Candida trichomonas vaginalis gardnerella vaginalis three-in-one antigen diagnostic reagent and kit

Abstract

The invention discloses a candida trichomonas vaginalis gardnerella vaginalis three-in-one antigen diagnostic reagent and a kit. A detection card and a reagent bottle filled with sample treatment liquid are arranged in the kit body; a candida detection test strip, a trichomonas vaginalis detection test strip and a gardnerella vaginalis detection test strip are arranged in a card body of the detection card; the candida detection test strip, the trichomonas vaginalis detection test strip and the gardnerella vaginalis detection test strip are respectively provided with an integrated sample pad treated by an integrated sample pad / conjugate pad treating fluid, a conjugate pad treated by the integrated sample pad / conjugate pad treating fluid, a nitrocellulose membrane and absorbent paper along the chromatography direction. The sample treatment liquid comprises 3-[3-(cholamidopropyl) dimethylamino] propanesulfonic acid inner salt and alginate oligosaccharide; the integrated sample pad / conjugate pad treating fluid comprises bovine serum albumin, lentinan and sodium laureth sulfate; the kit has the advantages of high sensitivity, high specificity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, in particular to a three-in-one antigen diagnostic reagent and kit for Candida vaginalis, Trichomonas vaginalis and Gardnerella vaginalis. Background Art

[0002] Vaginal infection is a common gynecological disease, among which bacterial vaginosis, candida albicans vaginitis and trichomoniasis are all common vaginal infections. Candida infection usually refers to candida albicans infection, which is a fungal infection. Candida albicans is one of the common flora in the vagina, but under certain conditions, such as decreased immunity, long-term use of antibiotics or changes in hormone levels, candida may overgrow and cause candidal vaginitis. The main symptoms of candidal vaginitis include vaginal itching, burning sensation, and increased discharge, which is usually cheese-like or curd-like. Vaginal Trichomonas infection, also known as Trichomonas vaginitis, is a sexually transmitted disease caused by Trichomonas vaginalis. Symptoms of Trichomonas vaginitis include increased vaginal discharge, which is purulent or foamy, and may be accompanied by bloody vaginal discharge, dysuria, frequent urination, hematuria, etc. Vaginal Gardnerella infection, also known as bacterial vaginitis, is a vaginal inflammation mainly caused by anaerobic bacteria such as Gardnerella. Symptoms of Gardnerella vaginitis include increased vaginal discharge, which is thin and light yellow with a distinct fishy odor, itching, redness and swelling of the vulva, etc.

[0003] Existing methods for detecting vaginal secretions mainly include leucorrhea appearance and pH detection, microscopic examination, culture method, biochemical enzymatic method, immunological method and nucleic acid detection method. Microscopic examination is simple but has low sensitivity; isolation and culture have high accuracy but are complicated and time-consuming; nucleic acid detection methods are too expensive, and immunofluorescence tests, enzyme immunoassays and nucleic acid detection methods are rarely used because they require corresponding instruments and equipment and the technical requirements of operators. In addition, the composition of vaginal secretions is complex and diverse, and interfering substances in the secretions will affect the detection. Therefore, in response to the problems existing in the existing technology, a three-in-one antigen diagnostic reagent and kit for Candida vaginalis and Trichomonas vaginalis is provided. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a three-in-one diagnostic reagent and kit for Candida vaginalis and Trichomonas vaginalis to solve the problems raised in the above technical background.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides a three-in-one antigen diagnostic reagent for Candida vaginalis and Trichomonas vaginalis, comprising a sample processing solution and an integrated sample pad / conjugate pad processing solution, wherein the sample processing solution comprises: 3.36-16.81 g / L citric acid, 10-100 g / L sodium citrate, 0.1-1 g / L 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (chaps), 0.5-1.5 g / L brown algae oligosaccharide, 5-20 g / L lauryl alcohol polyoxyethylene ether, 0.5-2 mL / L Proclin-300, and the pH is adjusted to 4.5-7.8.

[0006] Preferably, the integrated sample pad / conjugate pad treatment solution contains 4.32g~43.2g / L disodium hydrogen phosphate dodecahydrate, 1.12g~11.2g / L sodium dihydrogen phosphate dihydrate, 1.00~10.0g / L bovine serum albumin, 1.00~5.00g / L lentinan, 1.00mL~10.0mL / L sodium lauryl polyether sulfate, and the pH is adjusted to 7.5~8.5.

[0007] In a second aspect, the present invention provides a test kit, wherein a test card and a reagent bottle containing the above-mentioned sample processing liquid are provided in the test kit box body; the test card includes a card cover and a card body, and the card body is provided with a Candida detection test strip, a Trichomonas vaginalis detection test strip, and a Gardnerella vaginalis detection test strip, and the Candida detection test strip, the Trichomonas vaginalis detection strip, and the Gardnerella vaginalis detection strip comprise a plastic base, an integrated sample pad treated with an integrated sample pad / conjugation pad processing liquid, a conjugation pad treated with an integrated sample pad / conjugation pad processing liquid, a nitrocellulose membrane, and blotting paper, and an integrated sample pad treated with the above-mentioned integrated sample pad / conjugation pad processing liquid, a conjugation pad treated with the above-mentioned integrated sample pad / conjugation pad processing liquid, a nitrocellulose membrane, and blotting paper are provided on the plastic base along the chromatography direction.

[0008] A plastic base is preferred for supporting and bonding the various components of the immunochromatographic test strips. Both PVC (polyvinyl chloride) and PS (polystyrene) bases are suitable. Base thickness: 0.2mm-0.4mm; base width: 6cm-8cm.

