Tear sample eluate, dilution solution, and their manufacturing method, as well as tear collection and processing method

Abstract

A method for preparing a tear dilution solution is disclosed, and a tear sample elution solution and tear sample collection and processing device are provided. The use of an absorbent material allows for stable collection of small volumes of tear samples, and after elution with the sample elution solution provided herein, stable release of substances awaiting detection in the sample is possible.

Description

[Technical Field]

[0001] The present invention provides Priority of a prior application filed with the State Intellectual Property Office of China on September 30, 2022, with patent application number 2022112199260 and titled "Tear sample collection and processing device"; Priority of a prior application filed with the State Intellectual Property Office of China on September 30, 2022, with patent application number 2022112198906 and title "Tear sample eluate and use thereof"; Priority of a prior application filed with the State Intellectual Property Office of China on September 30, 2022, with patent application number 2022112198893 and titled "Method for producing tear diluent"; He claims, The entire contents of the above-referenced prior application are incorporated herein by reference.

[0002] The present invention relates to the field of biological sample collection, and in particular to a tear sample eluate, a dilution solution, a method for producing the same, and a tear collection and treatment device. [Background technology]

[0003] Tears are a slightly alkaline, clear liquid that primarily contains water, inorganic salts, and biologically active molecules, such as proteins, lipids, lysozyme, and the complement system. This makes them a highly complex system. The tear film is uniformly distributed within the conjunctival sac, forming a thin liquid film called the tear film. The tear film in front of the cornea is typically called the precorneal tear film (PCTF). Although the precorneal tear film is only 6–10 nm thick, it maintains the eye's moisture and improves eyeglass refraction by filling in minute corneal irregularities.

[0004] The secretion status of the human precorneal tear film is not only influenced by the metabolic state of the ocular surface, but analyzing its composition can also detect the presence of ocular surface diseases and the balance of the human body environment. Therefore, biochemical analysis of PCTF components can be used to detect the presence or progression of diseases and potentially apply to clinical treatment. Furthermore, 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 convenient option compared to collecting blood, urine, and fecal samples.

[0005] However, the amount of tear sample that can be collected is too small compared to the amount of clinically common samples such as blood, urine, and feces, which results in relatively poor detection rate, accuracy, reproducibility, and reliability of the test results, or requires significant efforts to ensure reliable results.

[0006] Current tear sample collection methods mainly include stimulated tear collection and capillary tear collection. The former requires stimulating tear secretion, typically achieved by applying irritating substances such as refreshing oil to the lower eyelid or stimulating the Jingming point with a needle, which tends to cause discomfort to the subject and does not allow for quantitative collection of samples, resulting in significant variability in test results. The latter requires using a capillary to aspirate tears from the ocular surface after reflex tears appear, placing significant demands on the operator. This can easily injure the subject's eye, and the limited capacity of the capillary means that the sample collected is too small, resulting in a disadvantage for subsequent testing. Furthermore, the stimulated tear collection method uses irritating substances, which increases the risk of sample contamination and prevents accurate representation of the tear condition.

[0007] At the same time, current immunoassay techniques, including fluorescent immunochromatography, colloidal gold immunochromatography, magnetic particle immunochemiluminescence, latex bead immunochromatography, and enzyme-linked immunosorbent assay (ELISA), all require large sample volumes and the coordination of multiple independent instruments and equipment to obtain more reliable test results. These complex and lengthy steps, high operator demands, and long processing times limit their application in testing small sample volumes. However, when these methods use small sample volumes, the results are unreliable, making it difficult to obtain clinically meaningful judgments.

[0008] Therefore, there is a strong demand in this field for a means to reliably collect and process small volumes of tear samples. Summary of the Invention

[0009] In view of the problems in the prior art, the inventors have found that by using an absorbent material, it is possible to stably collect a small volume of tear sample, and after eluting it with a sample eluent having a specific composition, it is possible to stably release the substance to be measured in the sample. This allows further qualitative, semi-quantitative, and quantitative analysis to be performed on the substance to be measured. In a separate step, it is also possible to realize analysis of various physiological parameters of the subject based on the analysis results of different substances to be measured. This completes the present invention.

[0010] Accordingly, in a first aspect, the present specification provides a tear sample eluate, the sample eluate comprising water as a solvent, a buffer, a surfactant, a stabilizer, a preservative and an osmotic adjuster, and having a pH value of 7.0 to 9.0.

