A Helicobacter pylori antigen colloidal gold detection kit and its application

Abstract

The present invention discloses a Helicobacter pylori antigen colloidal gold detection kit and its application. The detection kit includes a test strip, which comprises a bottom plate, and a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad arranged in sequence from the sample adding end on the surface of the bottom plate. The gold label pad contains a monoclonal antibody 1 against Helicobacter pylori labeled with colloidal gold, and the nitrocellulose membrane is coated with a detection line and a quality control line; the sample pad contains sodium alkylphenol polyoxyethylene ether sulfosuccinate and a copolymer of propylene oxide, ethylene oxide and vinyl diamine. On the one hand, the present invention can effectively increase the hygroscopicity of the glass fiber membrane and ensure the upward chromatography of the liquid; on the other hand, it can increase the sensitivity of the product, especially can improve the sensitivity when detecting fecal samples and saliva samples at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and particularly relates to a Helicobacter pylori antigen colloidal gold detection kit and its application. Background Art

[0002] Helicobacter pylori (HP) is the main pathogenic bacterium of upper gastrointestinal diseases. Its infection is not only closely related to the occurrence of type B gastritis, peptic ulcer, MALT lymphoma and gastric cancer, but also importantly related to the occurrence of diseases outside the gastrointestinal tract. It has been listed by the World Health Organization as a related pathogenic bacterium for the occurrence of malignant tumors such as gastric cancer [1-8]. The infection rate of HP in the general population in China is relatively high, reaching 50-80%, and it is still increasing at a rate of 1-2% per year. The number of newly infected cases exceeds 12 million per year. Therefore, the diagnosis and eradication treatment of HP have attracted increasing attention.

[0003] At present, the diagnostic methods for Helicobacter pylori infection include biochemical experiment detection, morphological detection, isolation and culture detection, immunological detection, nucleic acid detection and other methods. Among them, immunological detection is further divided into the method of detecting antigens and the method of serologically detecting antibodies. The detection of Helicobacter pylori antigen is mainly for fecal samples; there are a small number of literature reports on saliva, and Helicobacter pylori can be detected in saliva, and these detection reagents are all for single sample detection. At present, the detection methods for Helicobacter pylori antigen in feces include immunochromatographic assay kits, immunofluorescence assay kits and immunolacquer assay kits.

[0004] Colloidal gold labeling technology was developed in the 1980s of the last century. In 1971, Faulk and Taylor introduced colloidal gold into immunochemistry. Since then, this labeling method has been widely used in many fields such as drug detection and biomedicine. At present, there are about hundreds of commercial colloidal gold kits sold at home and abroad, such as the most common early pregnancy test strip, gonorrhea detection kit, etc. Colloidal gold rapid detection technology is a new detection technology, and it has been applied to many fields such as the detection of agricultural and veterinary drug residues, clinical disease detection, drug detection, etc. With the wide application, this technology has gradually tended to be mature. This project plans to use antibodies against Hp antigen as coating sources, and biological raw materials such as goat anti-mouse IgG as detection lines and quality control lines to be coated on carriers such as nitrocellulose membranes; together with biological raw materials such as gold-labeled anti-Hp monoclonal antibodies that have a competitive relationship with the coated antibodies and sample pads, filter papers and other reagents coated on polyester membranes to form a product detection reagent strip. This test strip needs to qualitatively detect the content of Helicobacter pylori in human feces within a determined threshold range.

[0005] For example, Chinese authorized patent CN104897892B discloses a Helicobacter pylori antigen colloidal gold detection kit. The detection kit is used to detect Helicobacter pylori antigen in a sample, and the sample is specifically human feces. The detection kit includes a test strip, which includes a base plate, and a sample pad, a gold label pad, a nitrocellulose membrane, and a water absorption pad arranged in sequence from the sample adding end on the surface of the base plate. The gold label pad contains a monoclonal antibody against Helicobacter pylori labeled with colloidal gold - 1. The nitrocellulose membrane is coated with a test line and a quality control line; the gold label pad is pretreated with a buffer solution, and the buffer solution is glycine buffer solution; the components added in the glycine buffer solution are NaCl and crown ether, the crown ether is 18 - crown - 6, the concentration of NaCl in the buffer solution is 0.25 - 0.5mg / ml, and the concentration of crown ether in the buffer solution is 4 - 8mg / ml.

[0006] During the detection process, first dilute the fecal specimen, and then add the diluted sample to the sample adding hole position of the product. The sample passes through the glass fiber pad and then chromatographs on the nitrocellulose membrane together with the gold - labeled antibody. If the sample does not contain the target substance, the gold - labeled antibody will chromatograph on the nitrocellulose membrane along with the solution and will not form an antibody - target substance - antibody sandwich with the antibody coated at the T - line position on the nitrocellulose membrane, so it cannot be captured by the T - line and thus cannot form a red band in the T - line area. If the sample contains the target substance, the target substance will first bind to the gold - labeled antibody and then chromatograph on the nitrocellulose membrane together. When chromatographing to the T - line area, the target position will bind to the antibody coated at the T - line position, thus forming a gold - labeled antibody - target substance - coated antibody sandwich conjugate, and thus forming a red band at the T - line position, which is a positive result.