[0009] Optimally, the integrated sample pad is made of fiberglass film, which has a smooth surface, uniform fiber distribution, and consistent thickness. The thickness is 0.19mm to 0.5mm; the crawling speed is 15 to 50mm / 60s; and the water storage capacity is 400g to 800g / m 2 It acts as a sample buffer in most chromatography processes and is also used in this product to immobilize nanoparticle-coupled antibodies.

[0010] Preferably, the connection between the integrated sample pad and the conjugate pad (marked in purple) is fixed with: In the Candida test strip, the junction between the integrated sample pad and the conjugate pad (marked in purple) is fixed with: red polystyrene nanoparticles coupled to rabbit anti-Candida polyclonal antibody 1 (abbreviated as: R-COO-Rabbit Pab 1 to Candida) and blue polystyrene nanoparticles coupled to mouse anti-genital mucosal antigen factor monoclonal antibody 1 (B-COO-Mouse Mab 1 to GMAF). In the described Trichomonas vaginalis test strip, the junction between the integrated sample pad and the binding pad (marked in purple) is fixed with: red polystyrene nanoparticles coupled to mouse anti-Trichomonas vaginalis monoclonal antibody 1 (abbreviated as: R-COO-Mouse Mab 1 to Trichomonas) and blue polystyrene nanoparticles coupled to mouse anti-genital mucosal antigen factor monoclonal antibody 1 (B-COO-Mouse Mab 1 to GMAF). In the vaginal Gardnerella test strip, the junction between the integrated sample pad and the binding pad (marked in purple) is fixed with: red polystyrene nanoparticles coupled to mouse anti-vaginal Gardnerella monoclonal antibody 1 (abbreviated as: R-COO-Mouse Mab 1 to Gardnerella) and blue polystyrene nanoparticles coupled to mouse anti-genital mucosal antigen factor monoclonal antibody 1 (B-COO-Mouse Mab 1 to GMAF). The preferred material for the binding pad is generally polyester cellulose film. Polyester cellulose film is formed using a specific process and, after special treatment, has excellent hydrophilicity, uniform fiber distribution, and fine texture. The fiber membrane is biologically inert and does not affect the normal reaction of biologically active substances. Thickness: 0.13mm~0.6mm; Climbing speed: 10s~40s / 4cm; Water storage capacity: 100g~700g / m 2 .

[0011] Preferably, the binding pad (marked in light blue) is fixed with: In the Candida test strip, the conjugate pad (marked by the light blue area) is fixed with: biotin molecular particles labeled rabbit anti-Candida polyclonal antibody 2 (abbreviated as: Biotin-Rabbit Pab 2 to Candida) In the Trichomonas vaginalis test strip, the binding pad (marked by the light blue area) is fixed with: biotin molecular particles labeled mouse anti-Trichomonas vaginalis monoclonal antibody 2 (abbreviated as: Biotin-Mouse Mab 2 to Trichomonas) In the vaginal Gardnerella detection test strip, the binding pad (marked by the light blue area) is fixed with: mouse anti-vaginal Gardnerella monoclonal antibody 2 labeled with biotin molecular particles (abbreviated as: Biotin-Mouse Mab 2 to Gardnerella).

[0012] The preferred nitrocellulose membrane (NC membrane) is made from organic chemical cellulose nitrate particles. Surfactants, polymers, salt ions, and forming agents are added to the mixture, which is dissolved to form a homogenate. This homogenate is then passed through a roller and pressed to form a thin film. This type of membrane is called nitrocellulose membrane. This membrane is a key consumable in biological experiments and is widely used in the production of immunochromatographic diagnostic test strips, serving as a carrier for C / T lines and as the site of immune reactions. Thickness ranges from 95µm to 225µm, and the creep speed is 90-170s / 4cm.

[0013] Preferably, the nitrocellulose membranes of the Candida detection test strip, the Trichomonas vaginalis detection test strip, and the Gardnerella vaginalis detection test strip are all provided with a detection line T line and a quality control line C line; the T line region of the nitrocellulose membrane is fixedly coated with streptavidin-SA, and the C line region is fixedly coated with mouse anti-genital mucosal antigen factor monoclonal antibody 2 (Mouse Mab 2 to GMAF).

[0014] Preferably, the absorbent paper is made of pure plant fiber through a special process. It has good water absorption capacity without chemical additives. The thickness is 0.35mm~2.3mm, the crawling speed is 4s~180s / 4cm, and the water storage capacity is 400~2400g / m 2 .

[0015] Preferably, the areas of the card cover corresponding to the Candida detection test strip, the Trichomonas vaginalis detection test strip, and the Gardnerella vaginalis detection test strip are respectively provided with a sample addition hole and an observation hole.

[0016] In a third aspect, the present invention provides a method for preparing a three-in-one diagnostic kit for Candida vaginalis and Trichomonas vaginalis, comprising the following steps: 1. Liquid preparation Activation coupling buffer (0.01-0.1 mol / L, pH 5.0-6.5, 100 mL): Weigh 0.195-1.95 g of morpholineethanesulfonic acid into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, adjust the pH to 5.0-6.5, and dilute to 100 mL.

[0017] Wash buffer (0.01-0.1 mol / L, pH 7.0-8.5, 100 mL): Weigh 0.121 g to 1.211 g of tris(hydroxymethyl)aminomethane into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, then add 0.05 mL to 0.5 mL of polysorbate 20 and 0.01 mL to 0.1 mL of sodium lauryl ether sulfate, adjust the pH to 7.0-8.5, and make up to 100 mL.