[0011] 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, or phosphoric acid, or an organic or inorganic base, such as sodium hydroxide or potassium hydroxide. The content of the pH adjuster is not particularly limited, and those skilled in the art can select its concentration and amount based on the final pH value of the sample eluate so that the sample eluate reaches the desired final pH value. In one embodiment, the sample eluate has a weak alkaline pH, for example, a pH value of about 7.1 to 8.5, preferably 7.5 to 8.2, more preferably 7.8 to 8.1, e.g., about 7.4, 7.6, or 8.0. Preferably, the final pH of the sample eluate is adjusted with an HCl solution.

[0012] In one embodiment, the buffer is selected from organic amine buffers, preferably organic amine buffers having a hydroxyl group. For example, the buffer may be selected from tris(hydroxymethyl)aminomethane, i.e., trometamol (Tris), and / or tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl). The content of the buffer in the sample eluate may be 10 mM to 100 mM, preferably 20 mM to 80 mM, more preferably 35 mM to 60 mM, and particularly about 50 mM.

[0013] In one embodiment, a combination of tris(hydroxymethyl)aminomethane as a buffering agent and HCl as a pH adjusting agent is preferred.

[0014] 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 nonionic surfactants. In one embodiment, the first surfactant is different from the second surfactant. Preferably, the first surfactant is selected from polysorbate surfactants, more preferably Tween series surfactants, such as Tween 20 and Tween 80. Preferably, the second surfactant is selected from block polyether surfactants, such as polyethylene glycol surfactants having terminal hydroxy groups, more preferably polyethylene glycol phenyl ether surfactants. In one example, the second surfactant is, for example, Triton TM The surfactant may be polyethylene glycol mono-4-octylphenyl ether, available under the trade name Tetronic 1307 (S9), or, for example, ethylenediaminetetrakis(ethoxylate-block-propoxylate) tetrol surfactant. For example, it is available under the trade name Tetronic 1307 (S9). The surfactant content in the sample eluate may be 0.1% to 5% by weight, preferably 0.5% to 4% by weight, and more preferably 1% to 3.5% by weight. The weight ratio of the first surfactant to the second surfactant may be 10:1 to 1:10, preferably 1:1 to 1:10, more preferably 1:1 to 1:8, e.g., 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 surfactants and the second surfactant is selected from the Triton series surfactants, preferably a combination of Tween 20 and Triton X-100, and the concentration ratio thereof 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.

[0015] In one embodiment, the stabilizer is selected from albumin, casein, gelatin, etc. Preferably, the stabilizer is casein. The content of the stabilizer in the sample eluate may be 0.1 wt % to 5 wt %, preferably 0.5 wt % to 4 wt %, more preferably 0.8 wt % to 2 wt %, for example, about 1 wt %.

[0016] In one embodiment, the sample eluate is free of particulate matter having a particle size greater than 0.50 μm, preferably particulate matter having a particle size greater than 0.45 μm, more preferably particulate matter having a particle size greater than 0.40 μm.

[0017] In one embodiment, the preservative is selected from Proclin-300, sodium azide, thimerosal, etc. The content of the preservative in the sample eluate may be 0.01 to 0.5 wt %, preferably 0.05 to 0.4 wt %, more preferably 0.1 to 0.3 wt %, for example, 0.1 wt %, 0.2 wt %, or 0.3 wt %.

[0018] In one embodiment, the tonicity adjusting agent is an alkali metal salt, such as an inorganic or organic salt, preferably a sodium or potassium salt, more preferably NaCl.

[0019] In one embodiment, the sample eluate may or may not contain an organic solvent. Preferably, the sample eluate does not contain an organic solvent. In a preferred embodiment, the sample eluate contains water as the only solvent.

[0020] In one embodiment, the sample elution solution may comprise about 10 mM to 100 mM of a buffering agent, about 0.1 wt % to 5 wt % of a surfactant, about 0.1 wt % to 5 wt % of a stabilizer, and optionally about 0.01 wt % to 0.5 wt % of a preservative and about 0.1 wt % to 5 wt % of an osmolality adjuster, and preferably the elution solution further has a pH value of about 7.0 to 9.0.

[0021] In one embodiment, the sample eluate may contain about 35 mM to 60 mM of buffer, about 1 wt % to 3.5 wt % of surfactant, about 0.8 wt % to 2 wt % of stabilizer, and optionally about 0.1 wt % to 0.3 wt % of preservative and about 0.5 wt % to 1.2 wt % of osmolality adjuster, wherein the surfactants include a first surfactant and a second surfactant, and the weight ratio of the first surfactant to the second surfactant may be 10:1 to 1:10, preferably 1:1 to 1:10, more preferably 1:1 to 1:8, and preferably the eluate further has a pH value of about 7.4 to 8.1.