[0007] However, the current colloidal gold test strips for detecting Helicobacter pylori antigen have low sensitivity; for home use, sometimes they cannot detect Helicobacter pylori in the sample and cannot play a good warning role. In addition, the current colloidal gold test strips for detecting Helicobacter pylori antigen usually only target a single fecal sample, and there are few reports on colloidal gold test strips for detecting Helicobacter pylori antigen that can simultaneously detect fecal and saliva samples. Summary of the Invention

[0008] The purpose of the present invention is to provide a colloidal gold test strip for detecting Helicobacter pylori antigen and its application to improve the detection sensitivity and be applicable to both fecal samples and saliva samples simultaneously. To achieve the purpose of the present invention, the following technical solutions are proposed:

[0009] The present invention provides a Helicobacter pylori antigen colloidal gold detection kit. The detection kit includes a test strip, and the test strip includes a base plate, and a sample pad, a gold conjugate pad, a nitrocellulose membrane, and a water absorption pad arranged in sequence from the sample adding end on the surface of the base plate. The gold conjugate pad contains a colloidal gold-labeled anti-Helicobacter pylori monoclonal antibody-1, and the nitrocellulose membrane is coated with a test line and a quality control line; the sample pad contains sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and a copolymer of propylene oxide-ethylene oxide-vinyl diamine.

[0010] There is no particular limitation on the sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester of the present invention. For example, it can be a surfactant such as emulsifier A103 / emulsifier YC103 and other single components with the content of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester being greater than 90%.

[0011] There is also no particular limitation on the copolymer of propylene oxide-ethylene oxide-vinyl diamine of the present invention. For example, it can be Tetronic 1307, Pluronic (poloxamer) 407 or Pluronic188.

[0012] In a preferred embodiment of the present invention, the sample pad is obtained by a treatment solution containing sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and a copolymer of propylene oxide-ethylene oxide-vinyl diamine. Among them, every 100 parts of the treatment solution contains 0.1 part - 1 part of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and 1 part - 1.5 parts of the copolymer of propylene oxide-ethylene oxide-vinyl diamine. It should be noted that this proportional range is the optimal range obtained through an orthogonal experiment on the amount added in each percentage solution of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and the copolymer of propylene oxide-ethylene oxide-vinyl diamine.

[0013] In a preferred embodiment of the present invention, the average particle size of the colloidal gold is 80 - 120 nm.

[0014] In a preferred embodiment of the present invention, the kit further contains a sample diluent. Every 100 parts of the diluent contains 0.5 - 0.7 parts of sodium dihydrogen phosphate, 6 - 10 parts of sodium chloride, 0.1 - 0.3 parts of potassium dihydrogen phosphate, and 0.01 - 0.03 parts of Proclin 300 for solution preparation, and the pH is adjusted to 7.5 - 8.5.

[0015] In a preferred embodiment of the present invention, the test strip is a lateral flow test strip or a vertical flow test strip; preferably a vertical flow test strip, and the kit is a reagent cup.

[0016] On the other hand, the present invention also relates to the application of the above kit in the preparation of a detection reagent for detecting Helicobacter pylori.

[0017] In a preferred embodiment of the present invention, the test sample of the detection reagent is feces and / or saliva.

[0018] The beneficial effects of the present invention are as follows: The sample pad of the present invention contains a combination of sodium alkylphenol polyoxyethylene ether sulfosuccinate and a surfactant of propylene oxide-ethylene oxide-vinyl diamine copolymer. On the one hand, it can effectively increase the hygroscopicity of the glass fiber membrane and ensure the upward chromatography of the liquid; on the other hand, it can increase the sensitivity of the product, especially can improve the sensitivity when detecting fecal samples and saliva samples simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic structural diagram of an embodiment of the present invention;

[0020] Figure 2 is an exploded view of an embodiment of the present invention;

[0021] Figure 3 is a front view of an embodiment of the present invention;

[0022] Figure 4 is Figure 3 the A-A sectional view of

[0023] Figure 5 is a schematic diagram of a partial structure of an embodiment of the present invention;

[0024] Figure 6 is Figure 5 a schematic structural diagram from another angle. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels.

[0027] Example 1:

[0028] Preparation process of the test strip:

[0029] 1 Preparation of colloidal gold

[0030] The colloidal gold prepared by this technology is a colloidal gold sol prepared by the sodium citrate reduction method, with a particle diameter of 100 nm. It has a uniform size and can effectively improve the binding of colloidal gold and HP-labeled antibodies. The purified water or water used in this step refers to ultrapure water.

