Latex immunodetection kit based on microcolumn gel card and application thereof

Abstract

The invention provides a latex immunodetection kit based on a microcolumn gel card and application of the latex immunodetection kit. The detection kit comprises gel microspheres, a gel buffer solution and a latex particle solution, the gel microspheres and the gel buffer solution are filled in a hole column of the micro-column gel card; the gel microspheres are cross-linked glucan microspheres; the gel buffer solution is a PBS (phosphate buffer solution) containing glycerol, sodium hyaluronate, alkyl glycoside and coconut oil acyl hydrolyzed oat protein potassium; the latex particle solution is a PBS buffer solution containing latex particles, and the surfaces of the latex particles are coated with antigens. According to the latex immunodetection kit based on the microcolumn gel card, the gel microspheres, the gel buffer solution and the latex particles coated with the antigen are matched with one another in the microcolumn gel card hole column, so that the technical effects of quickly, simply and conveniently detecting a sample and judging a detection result are achieved, and a negative towing phenomenon is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and relates to a latex immunoassay kit based on microcolumn gel and its application. Background Art

[0002] Commonly used clinical immunological detection methods include slide agglutination method and rate nephelometry. In the slide agglutination method, since the latex is white and the agglutination also appears as white particles, and the prozone or postzone phenomenon is likely to occur in vitro during the agglutination reaction, resulting in errors in result interpretation, and its specificity and sensitivity are poor. For the rate nephelometry, it uses the depth of turbidity formed by antigen-antibody in the medium for turbidity optical analysis, and a special protein analyzer is required for turbidity measurement, and there are many interfering factors. For example, light, water quality, and purity of the test substance will all affect the turbidity.

[0003] CN112255421A discloses a lipoprotein a detection kit and a detection method. The kit includes reagent R1, reagent R2, and a calibrator. The reagent R1 includes a first buffer solution at 50 - 250 mmol / L, an anti-interference agent at 1 - 2 wt%, a promoter at 1 - 2.5 wt%, and a preservative at 0.01 - 0.1 wt%. The reagent R2 includes a second buffer solution at 50 - 250 mmol / L, a stabilizer at 0.5 - 5 wt%, latex particles coated with anti-human lipoprotein a monoclonal antibody at 0.1 - 2 g / L, and a preservative at 0.02 - 0.2 wt%. Using the detection kit of the present invention can eliminate the problem of non-specific interference caused by factors such as hydrophobic interaction, complement, and rheumatoid factor, but it cannot solve the interference of light, water quality, and purity of the test substance.

[0004] Therefore, in view of the deficiencies of the immunological detection methods, it is urgent to develop a new detection technology with high sensitivity, good specificity, and convenient and rapid operation. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art and the actual needs, the present invention provides a latex immunoassay kit based on microcolumn gel and its application, achieving the effects of fast detection speed, easy judgment of detection results, and high accuracy.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a latex immunoassay kit based on a microcolumn gel card, including gel microspheres, a gel buffer solution, and a latex particle solution;

[0008] The gel microspheres and the gel buffer solution are filled in the pore columns of the microcolumn gel card;

[0009] The gel microspheres are cross-linked dextran microspheres;

[0010] The gel buffer is a PBS buffer containing glycerol, sodium hyaluronate, alkyl glycoside, and potassium coco-hydrolyzed oat protein;

[0011] The latex particle solution is a PBS buffer containing latex particles, and the surface of the latex particles is coated with an antigen.

[0012] In the present invention, the gel microspheres in the pore column of the microcolumn gel card act as molecular sieves. Under negative conditions, the latex particles that have not undergone immune binding pass through the gaps between the gel microspheres to reach the bottom of the pore column. Under positive conditions, the latex particles that have undergone strong immune binding are intercepted at the top of the gel microspheres to form an agglutination band, and the latex particles that have undergone medium or mild immune binding form an agglutination band in the middle of the pore column. The test result can be determined by visually observing the precipitation position of the latex particles in the pore column.

[0013] Glycerol, sodium hyaluronate, alkyl glycoside, and potassium coco-hydrolyzed oat protein in the gel buffer in the pore column have a lubricating effect on the gel microspheres and latex particles. Among them, glycerol has good solubility and lubricity for serum, enabling the negative serum that has not undergone immune agglutination to smoothly pass through the gaps between the gel microspheres and deposit at the bottom of the pore column, avoiding negative trailing. Glycerol is combined with sodium hyaluronate with a molecular weight of 10,000 - 50,000 Da to maintain the viscosity of the gel buffer within an appropriate range for the serum to fully contact the latex particles and flow in the pore column, improving the accuracy of the test result. Alkyl glycoside and potassium coco-hydrolyzed oat protein have good compatibility and stability in the same solution system, can reduce the liquid tension of the gel microspheres, and regulate the friction between the serum and the gel microspheres, so that the negative serum does not adhere to the gel microspheres, avoiding negative trailing. Each component cooperates synergistically to provide a liquid environment with lubrication and humidity, reducing the friction between the gel microspheres and the latex particles, enabling the latex particles that have not undergone immune binding to precipitate at the bottom of the pore column through the gaps between the gel microspheres under the action of centrifugal force, and helping the latex particles that have not undergone immune binding to reach the bottom of the pore column through the gaps between the gel microspheres under the action of an appropriate centrifugal force, avoiding the phenomenon of negative trailing.