[0022] In one embodiment, the sample eluate may comprise about 50 mM buffer, about 0.5% 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, and preferably the eluate further has a pH value of about 7.4.

[0023] As an example, the sample eluate 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 further having a pH value of about 7.4.

[0024] In one example, the sample eluate comprises about 50 mM Tris buffer, about 0.5% Tween 20, about 2.5% by weight Triton X-100, about 1% by weight casein, and about 0.2% by weight Proclin-300 and about 0.9% by weight NaCl, and preferably the eluate has a pH value of about 7.4.

[0025] In one embodiment, the sample elution liquid is for eluting the tear fluid adsorbed on the first absorbent sheet. In another embodiment, the first absorbent sheet and the sample elution liquid constitute a tear sample collection and processing device.

[0026] In one embodiment, the first absorbent sheet may have one or more dye-marking layers. The dye-marking layers may include a fluorescent material, such as sodium fluorescein. Preferably, the first absorbent sheet is a filter paper strip. As an example, the first absorbent sheet may be a filter paper strip used in a Schirmer tear production test. In one embodiment, the first absorbent sheet has one, two, or more readable markings starting from an end. Preferably, the end is the sampling end. The host material of the first absorbent sheet is not particularly limited. In one embodiment, the first absorbent sheet is a hydrophilic inert sheet, and the host material may include cellulose or a polymer, such as polyurethane.

[0027] 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.

[0028] In one embodiment, the amount of tear fluid contained in the predetermined length of the fully saturated portion 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 predetermined length of the fully saturated portion may be abbreviated as the "same tear sample," and because the first absorbent sheet is disposable, the tear sample collected in each collection operation is used for only one measurement process.

[0029] In one embodiment, the volume of sample eluate 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.

[0030] In a second aspect herein, there is provided herein a method for producing a tear diluent, comprising: a. contacting a first absorbent sheet with the ocular surface to collect tears and obtain a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a predetermined length of fully saturated portion or is completely saturated with tears; b. contacting a predetermined length of the fully saturated portion of the first absorbent sheet with a predetermined volume of the sample eluate according to the first aspect for a predetermined time to obtain a diluted tear solution, or immersing the first absorbent sheet fully saturated with tear fluid in a predetermined volume of the sample eluate to obtain a diluted tear solution.

[0031] In one embodiment, the predetermined time for contacting the first absorbent sheet with the sample eluate is 1 s to 5 min, preferably 3 s to 3 min, more preferably 5 s to 1 min. The first absorbent sheet can be contacted with the sample eluate by immersion and / or stirring.

[0032] In a third aspect of the present specification, there is further provided an immunological testing reagent kit including an immunological testing card, wherein the testing 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, wherein the absorbent pad and the sample receiving pad are spatially separated from each other, and the blotting membrane between them has a control line on the side closer to the absorbent pad and a testing line on the side closer to the sample receiving pad.

[0033] In one embodiment, the support is made of at least one material selected from polymers, glass and polyvinyl chloride (PVC), polystyrene (PS), or a combination thereof.

[0034] In one embodiment, the blotting membrane comprises at least one material selected from cellulose, nitrocellulose, and / or a combination thereof.

[0035] In one embodiment, the sample receiving pad is a glass cellulose film, nylon cellulose, or polyester cellulose. In one embodiment, the sample receiving pad contains one of fluorescent latex beads or colloidal gold probes. Preferably, the fluorescent latex beads are polystyrene latex beads that bind to fluorescently labeled probes.

[0036] In one embodiment, the immunoassay card further comprises a second absorbent sheet, which may be identical or different in size and structure to the first absorbent sheet, but which contains a defined amount of target substance. Preferably, the first and second absorbent sheets have the same host material and size.

[0037] In one embodiment, the immunoassay includes fluorescence immunochromatography, colloidal gold immunochromatography, latex bead immunochromatography, magnetic particle chemiluminescence and / or enzyme-linked immunosorbent, etc. In one embodiment, the immunoassay card is a fluorescence immunochromatography assay card or a colloidal gold immunochromatography assay card.

[0038] In one embodiment, the substance of interest is 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), lipocalin-1, cystatin S100, alpha-1 antitrypsin, alpha enolase, alpha-1-acid glycoprotein 1, S100 A8 (calgranulin A), S100 A9 (calgranulin B), S100 A4 and S100 A11 (calcium), prolactin-inducible protein (PIP), proline protein 4 (PRR4), PRR3, NACPP4, S100A6, annexin A1 (ANXA1), annexin A11 (ANXA11), cystatin-S (CST4), phospholipase A2, activating protein (PLAA), mammaglobin B, lipophilin A, matrix metalloproteinase (MMP)-9, anti-Ro / SSA, La / SSB antibodies, anti-α-fodrin antibodies, aquaporin 5 (AQP5), complement C-3, albumin, potassium ions, sodium ions, chloride ions, and the like may also be included.

