoprotein 1, S100A8 (calgranulin A), S100A9 (calgranulin B), S100A4 and S100A11 (calcium), prolactin-induced protein Protein (PIP), proline protein 4 (PRR4), PRR3, NACPP4, S100A6, annexin A1 (ANXA1), annexin A11 (ANXA11), cystatin-S (CST4), phospholipase A2, activating protein (PLAA), lactoglobin B, lipophilin A, matrix metalloproteinase (MMP)-9, anti-Ro\ / SSA, La\ / SSB antibodies, anti-α-fodrin antibody, aquaporin 5 (AQP5), complement C-3, albumin, potassium ions, sodium ions, chloride ions, etc.

[0046] In one embodiment, the test line (12) comprises mouse anti-human matrix metalloproteinase-9 IgG; preferably, the concentration of mouse anti-human matrix metalloproteinase-9 IgG in the test line (12) is about 5.0 mg / ml, 4.0 mg / ml, 3.0 mg / ml, 2.0 mg / ml, 1.0 mg / ml, 0.5 mg / ml, 0.25 mg / ml, or a range of any two of these values ​​as endpoints and any value included therein. In one embodiment, the control line (b) comprises goat anti-chicken IgY. In one embodiment, the fluorescent latex microspheres comprise conjugated mouse anti-human matrix metalloproteinase-9 IgG. In one embodiment, the test line (12) comprises mouse anti-human matrix metalloproteinase-9 IgG, the control line (11) comprises goat anti-chicken IgY, and the fluorescent latex microspheres comprise conjugated mouse anti-human matrix metalloproteinase-9 IgG; thus, the immunological test card can be used to detect human matrix metalloproteinases.

[0047] In a fourth aspect, the present disclosure further provides a method for preparing an immunological detection card, comprising the following steps:

[0048] (i) providing a blot membrane having a control line and a test line, and optionally drying the membrane at 37° C. for 8-20 hours;

[0049] (ii) providing a sample receiving pad, and providing fluorescent latex microspheres or colloidal gold probes on the sample receiving pad, and optionally drying the sample receiving pad at 37° C. for 8-20 hours;

[0050] (iii) placing a sample receiving pad on the second end of the blotting membrane near the detection line and contacting the two, and

[0051] (iv) Place an absorbent pad on the blotting membrane near the first end of the control line and bring the two into contact.

[0052] In one embodiment, fluorescent latex microspheres are provided on a sample receiving pad. Preferably, providing fluorescent latex microspheres on a sample receiving pad comprises the following steps:

[0053] (ii-1) treating blank fluorescent latex microspheres with activation solution;

[0054] (ii-2) treating the fluorescent latex microspheres obtained in step (ii-1) with a coupling buffer to obtain a fluorescent latex microsphere suspension;

[0055] (ii-3) treating the fluorescent latex microsphere suspension obtained in step (ii-2) with mouse anti-human matrix metalloproteinase-9 IgG;

[0056] (ii-4) treating the fluorescent latex microsphere suspension obtained in step (ii-3) with a blocking solution;

[0057] (ii-5) treating the fluorescent latex microsphere suspension obtained in step (ii-4) with a second washing solution;

[0058] (ii-6) Diluting the fluorescent latex microsphere suspension obtained in step (ii-5) with a microsphere diluent and spraying the diluted solution onto a blank sample receiving pad.

[0059] Preferably, before step (ii-1), the method further comprises step (ii-0) of washing the fluorescent latex microspheres with a first washing solution to obtain blank fluorescent latex microspheres. More preferably, the washing is performed under ultrasound, and even more preferably, the washing further comprises centrifugation after ultrasound treatment. Preferably, the first washing solution comprises morpholineethanesulfonic acid, preferably an aqueous solution of morpholineethanesulfonic acid.

[0060] Preferably, step (ii-1) of treating the blank fluorescent latex microspheres with an activation solution comprises treating the blank fluorescent latex microspheres with a first activation solution and a second activation solution. The first activation solution is a solution of sodium salt of N-hydroxysulfosuccinimide in a first washing solution, preferably, the concentration of sodium salt of N-hydroxysulfosuccinimide is 10 to 30 mg / mL. The second activation solution is a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in a first washing solution, preferably, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 10 to 30 mg / mL. The blocking solution comprises casein, bovine serum albumin, and a buffer.