[0031] (1) Prepare 2.5 ml of an aqueous solution of chloroauric acid at 0.1 g / ml and store it in the dark for later use.

[0032] (2) Prepare 6 ml of an aqueous solution of sodium citrate at 0.1 g / ml and store it in the dark for later use.

[0033] (3) Add water in an amount equal to the volume of the colloidal gold solution to be prepared to the beaker, e.g., 1 L of purified water; then place the beaker on a magnetic stirrer and heat it to boiling.

[0034] (4) Add 2 parts of the aqueous solution of chloroauric acid in (1) (volume ratio, V∶V) to every 100 parts of purified water while stirring magnetically and heating to boiling, and add the solution in (1) to the beaker; then add 1.5 parts of the solution in (2) to every 100 parts of the aqueous solution to the beaker with magnetic stirring and boiling to allow the two solutions to react, and continue stirring for 5 minutes.

[0035] (5) After stopping heating, wait for the solution to cool to room temperature. Use this solution as the primer solution for the next reaction.

[0036] (6) Add the solution and purified water to the beaker in an amount of 50 parts of the above primer solution to every 100 parts of purified water.

[0037] (7) Place it on a magnetic stirrer, stir and heat at the same time. When the temperature rises to 90 °C, add the solution in (1) to the beaker in an amount of 5 parts to every 100 parts of the solution, and continue stirring and heating. When the temperature rises to 100 °C, add the solution in (2) to the above beaker in an amount of 3 parts to every 100 parts of the solution for reaction. Time for 4 minutes, then immediately stop heating and continue stirring for 15 minutes.

[0038] (8) Then transfer the prepared colloidal gold solution to a cold water bath and cool it to room temperature while stirring. Obtain a 100-nm colloidal gold solution.

[0039] (9) Stop heating, and then under stirring conditions, let the reacted colloidal gold solution cool to room temperature, 2 - 8 °C, and store it in the dark for later use.

[0040] Labeling of 2HP-labeled antibody

[0041] The direct labeling method is adopted in this technology. That is, at room temperature, the commercially available anti-HP antibody is directly reacted with the colloidal gold solution, and then the required gold-labeled product is obtained by centrifugation. The specific method is as follows:

[0042] 2.1 Determination of the labeled pH:

[0043] (1) Take 1 ml of the colloidal gold solution prepared in 1 and put it into each of 10 EP tubes. Then, adjust the pH of the above colloidal gold solution to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 respectively with 0.1 M potassium carbonate and make marks. Then, add the HP-labeled antibody at a rate of 10 μg / mL respectively and vortex to mix evenly.

[0044] (2) Let the above solutions stand for 10 minutes, and add 10% sodium chloride aqueous solution into each EP tube at a rate of 100 μl / tube, and vortex to mix evenly.

[0045] (3) Let it stand for 15 minutes and observe the color change.

[0046] (4) Select the pH of the colloidal gold solution tube without precipitation, turning purple or black as the optimal labeled pH; that is, the pH of the solution that just does not change color and remains wine red is the optimal pH. That is, pH 8.0

[0047] 2.2 Determination of the labeled amount

[0048] (1) Take 1 ml of the colloidal gold solution prepared in 1 and put it into each of 10 EP tubes. Then, adjust the pH of the above colloidal gold solution to 7.5 respectively with 0.1 M potassium carbonate and make marks. Then, add the HP-labeled antibody at rates of 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 14 μg / mL, 16 μg / mL, 18 μg / mL, and 20 μg / mL respectively and vortex to mix evenly.

[0049] (2) Let it stand for 10 minutes, and then add BSA aqueous solution (0.85 parts of BSA per ten parts of water) into each tube at a rate of 24 μL / mL, and vortex to mix evenly.

[0050] (3) Let it stand for 15 minutes, and then add sodium chloride aqueous solution (1.5 parts of sodium chloride per ten parts of water) into the above tubes at a rate of 100 μL / mL, and vortex to mix evenly.

[0051] (4) Let it stand for 2 hours, and observe the sedimentation and color change in each tube. Select the amount of protein in the tube of the colloidal gold solution without precipitation, turning purple or black as the optimal labeled amount; that is, the amount of protein in the solution that just does not change color and remains wine red is the optimal labeled amount. That is, 8 μg / mL.

[0052] 2.3 Colloidal gold labeling of HP-labeled antibody

[0053] (1) Measure the colloidal gold solution according to the amount of one part of antibody per hundred parts of the colloidal gold solution. For example, take 100 ml of the colloidal gold solution and place it in a beaker.

[0054] (2) Adjust the pH of the colloidal gold solution to 8.0 with 0.1 M potassium carbonate solution. And place it on a magnetic stirrer and stir.