[0014] In the latex immunoassay kit based on the microcolumn gel card of the present invention, the gel microspheres, gel buffer, and latex particles coated with an antigen cooperate with each other in the pore column of the microcolumn gel card, achieving the technical effect of quickly and simply detecting a sample and judging the test result.

[0015] Preferably, the gel buffer is prepared from the following substances at the final concentrations:

[0016] Glycerol 100 - 200 mM

[0017] Sodium hyaluronate 50 - 100 mM

[0018] Alkyl glycoside 20 - 50 mM

[0019] Potassium coco hydrolyzed oat protein 1 - 5 mM

[0020] Disodium hydrogen phosphate 8 - 15 mM

[0021] Potassium dihydrogen phosphate 1 - 5 mM

[0022] Potassium chloride 1 - 5 mM

[0023] Sodium chloride 100 - 150 mM.

[0024] Among them, the final concentration of glycerol in the gel buffer is 100 - 200 mM, for example, it can be 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM, preferably 150 - 200 mM;

[0025] The final concentration of sodium hyaluronate in the gel buffer is 50 - 100 mM, for example, it can be 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, preferably 60 - 75 mM;

[0026] The final concentration of alkyl polyglycoside in the gel buffer is 20 - 50 mM, for example, it can be 20 mM, 30 mM, 40 mM or 50 mM, preferably 30 - 40 mM;

[0027] The final concentration of potassium coco hydrolyzed oat protein in the gel buffer is 1 - 5 mM, for example, it can be 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM, preferably 2 - 3.5 mM;

[0028] The final concentration of disodium hydrogen phosphate in the gel buffer is 8 - 15 mM, for example, it can be 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM or 15 mM, preferably 9 - 10.5 mM;

[0029] The final concentration of potassium dihydrogen phosphate in the gel buffer is 1 - 5 mM, for example, it can be 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM, preferably 1.5 - 2.5 mM;

[0030] The final concentration of potassium chloride in the gel buffer is 1 to 5 mM, for example, it can be 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or 5 mM, and preferably 2 to 3 mM;

[0031] The final concentration of sodium chloride in the gel buffer is 100 to 150 mM, for example, it can be 100 mM, 110 mM, 120 mM, 130 mM, 140 mM or 150 mM, and preferably 130 to 140 mM.

[0032] The molecular weight of sodium hyaluronate is 10,000 to 50,000 Da, for example, it can be 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da or 50,000 Da.

[0033] Preferably, the gel buffer is formulated from the following substances according to the final concentration:

[0034] Glycerol 150 to 200 mM

[0035] Sodium hyaluronate 60 to 75 mM

[0036] Alkyl polyglycoside 30 to 40 mM

[0037] Potassium coco-hydrolyzed oat protein 2 to 3.5 mM

[0038] Disodium hydrogen phosphate 9 to 10.5 mM

[0039] Potassium dihydrogen phosphate 1.5 to 2.5 mM

[0040] Potassium chloride 2 to 3 mM

[0041] Sodium chloride 130 to 140 mM.

[0042] In the present invention, glycerol, sodium hyaluronate, alkyl polyglycoside and potassium coco-hydrolyzed oat protein cooperate with each other synergistically under appropriate concentration conditions, providing a good lubricating environment for the latex particles, enabling the latex particles that have not undergone immune binding to completely precipitate at the bottom of the pore column through the gaps of the gel microspheres under the action of centrifugal force, completely avoiding the phenomenon of negative trailing, and ensuring the accuracy of the detection results.

[0043] Preferably, the pH of the gel buffer is 7.2 to 7.4.

[0044] Preferably, the particle size of the gel microspheres is 50 to 120 μm.

[0045] Preferably, the average particle size of the gel microspheres is 65 - 80 μm.

[0046] Preferably, the water absorption multiple of the gel microspheres is 8 - 12 mL / g, for example, it can be 8 mL / g, 9 mL / g, 10 mL / g, 11 mL / g or 12 mL / g.

[0047] In the present invention, gel microspheres with a particle size of 50 - 120 μm and a water absorption multiple of 8 - 12 mL / g are used as molecular sieves, and in combination with a gel buffer solution, it provides a suitable interstitial space for the latex particles, enabling the latex particles to precipitate entirely at the bottom of the pore column through the interstitial space of the gel microspheres under negative conditions, and being retained in the interstitial space of the gel microspheres without precipitating at the bottom of the pore column under positive conditions.

[0048] Preferably, the latex particles are polystyrene particles containing iron tetroxide.