[0061] Preferably, the coupling buffer of step (ii-2) comprises N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, preferably at a concentration of about 6 g / L, and preferably has a pH value of about 8.0.

[0062] Preferably, centrifugation is further included after the treatment in steps (ii-1), (ii-2), (ii-3), (ii-4) and / or (ii-5), preferably for 5 to 60 minutes, more preferably for 10 to 40 minutes, and even more preferably for 10 to 30 minutes.

[0063] Preferably, steps (ii-0) and / or (ii-4) further comprise using ultrasound-assisted washing.

[0064] Preferably, the spraying in step (ii-6) is performed at a usage amount of 5-10 μL / cm.

[0065] Preferably, in step (ii-3), the mass ratio of mouse anti-human matrix metalloproteinase-9 (MMP-9) IgG to the fluorescent latex microsphere suspension obtained in step (ii-2) is (0.1-0.5):(1-5). The treatment in step (ii-3) is carried out at 35-40°C, preferably 37°C, for at least 2 hours, preferably at least 3 hours.

[0066] Preferably, the treatment in step (ii-4) is carried out at 35 to 40° C., preferably 37° C., for 5 to 60 min, more preferably 10 to 40 min, and even more preferably 10 to 30 min.

[0067] As an example, the first wash solution is an aqueous solution of about 10.66 g / L morpholineethanesulfonic acid having a pH of about 6.1; the first activation solution is a solution of about 20 mg / mL N-hydroxysulfosuccinimide sodium salt in the first wash solution; the second activation solution is a solution of about 20 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the first wash solution; the coupling buffer is a solution of about 5.96 g / L N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid having a pH of about 8.0; the blocking solution is a solution containing 30 g / L casein, 10 g / L bovine serum albumin, and 0.05 mol / L Trisbase buffer; the second wash solution is a 6 g / L Trisbase buffer having a pH of about 8.0; and the microsphere diluent is a buffer of 30% sucrose, 0.5% casein, 1% glycine, and 50 mM Trisbase having a pH of about 8.0.

[0068] In a fifth aspect, this document also provides a method for detecting a target substance in tears.

[0069] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0070] a. contacting the first absorbent sheet with the ocular surface to collect tears, obtaining a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a fully saturated portion of a predetermined length or is completely saturated with tears;

[0071] b. contacting a fully saturated portion of a predetermined length of the first absorbent sheet with a predetermined volume of sample eluent for a predetermined time to obtain a tear diluent, or immersing the first absorbent sheet completely saturated with tears in a predetermined volume of sample eluent to obtain a tear diluent; and

[0072] c. The obtained tear fluid dilution is used for immunological detection to analyze the target substance in the tear fluid.

[0073] In one embodiment, step c of the method for detecting a target substance in tear fluid comprises contacting the obtained tear fluid dilution with the immunochromatographic test card of the third aspect of the present disclosure.

[0074] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0075] a '. The first absorbent sheet is brought into contact with the ocular surface to collect tears, obtaining a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a completely saturated portion;

[0076] b'. Bringing the first absorbent sheet into contact with the immunological test card of the third aspect of the present disclosure.

[0077] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0078] a. contacting at least two first absorbent sheets with the ocular surface of the same subject at different time points to collect tears, obtaining at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a fully saturated portion of a predetermined length or is completely saturated with tears;

[0079] b. contacting a fully saturated portion of a predetermined length of each first absorbent sheet with a predetermined volume of sample eluent for a predetermined time to obtain a tear diluent, or immersing each first absorbent sheet completely saturated with tears in a predetermined volume of sample eluent to obtain a tear diluent; and

[0080] c. Using the obtained tear fluid dilutions for immunological testing to analyze target substances in tears collected from the same subject at different time points.

[0081] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0082] a '. contacting at least two first absorbent sheets with the ocular surface of the same subject at different time points to collect tears, obtaining at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a completely saturated portion;

[0083] b'. bringing at least two first absorbent sheets into contact with the immunological test card of the third aspect of the present disclosure, respectively.