[0055] (3) Dilute the concentration of the HP-labeled antibody to 1 mg / ml with 2 mM Tris buffer solution at pH 7.4. Then take 0.8 ml of the diluted protein solution and add it to the colloidal gold solution. Add the above solution while stirring.

[0056] (4) Preparation of Tris buffer solution: Weigh Tris according to the amount of 2.2 parts of Tris per hundred parts of water, place it in a beaker, add 9 parts of water, then adjust the pH to 7.4 with 6 M hydrochloric acid solution, and then make up the volume to the required volume with purified water to obtain it.

[0057] (5) Stir on a magnetic stirrer for 25 minutes;

[0058] (6) Add 8.5% BSA aqueous solution to 100 ml of the above solution at a rate of 24 μl / ml, and continue to stir for 45 minutes.

[0059] (7) Transfer the above reacted solution to a centrifuge tube; at 4 °C, centrifuge at a speed of 8000 - 15000 rpm / min, for example, a centrifugation speed of 12000 rpm / min, and centrifuge for 35 minutes; discard the supernatant.

[0060] (8) Re-dissolve the precipitate with 2 mM Tris buffer solution containing 1 part per hundred parts, and the labeled gold-labeled HP antibody is obtained.

[0061] 2.4 Preparation of sample pad

[0062] 2.4.1 Preparation of sample pad treatment solution

[0063] (1) Weigh borax according to the amount of 4 parts of borax per hundred parts, and put it into a beaker. Then add 80 parts of water to the beaker, and place the beaker on a magnetic stirrer and stir.

[0064] (2) Add 0.3 part of ethylenediaminetetraacetate to the above beaker and continue to stir;

[0065] (3) Add 0.5 part of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester (commercial emulsifier YC103) to the above solution and continue to stir;

[0066] (4) Add 1 part of propylene oxide-ethylene oxide-vinyl diamine copolymer (commercially available Tetronic 1307) to the above solution, and continue stirring;

[0067] (5) Add 2 parts of Proclin 300 per ten thousand parts to the above solution, and continue stirring;

[0068] (6) Add 0.5 part of Tween 20 per ten thousand parts to the above solution, and continue stirring.

[0069] (7) After the solution is completely dissolved and clarified, adjust the pH of the solution to 8.2 with hydrochloric acid or sodium hydroxide, and then make up the volume to the required volume with purified water, and set aside at room temperature.

[0070] 2.4.2 Preparation of sample pad

[0071] (1) Prepare a commercially available glass fiber pad (length * width: 300 mm * 254 mm);

[0072] (2) Soak the glass fiber pad at a rate of 1 piece per hundred parts; first soak the glass fiber pad in the 2.4.1 solution for 2 minutes, then take it out and gently filter off the excess water with a glass rod. Turn it over, soak the glass fiber pad in the solution again for 2 minutes, and gently filter off the excess water with a glass rod. The sample pad is obtained.

[0073] (3) Put the obtained sample pad into an oven at 37 °C and dry it overnight (time: 17 - 20 hours).

[0074] (4) After drying, the sample pad is stored in a sealed manner and placed in a dry environment.

[0075] 2.5 Preparation of sheet

[0076] (1) Paste the nitrocellulose membrane on the PVC bottom plate;

[0077] (2) Preparation of coating solution:

[0078] (3) Detection line: Dilute the HP-coated antibody to 1 mg / ml with 0.01 M PB, and prepare the amount of the detection line at a rate of 2 μl / cm per piece.

[0079] (4) Control line: Dilute goat anti-mouse IgG to 0.5 mg / ml with 0.01 M PB, and prepare the amount of the control line at a rate of 2 μl / cm per piece.

[0080] (5) Coating: Coat the solution on the nitrocellulose membrane with a membrane coating agent.

[0081] (6) Put the coated sheet into an oven at 37 °C and dry it overnight.

[0082] (7) The dried sheet is stored airtight and placed in a dry place.

[0083] 2.6 Preparation of the gold-labeled strip

[0084] (1) Dilute the gold-labeled solution prepared in 2.3 with 0.01M PB buffer containing 1 part bovine serum albumin and 20 parts sucrose per 100 parts. The dilution ratio is 46 parts of the gold-labeled solution and 54 parts of the above 0.01M PB solution per 100 parts of the solution.

[0085] (2) Spray the diluted solution onto the substrate pad with a gold-labeling machine at a rate of 60 μl per strip. The substrate pad for spraying is a glass fiber pad (length * width: 300 mm * 84 mm).

[0086] (3) After spraying, place the gold-labeled strip in an oven at 37 °C and dry overnight.

[0087] (4) The dried gold-labeled strip is sealed in a self-sealing bag and then stored in a dry environment away from light.