[0049] Preferably, the density of the latex particles is 1.10 - 1.50 g / cm 3 , for example, it can be 1.10 g / cm 3 , 1.20 g / cm 3 , 1.30 g / cm 3 , 1.40 g / cm 3 or 1.50 g / cm 3 .

[0050] In the present invention, core - shell structured iron tetroxide - polystyrene microspheres are used as the solid phase carrier for the immune reaction, and in combination with gel microspheres and a gel buffer solution, it enables the latex particles under negative conditions to precipitate entirely at the bottom of the pore column through the interstitial space of the gel microspheres under the action of centrifugal force, solving the problem of negative carry - through.

[0051] Preferably, the particle size of the latex particles is 5 - 15 μm.

[0052] Preferably, the concentration of the latex particle solution is 3 - 10 mg / L, for example, it can be 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L or 10 mg / L.

[0053] When the concentration of latex particles is greater than 10 mg / L and a positive situation occurs, the latex particles precipitate at the bottom and middle of the pore column, interfering with the accurate judgment of the test result and wasting the latex particles; when the concentration of latex particles is less than 3 mg / L and a positive situation occurs, the precipitation lines of the latex particles aggregated on the top of the gel microspheres (strong agglutination) or inside the gel microspheres (moderate to mild agglutination) are thinner and not easy to observe, even causing false negative phenomena. In the present invention, PBS buffer is used to suspend the latex particles and maintain the concentration at 3-10 mg / L, making the result easy to observe and not causing waste of the latex particles.

[0054] Preferably, the surface of the latex particles is modified with a fluorescent label.

[0055] In the present invention, when the immune complex is formed, the precipitation line appears at the top of the gel microspheres (strong agglutination) or inside the gel microspheres (moderate to mild agglutination). To facilitate the observation of the antigen-antibody agglutinate, the latex particles coated with antigen are labeled with sodium fluorescein. 50-100 μL of sodium fluorescein solution is added to every 10 mL of the latex particle suspension coated with antigen, and the concentration of the sodium fluorescein solution is (0.01-0.05) g / mL.

[0056] In a second aspect, the present invention provides a method for using the detection kit described in the first aspect, including:

[0057] Adding a latex particle solution and a serum sample into the pore column of a micro-column gel card pre-filled with gel microspheres and a gel buffer;

[0058] After centrifugation, observe the micro-column gel card to judge the test result of the serum sample.

[0059] Preferably, the volume ratio of the gel microspheres to the gel buffer filled in the pore column of the micro-column gel card is (8-10):3, for example, it can be 8:3, 9:3 or 10:3.

[0060] Preferably, the volume of the gel microspheres and the gel buffer filled in the pore column of the micro-column gel card is 20-35 μL, for example, it can be 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, 30 μL, 31 μL, 32 μL, 33 μL, 34 μL or 35 μL, preferably 25-30 μL.

[0061] Preferably, the volume of the latex particle solution filled in the pore column of the micro-column gel card is 20-30 μL, for example, it can be 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL or 30 μL.

[0062] Preferably, the volume of the serum sample filled in the pore column of the microcolumn gel card is 25-35 μL, for example, it can be 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, 30 μL, 31 μL, 32 μL, 33 μL, 34 μL or 35 μL.

[0063] Preferably, a step of incubation is included before centrifugation.

[0064] Preferably, the temperature of the incubation is 35-40 °C, for example, it can be 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C.

[0065] Preferably, the time of the incubation is 15-30 min, for example, it can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min or 30 min.

[0066] Preferably, the centrifugal force of the centrifugation is 600-1500 g, for example, it can be 600 g, 700 g, 800 g, 900 g, 1000 g, 1100 g, 1200 g, 1300 g, 1400 g or 1500 g.

[0067] Preferably, the time of the centrifugation is 15-25 min, for example, it can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min or 25 min.

[0068] As a preferred technical solution, the method for using the detection kit described in the first aspect includes:

[0069] Pre-add gel microspheres and gel buffer to each pore column of the microcolumn gel card according to a volume ratio of 8:3. The total volume of the mixture of the gel microspheres and the gel buffer is 30 μL. After adding, use a card centrifuge to centrifuge, and the centrifugation conditions are: centrifuge at 3000 rpm for 3 min, and set aside after centrifugation;

[0070] Add 25 μL of latex particles coated with fluorescein sodium-labeled antigen and 30 μL of serum sample to each pore column of the microcolumn gel card, incubate at 37 °C for 20 min and then centrifuge;

[0071] Observe the position of the precipitation line in the pore column of the microcolumn gel card to judge the detection result of the serum sample.

[0072] In a third aspect, the present invention provides the use of the detection kit described in the first aspect for detecting anti-streptolysin O or rheumatoid factor.