[0084] In one embodiment, the same subject is in different states at different time points, such as a healthy state or a disease state, in particular, for example, in a healthy state or a disease state at different time points, or in a healthy state at at least one time point and in a disease state at at least another time point. In one embodiment, the present invention provides a qualitative, semi-quantitative or quantitative detection of a target substance in one or more tear samples collected from the same subject at different time points. In one embodiment, the present invention provides a quantitative detection method for a target substance in multiple tear samples from the same subject at different time points.

[0085] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0086] a. contacting the first absorbent sheet with the ocular surface of at least two different subjects to collect tears, obtaining at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a fully saturated portion of a predetermined length or is completely saturated with tears;

[0087] b. contacting a fully saturated portion of a predetermined length of each first absorbent sheet with a predetermined volume of sample eluent for a predetermined time to obtain a tear diluent, or immersing the first absorbent sheet completely saturated with tears in a predetermined volume of sample eluent to obtain a tear diluent; and

[0088] c. The obtained tear fluid dilutions are used for immunological testing to analyze target substances in tears collected from different subjects.

[0089] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0090] a '. bringing at least two first absorbent sheets into contact with the ocular surface of different subjects at different time points to collect tears, obtaining at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a completely saturated portion;

[0091] b'. bringing at least two first absorbent sheets into contact with the immunological test card of the third aspect of the present disclosure, respectively, to analyze target substances in tear fluid.

[0092] In one embodiment, different subjects are in the same or similar state, for example, they are in a healthy state or suffer from the same or similar disease. In one embodiment, different subjects are in different states, for example, they are in a healthy state or suffer from the same or similar disease. In one embodiment, the present invention provides a method for qualitatively, semi-quantitatively or quantitatively detecting a target substance in one or more tears of different subjects. In one embodiment, the present invention provides a method for quantitatively detecting a target substance in multiple tears of different subjects.

[0093] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0094] a. bringing the first absorbent sheet into contact with the ocular surface to collect tears, obtaining a first absorbent sheet at least partially saturated with tears;

[0095] b. Cutting a fully saturated portion of a predetermined length from the first absorbent sheet at least partially saturated with tear fluid;

[0096] c. contacting a fully saturated portion of a predetermined length with a predetermined volume of sample eluent for a predetermined time;

[0097] d. collecting the sample eluate after contact with the first absorbent sheet to obtain a tear dilution solution; and

[0098] e. The tear fluid diluted in step d is used for immunological detection to analyze the target substance in the tear fluid.

[0099] In one embodiment, the method for detecting a target substance in tear fluid comprises the following steps:

[0100] a. bringing the first absorbent sheet into contact with the ocular surface to collect tears, obtaining a first absorbent sheet at least partially saturated with tears, the first absorbent sheet at least partially saturated with tears having a fully saturated portion of a predetermined length;

[0101] b. contacting a fully saturated portion of a predetermined length with a predetermined volume of sample eluent for a predetermined time;

[0102] c. collecting the sample eluate after contact with the first absorbent sheet to obtain a tear dilution solution; and

[0103] d. using the tear fluid dilution collected in step c for immunological detection to analyze the target substance in the tear fluid.

[0104] In one embodiment, the present invention provides for the qualitative, semi-quantitative, or quantitative detection of different target substances. In one embodiment, the present invention provides for the qualitative, semi-quantitative, or quantitative detection of at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, or more different target substances in the same tear sample. In one embodiment, the same tear sample is a small volume sample, for example, a tear sample of less than about 0.0050 mL, preferably less than about 0.0040 mL, more preferably less than 0.0030 mL, and most preferably less than 0.0025 mL. In one embodiment, the present invention provides for the quantitative and comparative detection of at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, or more different target substances in the same tear sample. In one embodiment, provided herein are detection methods for comparing the relative amounts of at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, or more different target substances in the same tear fluid sample.

[0105] In a sixth aspect, a tear sample collection and processing device is provided herein, comprising:

[0106] a first package comprising a first absorbent sheet, and

[0107] a second package containing a sample eluent,

[0108] The sample eluent contains a buffer, a surfactant and a stabilizer, and has a pH value of 7.0 to 9.0.