[0088] 2.7 Assembly of the test strip

[0089] (1) Cut the prepared gold-labeled strip into small strips with a length of 300 mm and a width of 8 mm, and paste it on the lower edge of the nitrocellulose membrane of the sheet, pressing 1 - 2 mm over the nitrocellulose membrane;

[0090] (2) Cut the sample pad into small strips with a length of 300 mm and a width of 20 mm, and paste it on the lower end of the sheet; the lower edge of the sample pad extends 3 mm beyond the lower edge of the sheet, and the upper edge of the sample pad extends over the white edge of the gold-labeled strip.

[0091] (3) Cut the absorbent filter paper into small strips with a length of 300 mm and a width of 17 mm, align the upper edge of the absorbent filter paper with the upper edge of the sheet, and the lower edge presses about 2 mm over the nitrocellulose membrane.

[0092] (4) Press the assembled reagent card tightly. Then cut it into small strips with a width of 3.0 mm with a cutting machine.

[0093] (5) Preparation of the sample diluent: For every 100 parts of water, add 0.6 parts of sodium dihydrogen phosphate, 8 parts of sodium chloride, 0.2 parts of potassium dihydrogen phosphate, and 2 parts of Proclin 300 per 10,000 parts of water for solution preparation, and adjust the pH of the solution to 8.0.

[0094] Place the above test strip in the detection cup in the following examples:

[0095] As Figures 1-6As shown, the test cup includes a cup lid 1, a cup body 2, and a cup bottom 3. The cup lid and the cup bottom are respectively installed at both ends of the cup body, and the cup bottom is fixedly connected to the cup body. The cup lid 1 is used to seal the cup mouth of the cup body 2 in a threaded connection manner. A sampling rod for collecting samples is connected to the cup lid, and the sampling rod 4 can extend into the cup body. The cup body 2 is a hollow structure with openings at both ends, and the inner diameter of the cup body 2 gradually decreases from the large end to the small end, that is, the cup body 2 of the test cup is a conical cup with a narrow upper part and a wide lower part, which solves the problem that the existing integrated tube is prone to falling during the detection process, thus ensuring the stable effect of the detection result.

[0096] A vertical baffle 21 is provided inside the cup body 2, and both the left and right sides of the vertical baffle 21 are fixedly connected to the cup body 2 in a sealed manner; an upper spacer 22 is provided at the upper opening of the cup body 2, and a lower spacer 23 is provided at the lower opening of the cup body 2. Both the upper spacer 22 and the lower spacer 23 are fixedly connected to the cup body 2 in a sealed manner, that is, the upper side edge of the vertical baffle 21 is fixedly connected to the upper spacer 22 seamlessly, and the lower side edge of the vertical baffle 21 is fixedly connected to the lower spacer 23 seamlessly. The vertical baffle 21 divides the internal space of the cup body 2 into a collection and processing area and a detection area, and the collection and processing area and the detection area are respectively located on both sides of the vertical baffle 21. In other words, the inside of the test cup is divided into two independent chambers, a first chamber 11 corresponding to the collection and processing area, and a second chamber 12 corresponding to the detection area. At the same time, a communication chamber 10 is provided between the lower spacer 23 and the cup bottom, and the communication chamber 10 is connected to the detection area. An inner groove with a certain depth is formed on the cup bottom, and the cup bottom is fixedly connected to the cup body 2 in a sealed manner, so as to form a communication chamber 10 between the cup bottom, the cup body 2, and the lower spacer 23.

[0097] One end of the sampling rod 4 is fixedly connected to the cup lid, and the sampling rod 4 is connected to the middle position of the cup lid. The free end of the sampling rod 4 is a tip with spiral threads. The sampling rod 4 is used to collect fecal samples and penetrate the upper spacer 22 to extend into the collection and processing area, where a sample processing solution is placed. The upper spacer can be an integral sheet pad structure or composed of two pieces. When the upper spacer is a single piece, the periphery of the upper spacer is hermetically and fixedly connected to the cup body, and one end of the vertical baffle is hermetically and fixedly connected to the upper spacer, thereby dividing the upper spacer into two parts. When the upper spacer is composed of two pieces, one side of the first patch and the second patch are both hermetically and fixedly connected to the vertical baffle, and the other sides of the first patch and the second patch are respectively hermetically and fixedly connected to the cup body on both sides of the vertical baffle, but the material of the first spacer corresponding to the collection and processing area requires good sealing performance. A collection hole can be opened on the upper spacer 22 corresponding to the collection and processing area for inserting the collection rod. At this time, in order to prevent the processing solution from flowing out, an elastic sealing ring is provided at the collection hole, and the sealing ring is fixedly connected to the upper spacer. It is also possible not to preset a collection hole on the upper spacer 22 and open the hole on the upper spacer when needed. No matter which method is adopted, it is necessary to meet the requirement that it is convenient for the sampling rod 4 to be inserted into the collection and processing area inside the cup body 2. In addition, in order to ensure the chromatography effect of the test strip, an air flow hole 9 is opened on the upper spacer corresponding to the detection area, and the air flow hole is a through hole.