[0018] Blocking buffer (0.001-0.01 mol / L, pH 8.0-8.5, 100 mL): Weigh 0.012 g to 0.121 g of tris(hydroxymethyl)aminomethane into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, then weigh 5 g to 10 g of bovine serum albumin and 0.059 g to 0.585 g of lysine, adjust the pH to 8.0-8.5, and make up to 100 mL.

[0019] Nanosphere particle storage solution (0.02-0.05 mol / L, pH 7.5-8.0, 100 mL): Weigh 0.242 g to 2.422 g of tris(hydroxymethyl)aminomethane into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, then weigh and add 0.2 g to 0.8 g of polyethylene glycol 8000, 1 g to 5 g of bovine serum albumin, and 10 g to 20 g of sucrose, and weigh 0.02 mL to 0.2 mL of Proclin-300. Adjust the pH to 7.5-8.0 and make up to 100 mL.

[0020] Coating buffer (0.01mol / L~0.08mol / L, pH 7.0~8.0, 100mL): Weigh 0.591~4.728g disodium hydrogen phosphate dodecahydrate, 0.078~0.624g sodium dihydrogen phosphate dihydrate, 0.05~0.5g dextran, and 0.45~0.9g sodium chloride in a container of appropriate specifications. Dissolve in 90mL of ultrapure water. Add 0.02mL~0.2mL Proclin-300, adjust the pH to 7.0~8.0, and make up to 100mL.

[0021] Integrated sample pad / conjugate pad treatment solution (250 mL): Weigh 1.08g-10.8g disodium hydrogen phosphate dodecahydrate, 0.28g-2.8g sodium dihydrogen phosphate dihydrate, 0.25g-2.5g bovine serum albumin, and 0.25g-1.25g lentinan into a container of appropriate specifications. Dissolve in 200mL of ultrapure water. Add 0.25mL-2.5mL sodium laureth sulfate, adjust the pH to 7.5-8.5, and dilute to 250mL.

[0022] Phosphate buffer (100 mL): Weigh 2.33 g–23.3 g of disodium hydrogen phosphate dodecahydrate, 0.23 g–2.33 g of sodium dihydrogen phosphate dihydrate, and 0.878 g–8.78 g of sodium chloride. Dissolve in 90 mL of ultrapure water in a container of appropriate specifications. Adjust the pH to 6.5–8.0 and dilute to 100 mL.

[0023] Sample treatment solution (1000 mL): Weigh 3.36g-16.81g citric acid, 10g-100g sodium citrate, 0.1g-1g 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (chaps), 0.5-1.5g fucoidan, and 5g-20g lauryl alcohol polyoxyethylene ether into a container of appropriate specifications. Dissolve in 900mL of ultrapure water. Add 0.5mL-2mL Proclin-300, adjust the pH to 4.5-7.8, and make up to 1000mL.

[0024] 2. Preparation of integrated sample pad and conjugate pad 2.1 Integrated sample pad Place a glass fiber membrane (SB06 / 8964 / JG888) flat on a clean gauze. Measure 30-50 mL of the integrated sample pad / conjugate pad treatment solution and evenly blow it onto the glass fiber membrane. Roll the membrane back and forth with a roller 10-20 times to ensure that the liquid is fully and evenly distributed on the glass fiber membrane. Transfer the membrane to a drying room at 25-42°C and humidity ≤30% and dry it for 24-48 hours.

[0025] 2.2 Conjugate pad preparation Place a polyester fiber membrane (VL78 / 6613 / W75) flat on a clean gauze. Measure 40-60 mL of the integrated sample pad / conjugate pad treatment solution and evenly pipette it onto the polyester fiber membrane. Roll the membrane back and forth with a roller 10-20 times to ensure that the liquid is fully and evenly distributed on the polyester fiber membrane. Transfer the membrane to a drying room at 25-42°C and humidity ≤30% and dry it for 24-48 hours.

[0026] 3. Conjugation of Antibodies to NHS-Biotin 3.1 Biotin-Rabbit Pab 2 to Candida 3.1.1 Pipette 1 mg of rabbit anti-Candida polyclonal antibody 2 and dilute it with 0.5 mL to 5 mL of phosphate buffer. 3.1.2 Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffer. 3.1.3 Add 0.05 mL to 0.2 mL of biotin solution to the antibody solution, mix thoroughly, and incubate at 30-37°C for 1-4 hours. 3.1.4 Add the reaction solution to an ultrafiltration centrifuge tube and centrifuge in a refrigerated centrifuge at 4000-10000g for 10-25 minutes at 2-8°C. 3.1.5 Measure the volume of the liquid after centrifugation and dilute it to 0.1-0.5 mg / ml with phosphate buffer for later use.

[0027] 3.2 Biotin-Mouse Mab2 to Trichomonas 3.2.1 Pipette 1 mg of mouse anti-Trichomonas vaginalis monoclonal antibody 2 and dilute it with 0.5 mL to 5 mL of phosphate buffered saline; 3.2.2 Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffer. 3.2.3 Add 0.05 mL to 0.2 mL of biotin solution to the antibody solution, mix thoroughly, and incubate at 30-37°C for 1-4 hours. 3.2.4 Add the reaction solution to an ultrafiltration centrifuge tube and centrifuge in a refrigerated centrifuge at 4000-10000g for 10-25 minutes at 2-8°C. 3.2.5 Measure the volume of the liquid after centrifugation and dilute it to 0.1-0.5 mg / ml with phosphate buffer for later use.