[0039] In one embodiment, the test line (12) contains mouse anti-human matrix metalloproteinase-9 IgG, preferably at a concentration of 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 defined by any two of these values. In one embodiment, the control line (b) contains goat anti-chicken IgY. In one embodiment, the fluorescent latex beads contain mouse anti-human matrix metalloproteinase-9 IgG coupled thereto. In one embodiment, the test line (12) contains mouse anti-human matrix metalloproteinase-9 IgG, the control line (11) contains goat anti-chicken IgY, and the fluorescent latex beads contain couplings with mouse anti-human matrix metalloproteinase-9 IgG, so that the immunological test card can be used to test for human matrix metalloproteinase.

[0040] In a fourth aspect, the present disclosure further provides a method for manufacturing an immunoassay card, comprising: (i) providing a blotting membrane having a control line and a test line, and optionally drying it at 37°C for 8 to 20 hours; (ii) providing a sample receiving pad and providing fluorescent latex beads or colloidal gold probes on the sample receiving pad, and optionally drying it at 37°C for 8 to 20 hours; (iii) placing a sample receiving pad on the second end of the blotting membrane near the test line to contact the two; (iv) placing an absorbent pad on the first end of the blotting membrane near the control line to bring them into contact.

[0041] In one embodiment, the sample receiving pad is provided with fluorescent latex beads. Preferably, providing the sample receiving pad with fluorescent latex beads comprises: (ii-1) treating blank fluorescent latex beads with an activation solution; (ii-2) treating the fluorescent latex beads obtained in step (ii-1) with a coupling buffer to obtain a fluorescent latex bead suspension; (ii-3) treating the fluorescent latex bead suspension obtained in step (ii-2) with mouse anti-human matrix metalloproteinase-9 IgG; (ii-4) treating the fluorescent latex bead suspension obtained in step (ii-3) with a blocking solution; (ii-5) treating the fluorescent latex bead suspension obtained in step (ii-4) with a second washing solution; (ii-6) diluting the fluorescent latex bead suspension obtained in step (ii-5) with a bead diluent and then spraying it onto a blank sample receiving pad.

[0042] Preferably, the method further comprises, before step (ii-1), step (ii-0) of washing the fluorescent latex beads with a first washing solution to obtain blank fluorescent latex beads, more preferably the washing is performed under ultrasonication, and even more preferably the washing includes centrifugation after ultrasonic treatment. Preferably, the first washing solution comprises morpholinoethanesulfonic acid, preferably an aqueous solution of morpholinoethanesulfonic acid.

[0043] Preferably, treating the blank fluorescent latex beads with an activation solution in step (ii-1) includes treating the blank fluorescent latex beads with a first activation solution and a second activation solution. The first activation solution is a solution of N-hydroxysulfosuccinimide sodium salt in the first washing solution, and preferably, the concentration of N-hydroxysulfosuccinimide sodium salt is 10 to 30 mg / mL. The second activation solution is a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the first washing solution, and preferably, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 10 to 30 mg / mL. The blocking solution contains casein, bovine serum albumin, and a buffer.

[0044] Preferably, the coupling buffer in step (ii-2) comprises N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, preferably at a concentration of about 6 g / L, and preferably having a pH value of about 8.0.

[0045] Preferably, the method further comprises centrifuging the mixture after each of 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.

[0046] Preferably, steps (ii-0) and / or (ii-4) further comprise ultrasonically assisted cleaning.

[0047] Preferably, the spray application in step (ii-6) is carried out in an amount of 5 to 10 μL / cm.

[0048] Preferably, in step (ii-3), the mass ratio of mouse anti-human matrix metalloproteinase 9 (MMP-9) IgG to the fluorescent latex bead suspension obtained in step (ii-2) is (0.1 to 0.5):(1 to 5). The treatment in step (ii-3) is carried out at 35 to 40°C, preferably 37°C, for at least 2 hours, preferably at least 3 hours.

[0049] Preferably, the treatment in step (ii-4) is carried out at 35 to 40°C, preferably 37°C, for 5 to 60 minutes, more preferably 10 to 40 minutes, and even more preferably 10 to 30 minutes.

[0050] As an example, the first washing solution is an aqueous solution of about 10.66 g / L morpholinoethanesulfonic 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 washing solution, the second activation solution is a solution of about 20 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the first washing 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 washing solution is a 6 g / L Trisbase buffer having a pH of about 8.0, and the bead diluent is a solution of 30% sucrose, 0.5% casein, 1% glycine, and 50% glycerol having a pH of about 8.0. It is a buffer solution with 100 mM Trisbase.