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] (1) In the latex immunoassay kit based on a microcolumn gel card of the present invention, the gel microspheres in the pore column of the microcolumn gel card act as a molecular sieve, and cooperate with the gel buffer solution in the pore column to provide a lubricating liquid environment, reducing the friction between the gel microspheres and the latex particles. Moreover, the core-shell structured iron oxide-polystyrene microspheres are used as the solid-phase carrier for the immune reaction, avoiding the negative trailing phenomenon;

[0075] (2) The gel microspheres, gel buffer solution and latex particles coated with antigen in the present invention cooperate with each other in the pore column of the microcolumn gel card, achieving the technical effects of quickly and simply detecting samples and judging the detection results. Description of the Drawings

[0076] Figure 1A For the detection result of an anti-streptolysin O antibody positive serum sample using the detection kit of Example 1, Figure 1B For the detection result of an anti-streptolysin O antibody negative serum sample using the detection kit of Example 1.

[0077] Figure 2A For the detection result of an anti-rheumatoid factor antibody positive serum sample using the detection kit of Example 2, Figure 2B For the detection result of an anti-rheumatoid factor antibody negative serum sample using the detection kit of Example 2.

[0078] Figure 3A For the detection result of an anti-streptolysin O antibody positive serum sample using the detection kit of Example 3, Figure 3B For the detection result of an anti-streptolysin O antibody negative serum sample using the detection kit of Example 3.

[0079] Figure 4A For the detection result of an anti-rheumatoid factor antibody positive serum sample using the detection kit of Example 4, Figure 4B For the detection result of an anti-rheumatoid factor antibody negative serum sample using the detection kit of Example 4.

[0080] Figure 5A For the detection result of an anti-streptolysin O antibody positive serum sample using the detection kit of Example 5, Figure 5B For the detection result of an anti-streptolysin O antibody negative serum sample using the detection kit of Example 5.

[0081] Figure 6A The test results of a serum sample positive for anti - rheumatoid factor antibody detected using the test kit of Example 6 Figure 6B The test results of a serum sample negative for anti - rheumatoid factor antibody detected using the test kit of Example 6

[0082] Figure 7A The test results of a serum sample positive for anti - streptolysin O antibody detected using the test kit of Example 9 Figure 7B The test results of a serum sample negative for anti - streptolysin O antibody detected using the test kit of Example 9

[0083] Figure 8A The test results of a serum sample positive for anti - rheumatoid factor antibody detected using the test kit of Example 10 Figure 8B The test results of a serum sample negative for anti - rheumatoid factor antibody detected using the test kit of Example 10

[0084] Figure 9A Shown are the test results of a serum sample negative for anti - streptolysin O antibody detected using the test kit of Comparative Example 1 Figure 9B Shown are the test results of a serum sample negative for anti - streptolysin O antibody detected using the test kit of Comparative Example 2 Figure 9C Shown are the test results of a serum sample negative for anti - streptolysin O antibody detected using the test kit of Comparative Example 3 Figure 9D Shown are the test results of a serum sample negative for anti - streptolysin O antibody detected using the test kit of Comparative Example 4

[0085] Figure 10 The test results of a serum sample negative for anti - streptolysin O antibody detected using the test kit of Comparative Example 5 Detailed implementation mode

[0086] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific implementation modes described herein are only used to explain the present invention, rather than limiting the present invention

[0087] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels

[0088] Example 1

[0089] This embodiment provides a latex immunoassay kit based on a microcolumn gel card, and the kit includes a microcolumn gel card and a latex particle solution.

[0090] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and the gel buffer in a volume ratio of 8:3 to form a mixture, add 30 μL of the mixture into the pore column, centrifuge at 3000 rpm for 3 min, and then seal the card for later use;

[0091] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 10 mL / g;

[0092] The gel buffer is prepared from the following substances according to the final concentration with PBS buffer:

[0093] 183 mM glycerol, 70.5 mM sodium hyaluronate (molecular weight 15000 Da), 33.5 mM alkyl glycoside, 2.5 mM potassium coco-hydrolyzed oat protein.

[0094] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide - polystyrene microspheres three times, coat with anti-streptolysin O, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 8 mg / L. The density of the iron oxide - polystyrene microspheres is 1.10 g / cm 3 , and the particle size is 5 - 15 μm.

[0095] Add 30 μL of serum sample and 25 μL of latex particle solution into the pore column of the microcolumn gel card, incubate at 37°C for 20 min, then centrifuge at 1500 g for 20 min, and observe and judge the test results.

[0096] Example 2

[0097] This embodiment provides a latex immunoassay kit based on a microcolumn gel card, and the kit includes a microcolumn gel card and a latex particle solution.

[0098] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and the gel buffer in a volume ratio of 8:3 to form a mixture, add 30 μL of the mixture into the pore column, centrifuge at 3000 rpm for 3 min, and then seal the card for later use;

[0099] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 10 mL / g;

[0100] The gel buffer is prepared from the following substances according to the final concentration with PBS buffer:

[0101] 165 mM glycerol, 65 mM sodium hyaluronate (molecular weight 15000 Da), 38 mM alkyl glycoside, 3 mM potassium coco-hydrolyzed oat protein.