[0098] The vertical baffle 21 is located on one side of the central axis of the cup body 2, that is, the vertical baffle 21 is not located at the central axis of the cup body 2. This is to ensure that the vertical baffle 21 does not obstruct the sampling rod 4 on the cup lid 1 when the cup lid 1 is covered. A card slot 5 is provided on the side of the vertical baffle 21 close to the detection area, and a test strip 6 is installed in the card slot. The test strip 6 can be firmly clamped in the card slot. At the same time, the end of the test strip 6 extends to the communication cavity 10 and abuts against the bottom of the cup. In order to visually observe the test result, the cup body 2 is designed to be transparent, or at least the side of the cup body facing the test strip is transparent.

[0099] A raised part is provided at the position of the lower spacer 23 corresponding to the collection and processing area. At the same time, the length of the sampling rod 4 is set to be greater than the distance between the upper spacer 22 and the lower spacer 23, so as to facilitate the user to more easily pierce the lower spacer 23. The raised part is made of thin-layer polystyrene material.

[0100] A chuck 7 is provided on the cup lid, and a limiting structure is provided at the end of the cup body 2 where the upper spacer 22 is located, such as Figure 5As shown, the chuck 7 cooperates with the limiting structure, so as to control that when the cup lid is screwed to a certain position, the cup lid will be stuck in the limiting structure and will not continue to move downwards. Only by applying an external force to break the limiting structure can the bottle cap continue to be pressed downwards. In this embodiment, the limiting structure is a limiting groove 8, and the limiting groove at least includes a first section groove, a second section groove and a third section groove. When the detection has not started, the cup lid 1 is stuck in the first section groove, and at this time the sampling rod does not contact the lower spacer. There is a bump on the second section groove. Under the action of an external force, the bump is broken by the chuck 7 on the cup lid 1, so that the chuck 7 can freely slide through the second section groove, and then press the cup lid 1 downwards. The cup lid 1 falls along the third section groove, driving the sampling rod 4 to pierce the lower spacer 23 downwards.

[0101] 2.8 Fecal sample detection method:

[0102] Use the above detection cup for detection: Dip 0.05g - 0.5g of feces (i.e., feces the size of a soybean) with a sampling rod, then insert the sampling rod under the liquid level in the collection and processing area of the detection cup, stir it 3 - 5 times, and then tightly screw the cup lid. As the cup lid is tightened, the sampling rod moves downwards to the protrusion at the bottom, and its tip will pierce the protrusion. This allows the liquid to enter the microchannel formed by the first and second pieces of the base. Under the action of gravity and pressure difference, the liquid flows to the detection area and contacts the extremely hygroscopic sample pad through the opening of the first piece, so as to be absorbed onto the sample pad. The liquid undergoes chromatography upwards under the influence of capillary action through the sample pad. After passing through the gold label pad, the chromatography proceeds to the detection area on the nitrocellulose membrane, thereby realizing the detection of Helicobacter pylori. Observe the results and time for 10 minutes to observe the results.

[0103] 2.9 Saliva sample detection method:

[0104] Use the above detection cup for detection: Take 200uL of saliva with a graduated pipette and add it to the sample diluent in the detection area of the detection cup. Forcefully cover the lid. As the cup lid is tightened, the sampling rod moves downwards to the protrusion at the bottom, and its tip will pierce the protrusion. This allows the liquid to enter the microchannel formed by the first and second pieces of the base. Under the action of gravity and pressure difference, the liquid flows to the detection area and contacts the extremely hygroscopic sample pad through the opening of the first piece, so as to be absorbed onto the sample pad. The liquid undergoes chromatography upwards under the influence of capillary action through the sample pad. Observe the results and time for 10 minutes to observe the results.

[0105] Example 2:

[0106] Preparation process of the test strip:

[0107] 1. The preparation process of colloidal gold and the labeling process of HP antibody are the same as those in Example 1. Refer to Example 1.

[0108] 2. Preparation of the sample pad

[0109] 2.1 Preparation of Sample Pad Treatment Solution

[0110] (1) Weigh borax in an amount of 4 parts per hundred parts, put it into a beaker, then add 80 parts of water into the beaker, and place the beaker on a magnetic stirrer and stir.

[0111] (2) Add 0.3 part of ethylenediaminetetraacetate to the above beaker and continue stirring;

[0112] (3) Add 1 part of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester (commercially available emulsifier A103) to the above solution and continue stirring;

[0113] (4) Add 1 part of propylene oxide-ethylene oxide-vinyl diamine copolymer (commercially available Tetronic 1307) to the above solution and continue stirring;

[0114] (5) Add 2 parts of Proclin 300 per ten thousand parts to the above solution and continue stirring;

[0115] (6) Add 0.5 part of Tween 20 per ten thousand parts to the above solution and continue stirring.