[0051] In a fifth aspect, the present specification further provides a method for detecting a target substance in tear fluid.

[0052] In one embodiment, the method for detecting a target substance in tear fluid comprises: a. contacting a first absorbent sheet with the ocular surface to collect tears and obtain a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a predetermined length of fully saturated portion or is completely saturated with tears; b. contacting a predetermined length of a fully saturated portion of the first absorbent sheet with a predetermined volume of sample eluate for a predetermined time to obtain a diluted tear solution, or by immersing a first absorbent sheet that is fully saturated with tear fluid in a predetermined volume of sample eluate to obtain a diluted tear solution; c. Using the obtained diluted tear solution in an immunological assay to analyze the target substance in the tear.

[0053] In one embodiment, step c of the method for detecting a target substance in tear fluid comprises contacting the obtained diluted tear fluid with the immunochromatographic detection card of the third aspect of the present disclosure.

[0054] In one embodiment, the method for detecting a target substance in tear fluid comprises: a'. contacting a first absorbent sheet with the ocular surface to collect tears and obtain 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; b'. contacting the first absorbent sheet with the immunoassay card of the third aspect of the present disclosure.

[0055] In one embodiment, the method for detecting a target substance in tear fluid comprises: a. contacting at least two first absorbent sheets with the ocular surface of the same subject at different times to collect tears, thereby obtaining at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheets at least partially saturated with tears have a predetermined length of fully saturated portion or are completely saturated with tears; b. contacting a predetermined length of a fully saturated portion of each first absorbent sheet with a predetermined volume of sample eluate for a predetermined time to obtain a diluted tear solution, or by immersing each fully saturated first absorbent sheet in a predetermined volume of sample eluate to obtain a diluted tear solution; c. Using the obtained diluted tear solution in an immunological assay, the target substance in tears collected from the same subject at different time points is analyzed.

[0056] In one embodiment, the method for detecting a target substance in tear fluid comprises: a'. contacting at least two first absorbent sheets with the ocular surface of the same subject at different times to collect tears, thereby 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; b'. contacting at least two first absorbent sheets individually with the immunoassay card of the third aspect of the present disclosure.

[0057] In one embodiment, the same subject is in different states at different times, e.g., healthy or diseased, particularly, for example, all healthy or all diseased at different times, or healthy at at least one time point and diseased at at least one other time point. In one embodiment, provided herein is qualitative, semi-quantitative, or quantitative assay for one or more target substances in tear fluid samples collected from the same subject at different times. In one embodiment, provided herein is quantitative assay for target substances in multiple tear fluid samples collected from the same subject at different times.

[0058] In one embodiment, the method for detecting a target substance in tear fluid comprises: a. collecting tears by contacting a first absorbent sheet with the ocular surfaces of at least two different subjects to obtain at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheets at least partially saturated with tears have a predetermined length of fully saturated portion or are completely saturated with tears; b. contacting a predetermined length of a fully saturated portion of each first absorbent sheet with a predetermined volume of sample eluate for a predetermined time to obtain a diluted tear solution, or by immersing a first absorbent sheet that is fully saturated with tear fluid in a predetermined volume of sample eluate to obtain a diluted tear solution; c. Using the obtained diluted tear fluids in immunoassays, the target substance in the tears collected from different subjects is analyzed.

[0059] In one embodiment, the method for detecting a target substance in tear fluid comprises: a'. contacting at least two first absorbent sheets with the ocular surfaces of different subjects at different times to collect tears, thereby obtaining at least two first absorbent sheets at least partially saturated with tears, wherein the first absorbent sheets at least partially saturated with tears have a fully saturated portion; b'. The method includes a step of contacting at least two first absorbent sheets individually with the immunoassay card according to the third aspect of the present disclosure to analyze the target substance in the tear fluid.

[0060] In one embodiment, the different subjects are in the same or similar state, e.g., healthy or suffering from the same or similar disease. In one embodiment, the different subjects are in different states, e.g., healthy or suffering from the same or similar disease. In one embodiment, the different subjects are provided herein for qualitative, semi-quantitative, or quantitative detection of a target substance in one or more tears of different subjects. In one embodiment, the present disclosure provides for quantitative detection of a target substance in multiple tears of different subjects.