[0102] The preparation steps of the latex particle solution are as follows: Wash the Fe₃O₄-polystyrene microspheres with core-shell structure three times, coat them with rheumatoid factor, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 8 mg / L. The density of the Fe₃O₄-polystyrene microspheres is 1.20 g / cm 3 , and the particle size is 5 - 15 μm.

[0103] Add 30 μL of serum sample and 25 μL of latex particle solution into the pore column of the microcolumn gel card, incubate at 37 °C for 20 min, then centrifuge at 1500 g for 20 min, and observe and judge the test results.

[0104] Example 3

[0105] This example provides a latex immunoassay kit based on a microcolumn gel card. The kit includes a microcolumn gel card and a latex particle solution.

[0106] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and gel buffer in a volume ratio of 8:3 to form a mixture, add 30 μL of the mixture into the pore column, centrifuge at 3000 rpm for 3 min, and then seal the card for later use;

[0107] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 10 mL / g;

[0108] The gel buffer is prepared from the following substances at the final concentration with PBS buffer:

[0109] 150 mM glycerol, 60 mM sodium hyaluronate (molecular weight 20000 Da), 40 mM alkyl glycoside, 3.5 mM potassium coco-hydrolyzed oat protein.

[0110] The preparation steps of the latex particle solution are as follows: Wash the Fe₃O₄-polystyrene microspheres with core-shell structure three times, coat them with anti-streptolysin O, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 6 mg / L. The density of the Fe₃O₄-polystyrene microspheres is 1.30 g / cm 3 , and the particle size is 5 - 15 μm;

[0111] Add 100 μL of fluorescein sodium solution (concentration 0.01 g / mL) to 10 mL of latex particle solution to obtain a fluorescein sodium-labeled latex particle solution.

[0112] Add 30 μL of serum sample and 25 μL of fluorescein sodium-labeled latex particle solution into the well column of the microcolumn gel card. After incubation at 37 °C for 20 min, centrifuge at 1000 g for 20 min, and observe and judge the test result.

[0113] Example 4

[0114] This example provides a latex immunoassay kit based on a microcolumn gel card. The kit includes a microcolumn gel card and a latex particle solution.

[0115] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline. Mix the gel microspheres and gel buffer in a volume ratio of 8:3 to form a mixture. Add 30 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for standby.

[0116] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 9 mL / g.

[0117] The gel buffer is prepared from the following substances at the final concentrations with PBS buffer:

[0118] 200 mM glycerol, 75 mM sodium hyaluronate (molecular weight 15000 Da), 30 mM alkyl glycoside, 2 mM potassium coco-hydrolyzed oat protein.

[0119] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide-polystyrene microspheres three times, coat with rheumatoid factor, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 6 mg / L. The density of the iron oxide-polystyrene microspheres is 1.40 g / cm 3 and the particle size is 5 - 15 μm.

[0120] Add 100 μL of fluorescein sodium solution (concentration 0.01 g / mL) to 10 mL of the latex particle solution to obtain the fluorescein sodium-labeled latex particle solution.

[0121] Add 30 μL of serum sample and 25 μL of fluorescein sodium-labeled latex particle solution into the well column of the microcolumn gel card. After incubation at 37 °C for 20 min, centrifuge at 1500 g for 20 min, and observe and judge the test result.

[0122] Example 5

[0123] This example provides a latex immunoassay kit based on a microcolumn gel card. The kit includes a microcolumn gel card and a latex particle solution.

[0124] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and the gel buffer in a volume ratio of 8:3 to form a mixture, add 30 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for standby;

[0125] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 11 mL / g;

[0126] The gel buffer is prepared from the following substances in a final concentration by PBS buffer:

[0127] 125 mM glycerol, 80 mM sodium hyaluronate (molecular weight 20000 Da), 45 mM alkyl glycoside, 1.5 mM potassium coco-hydrolyzed oat protein.

[0128] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide-polystyrene microspheres three times, coat with anti-streptolysin O, and then add them to the PBS buffer to prepare a latex particle solution with a concentration of 7 mg / L. The density of the iron oxide-polystyrene microspheres is 1.50 g / cm 3 and the particle size is 5 - 15 μm;

[0129] Add 100 μL of sodium fluorescein solution (concentration 0.01 g / mL) to 10 mL of the latex particle solution to obtain a sodium fluorescein-labeled latex particle solution.

[0130] Add 30 μL of the serum sample and 25 μL of the sodium fluorescein-labeled latex particle solution into the well column of the microcolumn gel card, centrifuge at 1000 g for 20 min, and observe and judge the test result.

[0131] Example 6

[0132] This example provides a latex immunoassay kit based on a microcolumn gel card. The kit includes a microcolumn gel card and a latex particle solution.