[0109] In one embodiment, the sample eluent comprises water as a solvent; the sample eluent further comprises a preservative and / or an osmotic pressure regulator.

[0110] In one embodiment, the sample eluent comprises water as a solvent, a buffer, a surfactant, a stabilizer, a preservative, and an osmotic pressure regulator, and has a pH value of 7.0 to 9.0.

[0111] In one embodiment, the first absorbent sheet, sample eluent, buffer, surfactant, stabilizer, preservative and osmotic pressure regulator have the definitions described in the first aspect.

[0112] In a seventh aspect, the present invention also provides the use of the tear sample collection and processing device described in the sixth aspect for collecting and processing tears.

[0113] In the eighth aspect, an immunological sampling and detection kit is also provided herein, which comprises the first package, the second package and the immunological detection card in the sixth aspect; preferably, the immunological detection card is the immunological detection card described in the third aspect above.

[0114] Those skilled in the art will appreciate that the features in any aspect, embodiment or example of the present disclosure may be combined with each other as long as there is no conflict or incompatibility between these features.

[0115] Beneficial effects of the present invention

[0116] The method provided by the present disclosure can use a sheet of absorbent material, especially a fixed length portion or the entire length of a typical Schirmer tear test filter paper strip, as a device for collecting tear samples and quantifying them. After elution with the sample eluent provided by the present disclosure, the quantitative tear sample can be reproducibly and stably released. In addition, compared with existing tear collection and processing methods, the tear sample collection and processing device disclosed in the present disclosure greatly improves the convenience, accuracy, and stability of subsequent use of the collected tear samples for other tests. Moreover, the obtained tear dilution can be directly used in platforms such as fluorescent immunochromatography, colloidal gold immunochromatography, and latex chromatography immunochromatography to detect target substances in tears, making the sampling and detection process more convenient and reducing operational difficulty and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] FIG1 shows a calibration curve in Example 5 according to the present disclosure. Example

[0118] The preparation method of the present invention will be described in further detail below with reference to specific examples. It should be understood that the following examples are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technical solutions implemented based on the contents of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0119] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art; the reagents, raw materials, instruments, equipment, etc. used in the following examples are all available from commercial channels.

[0120] Example 1: Consistency test of absorbent sheet

[0121] Twenty commercially available Schirmer tear secretion test filter paper strips were provided, and sections of equal length were cut from each strip. The cut sections were then weighed using a precision analytical balance, and the data was recorded as the dry weight. After weighing, each cut section was fully soaked with tear fluid, and then immediately weighed using a precision analytical balance, and the data was recorded as the wet weight. The dry weight of each cut section was subtracted from its wet weight to obtain the mass of tear fluid absorbed. Assuming a tear specific gravity of 1 (i.e., a density of 1 g / mL), the volume of the soaked tear fluid was calculated, resulting in the results shown in Table 1 below.

[0122]

[0123] Example 2 Preparation of Fluorescent Immunochromatographic Detection Card

[0124] 1. Prepare the blot membrane: Dry the blank blot membrane with the control line and the test line at 37°C for 8-20 hours.