[0061] In one embodiment, the method for detecting a target substance in tear fluid comprises: a. contacting a first absorbent sheet with the ocular surface to collect tear fluid and obtain a first absorbent sheet at least partially saturated with tear fluid; b. cutting a length of fully saturated material from a first absorbent sheet that is at least partially saturated with tear fluid; c. contacting a predetermined length of fully saturated section with a predetermined volume of sample eluate for a predetermined time; d. collecting the sample eluate after contacting the first absorbent sheet to obtain a diluted tear solution; e. Using the diluted tear fluid collected in step d for immunoassay to analyze the target substance in the tear fluid.

[0062] In one embodiment, the method for detecting a target substance in tear fluid comprises: a. contacting a first absorbent sheet with an ocular surface to collect tears and obtain a first absorbent sheet at least partially saturated with tears, the first absorbent sheet being at least partially saturated with tears and having a predetermined length of fully saturated portion; b. contacting a predetermined length of fully saturated section with a predetermined volume of sample eluate for a predetermined time; c. collecting the sample eluate after contacting the first absorbent sheet to obtain a diluted tear solution; d. Using the diluted tear fluid collected in step c for immunoassay to analyze the target substance in the tear fluid.

[0063] In one embodiment, provided herein is qualitative, semi-quantitative, or quantitative detection of different target substances. In one embodiment, provided herein is 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, e.g., the tear sample volume is 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, provided herein is quantitative or 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 is a detection method 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.

[0064] In a sixth aspect, there is provided herein a tear sample collection and processing device, comprising: a first package including a first absorbent sheet; a second package containing a sample eluate; The sample eluate contains a buffer, a surfactant and a stabilizer, and has a pH value of 7.0 to 9.0.

[0065] In one embodiment, the sample eluate comprises water as a solvent, and said sample eluate further comprises a preservative and / or an osmolality adjusting agent.

[0066] In one embodiment, the sample elution solution contains water as a solvent, a buffer, a surfactant, a stabilizer, a preservative, and an osmotic pressure adjuster, and has a pH value of 7.0 to 9.0.

[0067] In one embodiment, the first absorbent sheet, sample eluate, buffer, surfactant, stabilizer, preservative and osmolality adjuster have the definitions as set out in the first aspect.

[0068] In a seventh aspect, there is further provided herein the use of the tear sample collection and processing device according to the sixth aspect for collecting and processing tear fluid.

[0069] In an eighth aspect, the present specification further provides an immunological sampling and testing reagent kit, which comprises a first package, a second package and an immunological testing card as described in the sixth aspect, and preferably the immunological testing card is the immunological testing card as described in the third aspect.

[0070] Those skilled in the art can understand that the features in any aspect, embodiment or example in the present disclosure may be combined with each other unless there is a contradiction or incompatibility between the features themselves. [Effects of the Invention]

[0071] The method provided herein allows a fixed-length or full-length portion of a water-absorbent material sheet, particularly a typical Schirmer tear testing filter paper strip, to be used as a device for collecting and quantifying tear samples, which can then be eluted with a sample elution solution provided herein, reproducibly and stably releasing a quantitative tear sample. Furthermore, the tear sample collection and processing device of the present disclosure significantly improves the convenience, accuracy, and stability of subsequent use of collected tear samples for other tests compared to conventional tear collection and processing methods. Furthermore, the resulting diluted tear solution can be directly used to detect target substances in tears using platforms such as fluorescent immunochromatography, colloidal gold immunochromatography, and latex chromatography immunotechnologies, making the sampling and testing process more convenient and less difficult and costly. [Brief explanation of the drawings]

[0072] [Figure 1] 1 shows a calibration curve in Example 5 according to the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0073] Example The manufacturing method of the present invention will be described in more detail below with reference to specific examples. It should be understood that the following examples are merely for illustrative purposes and should not be construed as limiting the scope of the claims of the present invention. Any technical solutions realized based on the content of the present invention are included within the scope of the claims of the present invention.

[0074] Unless otherwise specified, all experimental methods used in the following examples are conventional methods in the art, and all reagents, raw materials, instruments, equipment, etc. used in the following examples are commercially available.

[0075] Example 1: Consistency testing of absorbent sheets Twenty commercially available Schirmer tear secretion test filter paper strips were provided, and sections of the same length were cut from each strip. The cut sections were weighed on a precision analytical balance, and the data were recorded as dry weights. After each weighed section was thoroughly soaked with tear fluid, it was immediately weighed on a precision analytical balance and the data were recorded as wet weights. The dry weight of each cut section was subtracted from the wet weight to obtain the mass of the aspirated tear fluid. The volume of the wetting tear fluid was calculated assuming a specific gravity of 1 (i.e., a density of 1 g / mL), and the results in Table 1 below were obtained.