[0028] 3.3 Preparation of Biotin-Mouse Mab 2 to Gardnerella vaginalis 3.3.1 Pipette 1 mg of mouse anti-Gardnerella vaginalis monoclonal antibody 2 and dilute it with 0.5 mL to 5 mL of phosphate buffered saline. 3.3.2 Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffer. 3.3.3 Add 0.05 mL to 0.2 mL of biotin solution to the antibody solution, mix thoroughly, and incubate at 30-37°C for 1-4 hours. 3.3.4 Add the reaction solution to an ultrafiltration centrifuge tube and centrifuge in a refrigerated centrifuge at 4000-10000g for 10-25 minutes at 2-8°C. 3.3.5 Measure the volume of the liquid after centrifugation and dilute it to 0.1-0.5 mg / ml with phosphate buffer for later use.

[0029] 4. Red polystyrene nanoparticles (R-COOH) coupled to antibody solution 4.1 Preparation of Red Polystyrene Nanoparticles Conjugated to Rabbit Anti-Candida Polyclonal Antibody 1 (R-COO-Rabbit Pab 1 to Candida) 4.1.1 Weigh 20 mg of carbodiimide solid. 4.1.2 Add 4 mL of activation coupling buffer to fully dissolve the peptide to a concentration of 5 mg / mL. 4.1.3 Take 2 mg of red polystyrene nanospheres and add 0.89 mL to 0.95 mL of activation coupling buffer.

[0030] 4.1.4 Add 0.01mL~0.06mL of EDC solution.

[0031] 4.1.5 After mixing, incubate at 25-37°C for 10-50 minutes. 4.1.6 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.1.7 Add 1 mL of washing buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.1.8 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.1.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.1.10 Add 50 μg to 400 μg of rabbit anti-Candida polyclonal antibody 1, mix manually, and place on a mixer at 25-37°C for 1 to 6 hours. 4.1.11 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.1.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.1.13 Mix the mixture on a mixer for 1 to 6 hours.

[0032] 4.1.14 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.1.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 5-20 minutes. Set aside.

[0033] 4.2 Preparation of Red Polystyrene Nanoparticles-Conjugated Mouse Anti-Trichomonas Vaginalis Monoclonal Antibody 1 (R-COO-Mouse Mab 1 to Trichomonas) 4.2.1 Weigh 20 mg of carbodiimide solid. 4.2.2 Add 4 mL of activation coupling buffer to fully dissolve the mixture to a concentration of 5 mg / mL. 4.2.3 Take 2 mg of red polystyrene nanospheres and add 0.89 mL to 0.95 mL of activation coupling buffer.

[0034] 4.2.4 Add 0.01mL~0.06mL of EDC solution.

[0035] 4.2.5 After mixing, incubate at 25-37°C for 10-50 minutes. 4.2.6 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.2.7 Add 1 mL of washing buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.2.8 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.2.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.2.10 Add 50 μg to 400 μg of mouse anti-Trichomonas vaginalis monoclonal antibody 1, mix manually, and place on a mixer at 25-37°C for 1 to 6 hours. 4.2.11 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.2.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.2.13 Mix the mixture on a mixer for 1 to 6 hours.

[0036] 4.2.14 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.2.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 5-20 minutes. Set aside.

[0037] 4.3 Preparation of Red Polystyrene Nanoparticles-Conjugated Mouse Anti-Gardnerella Vaginalis Monoclonal Antibody 1 (R-COO-Mouse Mab 1 to Gardnerella) 4.3.1 Weigh 20 mg of carbodiimide solid. 4.3.2 Add 4 mL of activation coupling buffer to fully dissolve the peptide to a concentration of 5 mg / mL. 4.3.3 Take 2 mg of red polystyrene nanospheres and add 0.89 mL to 0.95 mL of activation coupling buffer.

[0038] 4.3.4 Add 0.01mL~0.06mL of EDC solution.

[0039] 4.3.5 After mixing, incubate at 25-37°C for 10-50 minutes. 4.3.6 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.3.7 Add 1 mL of washing buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.3.8 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.3.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.3.10 Add 50 μg to 400 μg of mouse anti-Gardnerella vaginalis monoclonal antibody 1, mix manually, and place on a mixer at 25-37°C for 1 to 6 hours. 4.3.11 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.3.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.3.13 Mix the mixture on a mixer for 1 to 6 hours.

[0040] 4.3.14 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.3.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 5-20 minutes. Set aside.

[0041] 4.4 Preparation of Blue Polystyrene Nanoparticles Conjugated to Mouse Anti-German Mucosal Antigen Factor Monoclonal Antibody 1 (B-COO-Mouse Mab 1 to GMAF) 4.4.1 Weigh 20 mg of carbodiimide solid. 4.4.2 Add 4 mL of activation coupling buffer to fully dissolve the mixture to a concentration of 5 mg / mL. 4.4.3 Take 2 mg of blue polystyrene nanospheres and add 0.89 mL to 0.95 mL of activation coupling buffer.

[0042] 4.4.4 Add 0.01mL~0.06mL of EDC solution.

[0043] 4.4.5 After mixing, incubate at 25-37°C for 10-50 minutes. 4.4.6 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.4.7 Add 1 mL of washing buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.4.8 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.4.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.4.10 Add 50 μg to 400 μg of mouse anti-genital mucosal antigen factor monoclonal antibody 1, mix manually, and place on a mixer at 25-37°C for 1 to 6 hours. 4.4.11 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.4.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic bath for 5–20 minutes. 4.4.13 Mix the mixture on a mixer for 1 to 6 hours.

[0044] 4.4.14 Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. 4.4.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 5-20 minutes. Set aside.