[0125] 2. Prepare the sample receiving pad: Add 0.001 g of fluorescent latex microspheres to 0.9 mL of the first wash solution (pH 6.1) containing 10.66 g / L morpholineethanesulfonic acid. Ultrasonicate and centrifuge at 10,000-15,000 rpm for 30 minutes, discarding the supernatant. Add 0.12 mL of a 20 mg / mL solution of N-hydroxysulfosuccinimide sodium salt in the first wash solution and 0.06 mL of 20 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the residue. Activate for 30 minutes, maintaining the temperature at 35-40°C and stirring at 100-150 rpm. Then, centrifuge at 10,000-15,000 rpm for 20 minutes, and discard the supernatant. To this residue, 1 mL of 5.96 g / L N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid solution at pH 8.0 was added, and the solution was sonicated until clear. The mixture was then centrifuged for 20 minutes, and the supernatant discarded. 0.0001 g of mouse anti-human matrix metalloproteinase-9 IgG was then added to the residue, mixed thoroughly, and the coupling reaction was continued. The resulting reaction mixture was centrifuged for 30 minutes, and the supernatant discarded. To the residue after the coupling reaction, 1 mL of a blocking solution containing 30 g / L casein, 10 g / L bovine serum albumin, and 0.05 mol / L Trisbase buffer was added for a blocking reaction, followed by centrifugation for 20 minutes, and the supernatant discarded. 1 mL of Trisbase buffer at pH 8.0 was added, and the solution was sonicated until clear. The solution was then centrifuged for 20 minutes, and the supernatant discarded. Then, 1 mL of a solution containing 30% sucrose, 0.5% casein, 1% glycine and 50 mM Trisbase at pH 8.0 was added to the residue to obtain a mixture, which was sprayed onto a glass cellulose membrane at a volume of 5-10 μL / cm to obtain a sample receiving pad.

[0126] 3. Place the sample receiving pad prepared in the previous step on the second end of the blotting membrane near the detection line in an overlapping manner;

[0127] 4. Place the absorbent pad in an overlapping manner on the blotting membrane near the first end of the control line.

[0128] Example 3 Preparation of colloidal gold detection card

[0129] 1. Prepare the blot membrane: Dry the blank blot membrane with the control line and the test line at 37°C for 8-20 hours.

[0130] 2. Prepare the sample receiving pad: Take 1 ml of 40 nm colloidal gold solution, add 10 μl of 50 mM potassium carbonate, vortex mix, and let it stand for 5 minutes. Take 10 μg of mouse anti-human matrix metalloproteinase-9 IgG, add it directly to the colloidal gold solution, vortex mix, and let it stand at room temperature for 30 minutes. After the reaction is completed, add 100 μl of 10% BSA (bovine serum albumin), vortex mix, and let it stand at room temperature for 30 minutes. After the reaction is completed, centrifuge at 10,000 rpm for 20 minutes and discard the supernatant. Then, add 0.1 mL of a solution containing 30% sucrose, 0.5% casein, 1% glycine, and 50 mM Trisbase at a pH of 8.0 to the residue to make up the volume. The mixture is then sprayed onto a glass cellulose membrane at a rate of 5-10 μL / cm to obtain a sample receiving pad.

[0131] 3. Place the sample receiving pad prepared in the previous step on the second end of the blotting membrane near the detection line in an overlapping manner;

[0132] 4. Place the absorbent pad in an overlapping manner on the blotting membrane near the first end of the control line.

[0133] Example 4: Test of sample elution rate

[0134] Pipette 100 μl of calibrator, add it to the sample well of the test card, let it stand horizontally, react in the dark for 15 minutes, and then read the results. The calibrator provided is the S0 to S5 solution of MMP-9 (matrix metalloproteinase-9), and the concentration gradient is set to 0 ng / ml, 0.3 ng / ml, 1 ng / ml, 10 ng / ml, 30 ng / ml, and 60 ng / ml.

[0135] The calibrators at different concentration gradients were loaded onto the test cards prepared in Examples 2 and 3, respectively, to verify their accuracy, and the results described in Tables 2 and 3 were obtained. Here, C represents the signal value of the control line, T represents the signal value of the test line, T / C represents the ratio of the test line to the control line, and CV represents the coefficient of variation. A coefficient of variation within 10% indicates stable tear release.

[0136] Table 2

[0137] Table 3

[0138] Example 5 Tear Detection Test (Dilution Method)

[0139] A commercially available Schirmer tear secretion test filter paper strip was placed in contact with the subject's ocular surface to collect tears, so that the filter paper strip was saturated with tears. Following the procedure of Example 1, a portion of the test filter paper strip of the same length that was completely saturated with tears was cut and immersed in 200 μl of sample eluent for 3 seconds, after which the liquid was collected to obtain a tear dilution solution, which was then loaded onto the sample receiving pad of the test card prepared in Example 2 for analysis.