[0076] [Table 1]

[0077] As can be seen from Table 1 above, the commercially available Schirmer tear secretion test filter paper strips showed a tear secretion rate of 9.8 × 10 -5A deviation of 0.01% and a coefficient of variation of 5% can be achieved, thereby confirming the volume stability when obtaining tear samples, enabling quantitative measurement of the samples using a reagent kit, and providing a basis for quantitative measurement of the concentration of target substances in tears through standard curve regression.

[0078] Example 2: Preparation of fluorescent immunochromatography test card 1. Preparation of blotting membrane: A blank blotting membrane with a control line and a test line was dried at 37°C for 8 to 20 hours.

[0079] 2. Preparation of the sample receiving pad: 0.001 g of fluorescent latex beads was added to 0.9 mL of the first washing solution (pH approximately 6.1) containing 10.66 g / L morpholinoethanesulfonic acid. After uniform mixing by ultrasonication, the mixture was centrifuged at 10,000-15,000 rpm for 30 minutes, and the supernatant was discarded. To the residue, 0.12 mL of a 20 mg / mL solution of N-hydroxysulfosuccinimide sodium salt in the first washing solution and 0.06 mL of a 20 mg / mL solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added sequentially. The mixture was activated for 30 minutes, stirred at 100-150 rpm while maintaining the temperature at 35-40°C, and then centrifuged at 10,000-15,000 rpm for 20 minutes. The supernatant was discarded. To this residue, 1 mL of a 5.96 g / L N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid solution at pH 8.0 was added, and the mixture was sonicated until a clear solution was obtained. The mixture was then centrifuged for 20 minutes, and the supernatant was discarded. 0.0001 g of mouse anti-human matrix metalloproteinase-9 IgG was then added to the residue, mixed uniformly, and the coupling reaction was continued. The resulting reaction mixture was centrifuged for 30 minutes, and the supernatant was 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. The mixture was then centrifuged for 20 minutes, and the supernatant was discarded. 1 mL of a pH 8.0 Trisbase buffer was added, and the mixture was sonicated until a clear solution was obtained. The mixture was then centrifuged for 20 minutes, and the supernatant was discarded. The residue was then diluted to volume with 1 mL of a solution containing 30% sucrose, 0.5% casein, 1% glycine, and 50 mM Trisbase at pH 8.0, to obtain a mixture, which was then spray-coated onto a glass cellulose film at a rate of 5–10 μL / cm to obtain a sample receiving pad.

[0080] 3. Place the sample receiving pad prepared in the previous step on the second end of the blotting membrane close to the test line; 4. An absorbent pad was placed over the first end of the blotting membrane near the control line.

[0081] Example 3: Preparation of colloidal gold testing card 1. Preparation of blotting membrane: A blank blotting membrane with a control line and a test line was dried at 37°C for 8 to 20 hours.

[0082] 2. Preparation of sample receiving pad: 1 mL of 40 nm colloidal gold solution was added with 10 μL of 50 mM potassium carbonate, vortexed to homogenize, and allowed to stand for 5 minutes. 10 μg of mouse anti-human matrix metalloproteinase-9 IgG was added directly to the colloidal gold solution, vortexed to homogenize, and allowed to react at room temperature for 30 minutes. After the reaction was complete, 100 μL of 10% bovine serum albumin (BSA) was added, vortexed to homogenize, and allowed to react at room temperature for 30 minutes. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 20 minutes and the supernatant was discarded. The residue was then diluted to volume with 0.1 mL of a solution containing 30% sucrose, 0.5% casein, 1% glycine, and 50 mM Trisbase (pH 8.0). This mixture was then sprayed onto a glass cellulose film at a volume of 5–10 μL / cm to obtain a sample receiving pad.

[0083] 3. Place the sample receiving pad prepared in the previous step on the second end of the blotting membrane close to the test line; 4. An absorbent pad was placed over the first end of the blotting membrane near the control line.

[0084] Example 4: Assay of sample dissolution rate 100 μL of the calibration sample was aspirated and added to the sample addition well on the test card. The sample was placed horizontally and allowed to react in the dark for 15 minutes before the results were read. The calibration samples provided were solutions of MMP-9 (matrix metalloproteinase-9) S0 to S5, with concentration gradients set at 0 ng / mL, 0.3 ng / mL, 1 ng / mL, 10 ng / mL, 30 ng / mL, and 60 ng / mL, respectively.

[0085] The accuracy of the calibration samples with different concentration gradients was confirmed by loading them onto the test cards manufactured in Examples 2 and 3, respectively, and the results are shown in Tables 2 and 3 below. The C value is the signal value of the control line, the T value is the signal value of the test line, T / C is the ratio between the test line and the control line, and CV is the coefficient of variation. Based on the difference test results, if the difference in the coefficient of variation is within 10%, it indicates a stable tear release effect.