[0116] (7) After the solution is completely dissolved and clarified, adjust the pH of the solution to 8.2 with hydrochloric acid or sodium hydroxide, and then make up the volume to the required volume with purified water and set aside at room temperature.

[0117] 2.2 Preparation of Sample Pad

[0118] (1) Prepare a commercially available glass fiber pad (length * width: 300 mm * 254 mm);

[0119] (2) Soak the glass fiber pad in an amount of 1 piece per hundred parts; first soak the glass fiber pad in the 2.1 solution for 2 minutes, then take it out and gently filter off the excess water with a glass rod. Turn it over and soak the glass fiber pad in the solution again for 2 minutes, and gently filter off the excess water with a glass rod. The sample pad is obtained.

[0120] (3) Put the obtained sample pad into an oven at 37 °C and dry it overnight (time: 17 - 20 hours).

[0121] (4) After drying, the sample pad is sealed and stored in a dry environment.

[0122] 3. Preparation of Sheet: Refer to the preparation of 2.5 sheet in Example 1.

[0123] 4. Preparation of Gold Label Strip: Refer to the preparation of 2.6 gold label strip in Example 1.

[0124] 5. Assembly of the test strip: Refer to the assembly of the test strip in 2.7 of Example 1.

[0125] 6. Testing

[0126] For the testing of fecal samples, refer to 2.8 in Example 1. For the testing of saliva samples, refer to 2.9 in Example 1.

[0127] Example 3

[0128] Preparation process of the test strip:

[0129] 1. The preparation process of colloidal gold and the labeling process of HP antibody are the same as those in Example 1. Refer to Example 1.

[0130] 2. Preparation of the sample pad

[0131] 2.1 Preparation of the sample pad treatment solution

[0132] (1) Weigh borax according to the amount of 4 parts per 100 parts, and put it into a beaker. Then add 80 parts of water into the beaker, and place the beaker on a magnetic stirrer and stir.

[0133] (2) Add 0.3 parts of ethylenediaminetetraacetate to the above beaker and continue stirring;

[0134] (3) According to the amount of adding 0.5 g of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester (commercial emulsifier A103) to 100 mL of solution, weigh 0.5 g of the above substance and add it to the solution in the beaker, and continue stirring;

[0135] (4) According to the amount of adding 1 g of propylene oxide-ethylene oxide-vinyl diamine copolymer (commercial Pluronic188) to 100 mL of solution, weigh 1 g of the above substance and add it to the solution in the beaker, and continue stirring;

[0136] (5) Add 2 parts per 10,000 parts of Proclin 300 to the above solution and continue stirring;

[0137] (6) Add 0.5 parts per 10,000 parts of Tween20 to the above solution and continue stirring.

[0138] (7) After the solution is completely dissolved and clarified, adjust the pH of the solution to 8.2 with hydrochloric acid or sodium hydroxide, and then make up the volume to the required volume with purified water and keep it at room temperature for standby.

[0139] 2.2 Preparation of the sample pad

[0140] (1) Prepare a commercially available glass fiber pad (length * width: 300 mm * 254 mm);

[0141] (2) Soak the glass fiber pad at a rate of 1 piece per 100 parts. First, soak the glass fiber pad in Solution 2.1 for 2 minutes, then take it out and gently filter off the excess moisture with a glass rod. Turn it over, soak the glass fiber pad in the solution again for 2 minutes, and gently filter off the excess moisture with a glass rod. The sample pad is thus obtained.

[0142] (3) Put the obtained sample pad into an oven at 37 °C and dry it overnight (for 17 - 20 hours).

[0143] (4) After drying, store the sample pad airtight and place it in a dry environment.

[0144] 3. Preparation of the sheet: Refer to the preparation of the sheet in 2.5 of Example 1.

[0145] 4. Preparation of the gold-labeled strip: Refer to the preparation of the gold-labeled strip in 2.6 of Example 1.

[0146] 5. Assembly of the test strip: Refer to the assembly of the test strip in 2.7 of Example 1.

[0147] 6. Testing

[0148] For the testing of fecal samples, refer to 2.8 in Example 1. For the testing of saliva samples, refer to 2.9 in Example 1.

[0149] Comparative Example 1:

[0150] It is the same as Example 1, except that the sample pad contains a propylene oxide - ethylene oxide - vinyl diamine copolymer and does not contain sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester.

[0151] Comparative Example 2:

[0152] It is the same as Example 1, except that the sample pad contains sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and does not contain a propylene oxide - ethylene oxide - vinyl diamine copolymer.