[0140] Where C is the signal value of the control line, T is the signal value of the test line, and T / C is the ratio of the test line to the control line. The concentration was calculated based on the calibration curve. Five tests were performed on two different subjects, and the results are shown in Tables 4 and 5 below.

[0141] The calibration curve fitting method is a four-parameter fitting, which is defined as follows: Y = (AD) / [1+(X / C)^B]+DA = 4.63846376754392 B = -1.00725333313379 C = 143.60632119348 D = 0.00936819607330606

[0142] Correlation coefficient R2: 0.999885514800651

[0143] Table 4 Test and calculation data of subject 1

[0144] Table 5 Test and calculation data of subject 2

[0145] Example 6 Tear Detection Test (Sandwich Method)

[0146] A commercially available Schirmer tear secretion test filter paper strip was brought into contact with the ocular surface to collect tears, saturating the filter paper strip with tear fluid. The filter paper strip was then placed on the sample receiving pad of the kit prepared in Example 3 for analysis. The results shown in Tables 6 and 7 below were obtained.

[0147] Table 6

[0148] Table 7

[0149] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A tear sample eluate, comprising: water as a solvent, a buffer, a surfactant, a stabilizer, a preservative, and an osmotic pressure regulator, and having a pH value of 7.0 to 9.

0.

2. The tear sample eluate according to claim 1, wherein The buffer is selected from organic amine buffers, preferably organic amine buffers with hydroxyl groups, and more preferably trishydroxymethylaminopropane.

3. The tear sample eluate according to claim 1 or 2, wherein: The surfactant includes a first surfactant and a second surfactant different from the first surfactant; preferably, the surfactant is selected from nonionic surfactants.

4. The tear sample eluate according to claim 3, wherein The first surfactant is selected from polysorbate surfactants; the second surfactant is selected from block polyether surfactants, preferably polyethylene glycol surfactants with terminal hydroxyl groups, or methyl ethylene oxide, 1,2-ethylenediamine and ethylene oxide polymer surfactants.

5. The tear sample eluate according to any one of claims 1 to 4, wherein: The stabilizer is selected from albumin, casein or gelatin.

6. The tear sample eluate according to any one of claims 1 to 5, wherein: The preservative is selected from Proclin-300, sodium azide or thimerosal.

7. The tear sample eluate according to any one of claims 1 to 6, wherein: The sample eluent may comprise about 10 mM to 100 mM of a buffer, about 0.1% to 5% by weight of a surfactant, about 0.1% to 5% by weight of a stabilizer, and optionally about 0.01% to 0.5% by weight of a preservative and about 0.1% to 5% by weight of an osmotic pressure regulator; preferably, the eluent also has a pH value of about 7.0 to 9.0; Preferably, the sample eluent comprises about 50 mM Tris buffer, about 0.5% Tween 20, about 2.5% by weight of Triton X-100, about 1% by weight of casein, about 0.2% by weight of Proclin-300 and about 0.9% by weight of NaCl, and has a pH of about 7.

4.

8. A method for preparing a tear diluent, comprising the following steps: a. contacting the first absorbent sheet with the ocular surface to collect tears, obtaining a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a fully saturated portion of a predetermined length or is completely saturated with tears; and b. contacting a completely saturated portion of a predetermined length of the first absorbent sheet with a predetermined volume of a sample eluent for a predetermined time to obtain a tear diluent, or immersing the first absorbent sheet completely saturated with tears in a predetermined volume of the sample eluent according to any one of claims 1 to 7 to obtain a tear diluent; Preferably, the predetermined time for the first absorbent sheet to be in contact with the sample eluent is 1 second to 5 minutes, preferably 3 seconds to 3 minutes, more preferably 5 seconds to 1 minute.

9. A tear sample collection and processing device, comprising: a first package comprising a first absorbent sheet, and a second package containing a sample eluent, Wherein, the sample eluent comprises a buffer, a surfactant and a stabilizer, and has a pH value of 7.0 to 9.0; the buffer, surfactant and stabilizer have the definitions as described in any one of claims 1 to 7; Preferably, the sample eluent is the sample eluent according to any one of claims 1 to 7.

10. Use of the tear sample collection and processing device according to claim 9 for collecting and processing tear fluid.

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