[0045] 5. Preparation of Biotinylated Antibody-Conjugate Membrane Cut the bonding pad into strips of 6mm~10mm*300mm.

[0046] Candida: Spray biotin-labeled rabbit anti-Candida polyclonal antibody 2 (Biotin-Rabbit Pab2 to Candida) evenly onto the conjugate pad at a rate of 30-90 μL / strip. Dry for 2-4 hours at 20-30°C and humidity ≤30%.

[0047] Trichomonas vaginalis: Spray 30uL-90uL of biotin-labeled mouse anti-Trichomonas monoclonal antibody 2 solution evenly onto the conjugate pad. Dry for 2-4 hours at 20°C-30°C and humidity ≤30%.

[0048] Gardnerella vaginalis: Prepare biotin-labeled mouse anti-Gardnerella vaginalis monoclonal antibody 2 (Biotin-Mouse Mab 2 to Gardnerella). Spray the solution evenly onto the conjugate pad at a rate of 30uL-90uL per strip. Dry for 2-4 hours at a temperature of 20°C-30°C and a humidity of ≤30%.

[0049] 6. Preparation of Nanosphere Particle-labeled Conjugate Film 6.1 Preparation of Nanoparticle Working Solution 6.1.1 Preparation of Candida albicans Nanoparticles Working Solution: Mix the R-COO-Rabbit Pab 1 to Candida solution and the B-COO-Mouse Mab 1 to GMAF solution in a volume ratio of 1:1 to 1:4 and set aside.

[0050] 6.1.2 Preparation of Trichomonas vaginalis Nanoparticles Working Solution: Mix the R-COO-Mouse Mab 1 to Trichomonas solution and the B-COO-Mouse Mab 1 to GMAF solution in a volume ratio of 1:1 to 1:4 and set aside.

[0051] 6.1.3 Preparation of Gardnerella vaginalis Nanoparticles Working Solution: Mix the R-COO-Mouse Mab 1 to Gardnerella solution and the B-COO-Mouse Mab 1 to GMAF solution in a volume ratio of 1:1 to 1:4 and set aside.

[0052] 6.2 Immobilization of Nanosphere-Labeled Complexes Cut the integrated sample pad into strips of 18mm~22mm*300mm.

[0053] Candida: Spray the Candida nanosphere working solution evenly onto the integrated sample pad at a rate of 1.5 mL to 2.5 mL per strip. Dry for 4 to 8 hours at a temperature of 25°C to 42°C and a humidity of ≤30%.

[0054] Trichomonas vaginalis: Spray the Trichomonas vaginalis nanosphere working solution evenly onto the integrated sample pad at a concentration of 1.5 mL to 2.5 mL per strip. Dry for 4 to 8 hours at a temperature of 25°C to 42°C and a humidity of ≤30%.

[0055] Gardnerella vaginalis: Spray the working solution of Gardnerella vaginalis nanosphere particles evenly onto the integrated sample pad at a concentration of 1.5 mL to 2.5 mL per strip. Dry for 4 to 8 hours at a temperature of 25°C to 42°C and a humidity of ≤30%.

[0056] 7. Coating membrane preparation 7.1 Back plate-Nitrocellulose Membrane-absorbent: Bp-NC-Abs) Paste the cut nitrocellulose membrane (2.0cm~2.5cm*30cm) to the plastic base (6cm~8cm*30cm), and then affix absorbent paper (1.7cm~2.2cm*30cm).

[0057] 7.2 Preparation of membrane coating working solution Preparation of membrane coating working solution: Preparation of T-line membrane coating working solution: Take a certain amount of streptavidin and dilute it with coating buffer to 1mg / mL~2mg / mL.

[0058] Preparation of C-line membrane coating working solution: Take a certain amount of mouse genital mucosal antigen factor monoclonal antibody 2 (MouseMab 2 to GMAF) and dilute it to 1mg / mL~2mg / mL with coating buffer.

[0059] 7.3 Coating 7.3.1 Film Scraping Instrument Debugging Place the fixed nitrocellulose membrane plate (Bp-NC-Abs) on the work surface of the membrane stripper, set the T-line and C-line spray volume to 0.4uL / cm~1.2uL / cm, the speed to 25mm / s~200mm / s, and start the device to start membrane stripping.

[0060] 7.3.2 Drying Dry for 24 to 48 hours at a temperature of 25°C to 42°C and a humidity of ≤30%.

[0061] 8. Assembly Candida: The upper edge of the biotin-labeled antibody Biotin-Rabbit Mab 2 to Candida conjugate membrane is pressed 1 to 2.5 mm against the lower edge of the Candida-coated nitrocellulose membrane (NC membrane) and then pressed flush with the lower edge of the Candida nanosphere particle-labeled conjugate membrane against the plastic base, and marked on absorbent paper: VVC.

[0062] Trichomonas vaginalis: Press the upper edge of the biotin-labeled antibody Biotin-Mouse Mab 2 to Trichomonas conjugate membrane against the lower edge of the nitrocellulose membrane (NC membrane) of the Trichomonas vaginalis membrane 1 to 2.5 mm, and then press the lower edge of the Trichomonas vaginalis nanosphere particle labeled conjugate membrane flush with the plastic base and mark it on the absorbent paper: TV.

[0063] Gardnerella vaginalis: The upper edge of the biotin-mouse Mab 2 to Gardnerella conjugate membrane, labeled with biotin molecules, is pressed against the lower edge of the nitrocellulose membrane (NC membrane) coated with the Gardnerella vaginalis membrane 1 to 2.5 mm, and then the lower edge of the Gardnerella vaginalis nanosphere particle-labeled conjugate membrane is pressed flush with the plastic base plate and marked on absorbent paper: GV.