[0133] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and the gel buffer in a volume ratio of 8:3 to form a mixture, add 30 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for standby;

[0134] Among them, the particle size of the gel microspheres is 40 - 70 μm, and the water absorption multiple is 8 mL / g;

[0135] The gel buffer is prepared from the following substances in a final concentration by PBS buffer:

[0136] 110 mM glycerol, 88.5 mM sodium hyaluronate (molecular weight 20,000 Da), 46.5 mM alkyl polyglycoside, 4 mM potassium coco-hydrolyzed oat protein.

[0137] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide-polystyrene microspheres three times, coat them with rheumatoid factor, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 10 mg / L. The density of the iron oxide-polystyrene microspheres is 1.20 g / cm 3 , and the particle size is 5 - 15 μm;

[0138] Add 100 μL of sodium fluorescein solution (concentration 0.01 g / mL) to 10 mL of the latex particle solution to obtain a sodium fluorescein-labeled latex particle solution.

[0139] Add 30 μL of serum sample and 25 μL of sodium fluorescein-labeled latex particle solution into the well column of the microcolumn gel card, incubate at 37°C for 20 min, then centrifuge at 1500 g for 20 min, and observe and judge the test results.

[0140] Example 7

[0141] This example provides a latex immunoassay kit based on a microcolumn gel card. The kit includes a microcolumn gel card and a latex particle solution.

[0142] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and gel buffer in a volume ratio of 9:3 to form a mixture, add 30 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for later use;

[0143] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 12 mL / g;

[0144] The gel buffer is prepared from the following substances according to the final concentration with PBS buffer:

[0145] 100 mM glycerol, 100 mM sodium hyaluronate (molecular weight 10,000 Da), 20 mM alkyl polyglycoside, 1 mM potassium coco-hydrolyzed oat protein.

[0146] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide-polystyrene microspheres three times, coat them with anti-streptolysin O, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 10 mg / L. The density of the iron oxide-polystyrene microspheres is 1.10 g / cm 3 , and the particle size is 5 - 15 μm;

[0147] Add 100 μL of sodium fluorescein solution (concentration 0.01 g / mL) to 10 mL of latex particle solution to obtain a sodium fluorescein-labeled latex particle solution.

[0148] Add 30 μL of serum sample and 25 μL of sodium fluorescein-labeled latex particle solution into the well column of a microcolumn gel card, incubate at 37 °C for 20 min, then centrifuge at 1500 g for 20 min, and observe and judge the test result.

[0149] Example 8

[0150] This example provides a latex immunoassay kit based on a microcolumn gel card, and the kit includes a microcolumn gel card and a latex particle solution.

[0151] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and gel buffer in a volume ratio of 8:3 to form a mixture, add 20 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for standby;

[0152] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 10 mL / g;

[0153] The gel buffer is prepared from the following substances according to the final concentration with PBS buffer:

[0154] 100 mM glycerol, 50 mM sodium hyaluronate (molecular weight 30000 Da), 50 mM alkyl glycoside, 5 mM potassium coco-hydrolyzed oat protein.

[0155] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide-polystyrene microspheres three times, coat with rheumatoid factor, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 6.5 mg / L. The density of the iron oxide-polystyrene microspheres is 1.20 g / cm 3 and the particle size is 5 - 15 μm;

[0156] Add 100 μL of sodium fluorescein solution (concentration 0.01 g / mL) to 10 mL of latex particle solution to obtain a sodium fluorescein-labeled latex particle solution.

[0157] Add 30 μL of serum sample and 25 μL of sodium fluorescein-labeled latex particle solution into the well column of a microcolumn gel card, incubate at 37 °C for 20 min, then centrifuge at 1500 g for 20 min, and observe and judge the test result.

[0158] Example 9

[0159] This embodiment provides a latex immunoassay kit based on a microcolumn gel card, and the kit includes a microcolumn gel card and a latex particle solution.

[0160] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and the gel buffer in a volume ratio of 8:3 to form a mixture, add 35 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for standby;

[0161] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 5 mL / g;

[0162] The gel buffer is prepared from the following substances according to the final concentration with PBS buffer:

[0163] 95 mM glycerol, 105 mM sodium hyaluronate (molecular weight 50000 Da), 15 mM alkyl glycoside, 7 mM potassium coco-hydrolyzed oat protein.

[0164] The preparation steps of the latex particle solution are as follows: Wash the core-shell structured iron oxide-polystyrene microspheres three times, coat with anti-streptolysin O, and then add them to the PBS buffer to prepare a latex particle solution with a concentration of 5 mg / L. The density of the iron oxide-polystyrene microspheres is 1.25 g / cm 3 and the particle size is 5 - 15 μm.

[0165] Add 35 μL of the serum sample and 30 μL of the latex particle solution into the well column of the microcolumn gel card, incubate at 40°C for 15 min, then centrifuge at 1200 g for 15 min, and observe and judge the test result.

[0166] Example 10

[0167] This embodiment provides a latex immunoassay kit based on a microcolumn gel card, and the kit includes a microcolumn gel card and a latex particle solution.