[0086] [Table 2]

[0087] [Table 3]

[0088] Example 5 Tear fluid measurement test (dilution method) A commercially available Schirmer tear secretion test filter paper strip was brought into contact with the ocular surface of the subject to collect tears. The filter paper strip was saturated with tears. According to the procedure of Example 1, a section of the same length that was completely saturated with tears was cut from the test filter paper strip and immersed in 200 μL of sample dissolution solution for 3 seconds. The liquid was then collected to obtain a diluted tear solution, which was then loaded onto the sample receiving pad of the test card prepared in Example 2 for analysis.

[0089] The C value is the signal value at the control line, the T value is the signal value at the test line, and T / C is the ratio between the test line and the control line. The concentration was calculated based on the calibration curve. Two different subjects were tested five times each, and the results are shown in Tables 4 and 5 below.

[0090] Among them, the calibration curve fitting method is four-parameter fitting, which is defined as follows:

[0091] Y=(AD) / [1+(X / C)^B]+D A=4.63846376754392 B=-1.00725333313379 C=143.60632119348 D=0.00936819607330606 Correlation coefficient R2: 0.999885514800651

[0092] [Table 4]

[0093] [Table 5]

[0094] Example 6 Tear Measurement Test (Sandwich Method) A commercially available Schirmer tear secretion test filter paper strip was placed in contact with the ocular surface to collect tears, and the filter paper strip was saturated with tears. The filter paper strip was then placed on the sample receiving pad of the reagent kit prepared in Example 3 for analysis. The results shown in Tables 6 and 7 below were obtained.

[0095] [Table 6]

[0096] [Table 7]

[0097] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the scope of the spirit and principles of the present invention should be included within the scope of the claims 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 adjuster, and having a pH value of 7.0 to 9.

0.

2. 2. The tear sample eluate according to claim 1, wherein the buffer is selected from organic amine buffers, preferably an organic amine buffer having a hydroxy group, more preferably tris(hydroxymethyl)aminomethane.

3. 3. The tear sample eluate according to claim 1, wherein the surfactant comprises a first surfactant and a second surfactant different from the first surfactant, and 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 and the second surfactant is selected from block polyether surfactants, preferably a polyethylene glycol surfactant having a terminal hydroxy group or an ethylenediaminetetrakis(ethoxylate-block-propoxylate)tetrol surfactant.

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

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

7. The sample eluate may comprise about 10 mM to 100 mM of a buffering agent, about 0.1 wt % to 5 wt % of a surfactant, about 0.1 wt % to 5 wt % of a stabilizer, and optionally about 0.01 wt % to 0.5 wt % of a preservative and about 0.1 wt % to 5 wt % of an osmolality adjusting agent, preferably the eluate further has a pH value of about 7.0 to 9.0; Preferably, the sample eluate comprises about 50 mM Tris buffer, about 0.5% Tween 20, about 2.5% by weight Triton X-100, about 1% by weight casein, and about 0.2% by weight Proclin-300 and about 0.9% by weight NaCl, and has a pH value of about 7.

4. A tear sample eluate according to any one of claims 1 to 6.

8. 1. A method for producing a tear diluent, comprising: a. contacting a first absorbent sheet with the ocular surface to collect tears and obtain a first absorbent sheet at least partially saturated with tears, wherein the first absorbent sheet at least partially saturated with tears has a predetermined length of fully saturated portion or is completely saturated with tears; b. contacting a predetermined length of the fully saturated portion of the first absorbent sheet with a predetermined volume of sample eluate for a predetermined time to obtain a diluted tear solution, or immersing the first absorbent sheet fully saturated with tear fluid in a predetermined volume of the sample eluate according to any one of claims 1 to 7 to obtain a diluted tear solution; Including, Preferably, the predetermined time for contacting the first absorbent sheet with the sample eluate is 1 s to 5 min, preferably 3 s to 3 min, more preferably 5 s to 1 min. method.

9. a first package including a first absorbent sheet; a second package containing a sample eluate, Wherein, the sample elution solution comprises a buffer, a surfactant and a stabilizer, and has a pH value of 7.0 to 9.0, and the buffer, surfactant and stabilizer have the definitions set forth in any one of claims 1 to 7; Preferably, the sample eluate is a sample eluate according to any one of claims 1 to 7. Tear sample collection and processing equipment.

10. Use of the tear sample collection and processing device of claim 9 for collecting and processing tears.

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