[0064] 9. Cut into strips Cut the assembled test strip sheet into strips with a width of 3.8~4.2mm.

[0065] 10. Card installation Place the qualified test strips into the corresponding card shells in turn, cover the card cover, fasten it, and press the shell.

[0066] Compared with the prior art, the present invention has the following beneficial effects: 1. The sample processing solution provided by the present invention, based on the pH buffering of citric acid buffer, improves the extraction efficiency of target analytes through the synergistic effect of 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt and brown algae oligosaccharides, thereby significantly improving the sensitivity and specificity of the kit. Among them, 3-[3-(cholamidopropyl)dimethylamino]3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (chaps), a zwitterionic detergent, can dissolve membrane proteins, stabilize various protein-DNA complexes, and retain the biological activity of proteins in solution; brown algae oligosaccharides prevent antigen degradation and deformation.

[0067] 2. The brown algal oligosaccharides contained in the sample processing solution provided by the present invention work synergistically with the lentinan in the integrated sample pad / conjugate pad processing solution to improve sensitivity. Lentinan effectively detects trace components by promoting antigen-antibody reactions or inhibiting nonspecific reactions.

[0068] The present invention provides a three-in-one diagnostic kit for Candida, Trichomonas, and Gardnerella vaginalis antigens, demonstrating both high sensitivity and specificity, enabling rapid detection of Candida, Trichomonas, and Gardnerella vaginalis antigens. Furthermore, the kit offers advantages such as rapid and easy operation, accurate results, and affordability. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is a product picture of the test card in the present invention; Figure 2 Schematic diagram of the structure of the test strip in the present invention; Figure 3 The sensitivity, specificity and efficacy bar graphs of Candida detected in Example 1 and Comparative Examples 1-4 are shown; Figure 4 The sensitivity, specificity and efficacy bar graphs of Trichomonas vaginalis detected in Example 1 and Comparative Examples 1-4 are shown; Figure 5 This is a bar graph of the sensitivity, specificity, and efficacy of Gardnerella vaginalis detected in Example 1 and Comparative Examples 1-4; Figure 6 It is a bar graph showing the detection sensitivity of Example 1, Comparative Example 2 and Comparative Example 5 to Candida antigen.

[0070] In the figure, 1. Plastic base plate; 2. Integrated sample pad; 3. Conjugate pad; 4. Nitrocellulose membrane; 5. Absorbent paper. DETAILED DESCRIPTION

[0071] The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0072] In the present invention, Antibody 1 and Antibody 2 represent two primary antibodies, or represent the antibodies used on the test strip conjugate pad and the detection line, respectively. For example, Rabbit Anti-Candida Polyclonal Antibody 1 and Rabbit Anti-Candida Polyclonal Antibody 2 are both rabbit anti-Candida polyclonal antibodies. They are only identified by the numbers 1 and 2 at the end for the purpose of distinction.

[0073] In the present invention, some of the raw materials are: NHS-biotin: Shanghai Yuanye Biotechnology Co., Ltd. (1 g / tube), rabbit anti-Candida polyclonal antibody 2: Leinco Technologies, Inc. (1 mg / tube), mouse anti-Trichomonas vaginalis monoclonal antibody 2: Leinco Technologies, Inc. (1 mg / tube), mouse anti-Gardnerella vaginalis monoclonal antibody 2: Bioss Inc. (1 mg / tube); Red polystyrene nanoparticles: BangsLabs (10 mg / ml), rabbit anti-Candida polyclonal antibody 1: Santa Cruz Biotechnology, Inc. (1 mg / tube), mouse anti-Trichomonas vaginalis monoclonal antibody 1: LeincoTechnologies, Inc. (1 mg / tube), mouse anti-Gardnerella vaginalis monoclonal antibody 1: Bioss Inc. (1 mg / tube); Blue polystyrene nanoparticles: BangsLabs (10 mg / ml), mouse anti-genital mucosal antigen factor monoclonal antibody 1: Holmesbio (1 mg / vial); Streptavidin: Novazon (1 mg / vial), mouse genital mucosal antigen factor monoclonal antibody 2: Holmesbio (1 mg / vial).

[0074] The present invention provides a three-in-one antigen diagnostic kit for Candida vaginalis and Trichomonas vaginalis Gardnerella vaginalis. The kit box body is provided with a test card and a reagent bottle containing a sample processing liquid. The test card comprises a card cover and a card body. The card body is provided with a Candida detection test strip, a Trichomonas vaginalis detection test strip, and a Gardnerella vaginalis detection test strip. The Candida detection test strip, the Trichomonas vaginalis detection strip, and the Gardnerella vaginalis detection strip comprise a plastic base plate 1, an integrated sample pad 2, a conjugation pad 3, a nitrocellulose membrane 4, and a blotting paper 5. The integrated sample pad 2 treated with an integrated sample pad / conjugation pad processing liquid, the conjugation pad 3 treated with an integrated sample pad / conjugation pad processing liquid, the nitrocellulose membrane 4, and the blotting paper 4 are all provided on the plastic base plate 1 along the chromatography direction. Figure 1 The card cover and the areas corresponding to the Candida test strip, the Trichomonas vaginalis test strip and the Gardnerella vaginalis test strip are respectively provided with a sample addition hole and an observation hole, as shown. Figure 2 shown.