[0168] The preparation steps of the microcolumn gel card are as follows: Wash the cross-linked dextran gel microspheres three times with physiological saline, mix the gel microspheres and the gel buffer in a volume ratio of 10:3 to form a mixture, add 30 μL of the mixture into the well column, centrifuge at 3000 rpm for 3 min, and then seal the card for standby;

[0169] Among them, the particle size of the gel microspheres is 50 - 120 μm, and the water absorption multiple is 15 mL / g;

[0170] The gel buffer is prepared from the following substances according to the final concentration with PBS buffer:

[0171] 210 mM glycerol, 45 mM sodium hyaluronate (molecular weight 60,000 Da), 55 mM alkyl polyglycoside, 0.5 mM potassium coco-hydrolyzed oat protein.

[0172] The preparation steps of the latex particle solution are as follows: Wash the Fe₃O₄-polystyrene microspheres with a core-shell structure three times, coat them with rheumatoid factor, and then add them to PBS buffer to prepare a latex particle solution with a concentration of 5 mg / L. The density of the Fe₃O₄-polystyrene microspheres is 1.35 g / cm 3 ³, and the particle size is 5 - 15 μm.

[0173] Add 25 μL of serum sample and 20 μL of latex particle solution into the well column of the microcolumn gel card, incubate at 35 °C for 30 min, and then centrifuge at 600 g for 25 min to observe and judge the test results.

[0174] Comparative Example 1

[0175] Compared with Example 1, the gel buffer of Comparative Example 1 is prepared from the following substances at the final concentration with PBS buffer: 184 mM glycerol, 71 mM sodium hyaluronate, 34.5 mM alkyl polyglycoside, and other conditions are the same as those in Example 1.

[0176] Comparative Example 2

[0177] Compared with Example 1, the gel buffer of Comparative Example 2 is prepared from the following substances at the final concentration with PBS buffer: 198 mM glycerol, 85.5 mM sodium hyaluronate, 6 mM potassium coco-hydrolyzed oat protein, and other conditions are the same as those in Example 1.

[0178] Comparative Example 3

[0179] Compared with Example 1, the gel buffer of Comparative Example 3 is prepared from the following substances at the final concentration with PBS buffer: 213 mM glycerol, 63.5 mM alkyl polyglycoside, 13 mM potassium coco-hydrolyzed oat protein, and other conditions are the same as those in Example 1.

[0180] Comparative Example 4

[0181] Compared with Example 1, the gel buffer of Comparative Example 4 is prepared from the following substances at the final concentration with PBS buffer: 170.5 mM sodium hyaluronate, 113.5 mM alkyl polyglycoside, 5.5 mM potassium coco-hydrolyzed oat protein, and other conditions are the same as those in Example 1.

[0182] Comparative Example 5

[0183] Compared with Example 1, the latex particles in Comparative Example 5 are polystyrene particles without Fe₃O₄, and the density is 1.05 g / cm3 , with other conditions being the same as those in Example 1.

[0184] Test results

[0185] Figure 1A and Figure 1B As shown in the test results of Example 1, the patient's serum contains anti-streptolysin O antibody. The latex particles undergo antigen-antibody immune binding reaction and aggregate at the top of the pore column to form a precipitation line. The healthy serum does not contain anti-streptolysin O antibody, and the latex particles all precipitate at the bottom of the pore column through the gaps between the gel microspheres, without negative trailing phenomenon.

[0186] Figure 2A and Figure 2B As shown in the test results of Example 2, the patient's serum contains anti-rheumatoid factor antibody. The latex particles undergo antigen-antibody immune binding reaction and aggregate at the top of the pore column to form a precipitation line. The healthy serum does not contain anti-rheumatoid factor antibody, and the latex particles all precipitate at the bottom of the pore column through the gaps between the gel microspheres, without negative trailing phenomenon.

[0187] Figure 3A and Figure 3B As shown in the test results of Example 3, they are consistent with the test results of Example 1. Figure 4A and Figure 4B As shown in the test results of Example 4, they are consistent with the test results of Example 2.

[0188] Figure 5A and Figure 5B As shown in the test results of Example 5, after adding the latex particle solution and the serum sample, the incubation step was omitted, resulting in a negative trailing phenomenon compared with the test results of Examples 1 - 4. This shows that incubation is beneficial for the latex particles to pass through the gaps between the gel microspheres, facilitating direct visual observation and determination of the results, avoiding the occurrence of negative trailing phenomenon, and thus ensuring the accuracy of the results.

[0189] Figure 6A and Figure 6B As shown in the test results of Example 6, gel microspheres with a diameter range of 40 - 70 μm were used. The gaps between the gel microspheres were too small. Under the action of centrifugal force, individual latex microspheres could not pass through the gaps between the gel microspheres and were thus trapped inside the gel microspheres, causing negative trailing; the test results of Example 7 and the test results of Example 8 are generally consistent with the test results of Example 1 or 2.