[0153] Comparison of the detection effects of the three sample pads

[0154] Prepare test strips by using three sample pads containing two kinds of surface active components (Sample Pad 1 of Example 1), a propylene oxide - ethylene oxide - vinyl diamine copolymer (Sample Pad 2 of Comparative Example 1), and sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester (Sample Pad 3 of Comparative Example 2) together with the gold label, sheet, absorbent paper, etc. prepared according to the method in 2, and conduct tests. The comparison test results of the three types of sample pads for cultures and fecal samples are as follows:

[0155]

[0156]

[0157] Note: "-" indicates negative; "+" indicates positive, and the positive degree increases from +→++++; "+" indicates weakly positive.

[0158] From the above results, it can be seen that the test strip prepared with the sample pad containing only the copolymer of propylene oxide - ethylene oxide - vinyl diamine has low sensitivity; while the sample pad containing only sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester also has low sensitivity and is prone to false positives. After the two are used in combination, the false positive phenomenon is eliminated and the sensitivity of the product is enhanced.

[0159] Comparison of test results of clinical saliva samples for three sample pads

[0160]

[0161] Result detection and comparison with commercially available reagents

[0162] The reagent of the present invention and a commercially available kit were used for comparative testing, and Helicobacter pylori bacterial cultures and real clinical samples were detected. The test results are as follows:

[0163]

[0164]

[0165] Comparison results of fecal sample detection

[0166] Note: "-" indicates negative; "+" indicates positive, and the positive degree increases from +→++++; "+" indicates weakly positive.

[0167] Saliva sample

[0168] Saliva sample detection method: Use a graduated pipette to take 200 μL of saliva and add it to the sample diluent in the detection area of the detection cup. Observe the results and time for 10 minutes to observe the results. The results are shown in the following table. It should be noted that since no commercially available colloidal gold immunochromatographic reagent for detecting saliva products was found, no comparative analysis was carried out.

[0169]

[0170]

[0171] Detection results of Example 2 and comparison with commercially available results

[0172] Fecal sample detection results

[0173]

[0174]

[0175] Saliva sample detection results

[0176]

[0177] Detection results of Example 3 and comparison results with commercially available reagents

[0178] Detection results of fecal samples

[0179]

[0180]

[0181] Detection results of saliva samples

[0182]

[0183] From the above detection results, it can be seen that the present invention has a high detection sensitivity for Helicobacter pylori antigen in feces and saliva.

[0184] Example 4:

[0185] Using the test strip of the present invention, fecal samples were detected respectively according to horizontal chromatography and vertical chromatography, and the comparison of the detection results is as follows:

[0186] Thus, it can be seen that the test strips prepared in Examples 1 - 3 of this patent can be placed in a kit for horizontal lateral chromatography, and can also be placed in a test reagent cup for vertical chromatography. However, for the test strip of this application, it is more suitable for vertical chromatography. For test strips under the same conditions, the color development of vertical chromatography is slightly stronger than that of horizontal chromatography.

[0187] The preferred embodiments of the present invention have been described above, but they are not intended to limit the present invention. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A kit for detecting Helicobacter pylori antigen by colloidal gold, the kit comprising a test strip, the test strip including a base plate, and a sample pad, a gold label pad, a nitrocellulose membrane and an absorbent pad arranged in sequence from the sample adding end on the surface of the base plate, the gold label pad containing a colloidal gold-labeled anti-Helicobacter pylori monoclonal antibody, and the nitrocellulose membrane being coated with a test line and a quality control line; the sample pad containing sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and a copolymer of propylene oxide-ethylene oxide-vinyl diamine.

2. The kit according to claim 1, wherein the sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester is selected from emulsifier A103 and / or emulsifier YC103.

3. The kit according to claim 1, wherein the copolymer of propylene oxide-ethylene oxide-vinyl diamine is Tetronic 1307.

4. The kit according to any one of claims 1-3, wherein the sample pad is obtained by treating with a treatment liquid containing sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and a copolymer of propylene oxide-ethylene oxide-vinyl diamine, wherein, every 100 parts of the treatment liquid contains 0.1 part - 1 part of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester and 1 part - 1.5 parts of the copolymer of propylene oxide-ethylene oxide-vinyl diamine.

5. The kit according to claim 1, wherein the average particle size of the colloidal gold is 80 - 120 nm.

6. The kit according to claim 1, wherein the kit further contains a sample diluent, and every 100 parts of the diluent contains 0.5 - 0.7 part of sodium dihydrogen phosphate, 6 - 10 parts of sodium chloride, 0.1 - 0.3 part of potassium dihydrogen phosphate and 0.01 - 0.03 part of Proclin 300 for solution preparation, and the pH is adjusted to 7.5 - 8.

5.

7. The kit according to claim 1, wherein the test strip is a lateral flow test strip or a vertical flow test strip.

8. Use of the kit according to any one of claims 1-7 in the preparation of a detection reagent for detecting Helicobacter pylori.

9. The use according to claim 8, wherein the detection sample of the detection reagent is feces and / or saliva.

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