[0075] Example 1

[0076] A preparation method of a three-in-one antigen diagnostic kit for Candida vaginalis Trichomonas vaginalis Gardnerella vaginalis, comprising 1. Liquid preparation Activation coupling buffer (0.01 mol / L, pH 5.5, 100 mL): Weigh 0.195 g of morpholineethanesulfonic acid into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, adjust the pH to 5.5, and dilute to 100 mL.

[0077] Wash buffer (0.01 mol / L, pH 7.4, 100 mL): Weigh 0.121 g of tris(hydroxymethyl)aminomethane into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, then add 0.05 mL of polysorbate 20 and 0.01 mL of sodium lauryl polyether sulfate, adjust the pH to 7.4, and make up to 100 mL.

[0078] Blocking buffer (0.001 mol / L, pH 8.2, 100 mL): Weigh 0.012 g of tris(hydroxymethyl)aminomethane into a container of appropriate specifications, dissolve in 90 mL of ultrapure water, then weigh and add 5 g of bovine serum albumin and 0.059 g of lysine, adjust the pH to 8.2, and make up to 100 mL.

[0079] Nanosphere particle storage solution (0.02 mol / L, pH 7.8, 100 mL): Weigh 0.242 g of tris(hydroxymethyl)aminomethane into a container of appropriate specifications, dissolve it in 90 mL of ultrapure water, then weigh and add 0.2 g of polyethylene glycol 8000, 1 g of bovine serum albumin, 10 g of sucrose, and 0.02 mL of Proclin-300. Adjust the pH to 7.8 and make up to 100 mL.

[0080] Coating buffer (0.01 mol / L, pH 7.4, 100 mL): Weigh 0.591 g of disodium hydrogen phosphate dodecahydrate, 0.078 g of sodium dihydrogen phosphate dihydrate, 0.05 g of dextran, and 0.45 g of sodium chloride in a container of appropriate specifications. Dissolve them in 90 mL of ultrapure water. Add 0.02 mL of Proclin-300, adjust the pH to 7.4, and make up to 100 mL.

[0081] Integrated sample pad / conjugate pad treatment solution (250 mL): Weigh 1.08 g disodium hydrogen phosphate dodecahydrate, 0.28 g sodium dihydrogen phosphate dihydrate, 0.25 g bovine serum albumin, and 0.25 g lentinan into a container of appropriate specifications. Dissolve them in 200 mL of ultrapure water. Then, add 0.25 mL of sodium laureth sulfate, adjust the pH to 7.8, and make up to 250 mL.

[0082] Phosphate buffer (100 mL): Weigh 2.33 g disodium hydrogen phosphate dodecahydrate, 0.23 g sodium dihydrogen phosphate dihydrate, and 0.878 g sodium chloride. Dissolve in 90 mL of ultrapure water in a container of appropriate specifications. Adjust the pH to 7.4 and make up to 100 mL.

[0083] Sample treatment solution (1000 mL): Weigh 3.36 g citric acid, 10 g sodium citrate, 0.1 g 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (chaps), 0.5 g brown algae oligosaccharide, and 5 g lauryl alcohol polyoxyethylene ether into a container of appropriate specifications. Dissolve in 900 mL of ultrapure water. Then add 0.5 mL of Proclin-300, adjust the pH to 6.8, and make up to 1000 mL.

[0084] Brown algae oligosaccharide, CAS: 9005-38-3, was purchased from Hunan Guangyi Biotechnology Co., Ltd.; Lentinan, CAS: 37339-90-5, was purchased from Xi'an Tianbao Biotechnology Co., Ltd.

[0085] 2. Preparation of integrated sample pad and conjugate pad 2.1 Integrated sample pad Place an SB06 glass fiber membrane flat on a clean gauze net. Measure 40 mL of the integrated sample pad / conjugate pad treatment solution and evenly blow it onto the glass fiber membrane. Roll the membrane back and forth 15 times with a roller to ensure that the liquid is fully and evenly distributed on the glass fiber membrane. Transfer the membrane to a drying room at 35°C and humidity ≤30% and dry it for 36 hours.

[0086] 2.2 Conjugate pad preparation Place a VL78 polyester fiber membrane flat on a clean gauze net. Measure 50 mL of the integrated sample pad / conjugate pad treatment solution and evenly pipette it onto the polyester fiber membrane. Roll the membrane back and forth 15 times with a roller to ensure that the liquid is fully and evenly distributed on the polyester fiber membrane. Transfer the membrane to a drying room at 35°C and humidity ≤30% and dry it for 36 hours.

[0087] 3. Conjugation of Antibodies to NHS-Biotin 3.1 Biotin-Rabbit Pab 2 to Candida 3.1.1 Pipette 1 mg of rabbit anti-Candida polyclonal antibody 2 and dilute it with 2.5 mL of phosphate buffered saline. 3.1.2 Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffered saline. 3.1.3 Add 0.5 mL of biotin solution to the antibody solution, mix thoroughly, and incubate the mixture at 37°C for 2 hours. 3.1.4 Add the reacted solution to an ultrafiltration centrifuge tube and centrifuge at 8000 g for 15 minutes at 4°C in a refrigerated centrifuge. 3.1.5 Determine the volume of the liquid after centrifugation and dilute it to 0.2 mg / mL with phosphate buffered saline for later use.