[0190] Figure 7A and Figure 7B As shown in the test results of Example 9, Figure 8A and Figure 8BThe detection results of Example 10 are shown. The concentrations of various substances in the gel buffer slightly affect the lubricity of the gel buffer. Under negative conditions, part of the latex particles precipitate at the bottom of the pore column, showing a negative trailing phenomenon.

[0191] Figure 9A The detection results of Comparative Example 1 are shown. Figure 9B The detection results of Comparative Example 2 are shown. Figure 9C The detection results of Comparative Example 3 are shown. Figure 9D The detection results of Comparative Example 4 are shown. The gel buffers of Comparative Examples 1, 2, 3, and 4 lack glycerol, sodium hyaluronate, alkyl glycoside, and potassium coco-hydrolyzed oat protein components respectively, which significantly affect the lubricity of the gel buffer. Under negative conditions, the latex particles cannot completely precipitate at the bottom of the pore column after centrifugation, showing an obvious trailing phenomenon.

[0192] Figure 10 The detection results of Comparative Example 5 are shown. The density of the latex particles is small. Under negative conditions, the latex particles cannot completely precipitate at the bottom of the pore column after centrifugation, showing an obvious trailing phenomenon.

[0193] Clinical sample detection results

[0194] Seventy-two clinically confirmed positive sera of anti-streptolysin O antibody were taken and detected using the detection kit of Example 1. Clear precipitation lines appeared when the serum titer was 1 / 40 - 1 / 80. The critical value of the detection method of the present invention is 1 / 80, which is the lowest detection limit and also serves as the positive quality control product of this method, meeting the requirements of true and reliable clinical test data.

[0195] One hundred and two normal human sera were taken and detected using the detection kit of Example 1. The serum titer was 1 / 40, and no precipitation lines were found in the detection results, with a coincidence rate of 100%.

[0196] The applicant declares that the present invention uses the above examples to illustrate the detailed method of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A latex immunoassay kit based on a microcolumn gel card, characterized in that, It includes gel microspheres, gel buffer solution and latex particle solution; The gel microspheres and the gel buffer solution are filled in the pore columns of a microcolumn gel card; The gel microspheres are cross-linked dextran microspheres; The latex particle solution is a PBS buffer solution containing latex particles, and the surface of the latex particles is coated with an antigen; The gel buffer solution is prepared from the following substances in a final concentration by PBS buffer solution: Glycerol 100 - 200 mM Sodium hyaluronate 50 - 100 mM Alkyl glycoside 20 - 50 mM Potassium coco-hydrolyzed oat protein 1 - 5 mM; The molecular weight of the sodium hyaluronate is 10,000 - 50,000 Da.

2. The detection kit according to claim 1, wherein The gel buffer solution is prepared from the following substances in a final concentration by PBS buffer solution: Glycerol 150 - 200 mM Sodium hyaluronate 60 - 75 mM Alkyl glycoside 30 - 40 mM Potassium coco-hydrolyzed oat protein 2 - 3.5 mM; The molecular weight of the sodium hyaluronate is 10,000 - 50,000 Da.

3. The detection kit according to claim 1, wherein The pH of the gel buffer solution is 7.2 - 7.

4.

4. The detection kit according to claim 1, wherein The particle size of the gel microspheres is 50 - 120 μm; The water absorption multiple of the gel microspheres is 8 - 12 mL / g.

5. The detection kit according to claim 1, characterized in that The latex particles are polystyrene particles containing iron oxide; The density of the latex particles is 1.1~1.5 g / cm 3 ; The particle size of the latex particles is 5 - 15 μm; The concentration of the latex particle solution is 3 - 10 mg / L; The surface of the latex particles is modified with a fluorescent label.

6. A method for using the detection kit according to any one of claims 1-5, characterized in that, It includes: Adding the latex particle solution and a serum sample into the pore columns of a microcolumn gel card pre-filled with gel microspheres and gel buffer solution; After centrifugation, observing the microcolumn gel card to judge the detection result of the serum sample.

7. The usage method according to claim 6, characterized in that, The volume ratio of the gel microspheres and the gel buffer solution filled in the pore columns of the microcolumn gel card is (8 - 10):3; The volume of the gel microspheres and the gel buffer solution filled in the pore columns of the microcolumn gel card is 20 - 35 μL; The volume of the latex particle solution filled in the pore columns of the microcolumn gel card is 20 - 30 μL; The volume of the serum sample filled in the pore columns of the microcolumn gel card is 25 - 35 μL.

8. The usage method according to claim 6, characterized in that, Before centrifugation, it includes an incubation step; The temperature of the incubation is 35 - 40 °C; The time of the incubation is 15 - 30 min.

9. The usage method according to claim 6, characterized in that, The centrifugal force of the centrifugation is 600 - 1500 g; The time of the centrifugation is 15 - 25 min.

10. Use of the detection kit according to any one of claims 1 - 5 in detecting anti-streptolysin O or rheumatoid factor.

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