[0088] 3.2 Biotin-Mouse Mab2 to Trichomonas 3.2.1 Pipette 1 mg of mouse anti-Trichomonas vaginalis monoclonal antibody 2 and dilute it with 2.5 mL of phosphate buffered saline. 3.2.2 Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffered saline. 3.2.3 Add 0.5 mL of biotin solution to the antibody solution, mix thoroughly, and incubate the mixture at 37°C for 2 hours. 3.2.4 Add the reacted solution to an ultrafiltration centrifuge tube and centrifuge at 8000 g in a refrigerated centrifuge at 4°C for 15 minutes. 3.2.5 Determine the volume of the liquid after centrifugation and dilute it to 0.2 mg / mL with phosphate buffered saline for later use.

[0089] 3.3 Preparation of Biotin-Mouse Mab 2 to Gardnerella vaginalis 3.3.1 Pipette 1 mg of mouse anti-Gardnerella vaginalis monoclonal antibody 2 and dilute it with 2.5 mL of phosphate buffered saline. 3.3.2 Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffered saline. 3.3.3 Add 0.5 mL of biotin solution to the antibody solution, mix thoroughly, and incubate the mixture at 37°C for 2 hours. 3.3.4 Add the reacted solution to an ultrafiltration centrifuge tube and centrifuge at 8000 g in a refrigerated centrifuge at 4°C for 15 minutes. 3.3.5 Determine the volume of the liquid after centrifugation and dilute it to 0.2 mg / mL with phosphate buffered saline for later use.

[0090] 4. Red polystyrene nanoparticles (R-COOH) coupled to antibody solution 4.1 Preparation of Red Polystyrene Nanoparticles Conjugated to Rabbit Anti-Candida Polyclonal Antibody 1 (R-COO-Rabbit Pab 1 to Candida); 4.1.1 Weigh 20 mg of solid carbodiimide; 4.1.2 Dissolve thoroughly in 4 mL of activation coupling buffer to a concentration of 5 mg / mL; 4.1.3 Take 2 mg of red polystyrene nanoparticles and add 0.95 mL of activation coupling buffer. 4.1.4 Add 0.05 mL of EDC solution. 4.1.5 After mixing, incubate at 37°C for 30 min; 4.1.6 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.1.7 Add 1 mL of washing buffer and resuspend in an ultrasonic cleaner for 20 min; 4.1.8 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.1.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic cleaner for 20 min; 4.1.10 Add 1,200 μg of rabbit anti-Candida polyclonal antibody, mix manually, place on a mixer and incubate at 37°C for 4 h; 4.1.11 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.1.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic cleaner for 20 min; 4.1.13 Incubate on a mixer and incubate for 4 h. 4.1.14 Centrifuge at 12000 rpm, 4°C for 20 min and discard the supernatant. 4.1.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 20 min. Set aside.

[0091] 4.2 Preparation of Red Polystyrene Nanoparticles-Conjugated Mouse Anti-Trichomonas Vaginalis Monoclonal Antibody 1 (R-COO-Mouse Mab 1 to Trichomonas) 4.2.1 Weigh 20 mg of solid carbodiimide; 4.2.2 Add 4 mL of activation coupling buffer to fully dissolve to a concentration of 5 mg / mL; 4.2.3 Take 2 mg of red polystyrene nanospheres and add 0.95 mL of activation coupling buffer. 4.2.4 Add 0.05 mL of EDC solution. 4.2.5 After mixing, incubate at 37°C for 30 min; 4.2.6 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.2.7 Add 1 mL of washing buffer and resuspend in an ultrasonic cleaner for 20 min; 4.2.8 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.2.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic cleaner for 20 min; 4.2.10 Add 1,200 μg of mouse anti-Trichomonas vaginalis monoclonal antibody, mix manually, place on a mixer, and incubate at 37°C for 4 h; 4.2.11 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.2.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic cleaner for 20 min; 4.2.13 Incubate on a mixer for 4 h. 4.2.14 Centrifuge at 12000 rpm, 4°C for 20 min and discard the supernatant. 4.2.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 20 min. Set aside.

[0092] 4.3 Preparation of Red Polystyrene Nanoparticles-Conjugated Mouse Anti-Gardnerella Vaginalis Monoclonal Antibody 1 (R-COO-Mouse Mab 1 to Gardnerella) 4.3.1 Weigh 20 mg of carbodiimide solid; 4.3.2 Add 4 mL of activation coupling buffer to fully dissolve to a concentration of 5 mg / mL; 4.3.3 Take 2 mg of red polystyrene nanospheres and add 0.95 mL of activation coupling buffer. 4.3.4 Add 0.05 mL of EDC solution. 4.3.5 After mixing, incubate at 37°C for 30 min; 4.3.6 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.3.7 Add 1 mL of washing buffer and resuspend in an ultrasonic cleaner for 20 min; 4.3.8 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.3.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic cleaner for 20 min; 4.3.10 Add 1,200 μg of mouse anti-Gardnerella vaginalis monoclonal antibody, mix manually, place on a mixer, and incubate at 37°C for 4 h; 4.3.11 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.3.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic cleaner for 20 min; 4.3.13 Incubate on a mixer for 4 h. 4.3.14 Centrifuge at 12000 rpm, 4°C for 20 min and discard the supernatant. 4.3.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 20 min. Set aside.

[0093] 4.4 Preparation of Blue Polystyrene Nanoparticles Conjugated to Mouse Anti-German Mucosal Antigen Factor Monoclonal Antibody 1 (B-COO-Mouse Mab 1 to GMAF) 4.4.1 Weigh 20 mg of solid carbodiimide; 4.4.2 Add 4 mL of activation coupling buffer to fully dissolve to a concentration of 5 mg / mL; 4.4.3 Take 2 mg of blue polystyrene nanospheres and add 0.95 mL of activation coupling buffer. 4.4.4 Add 0.05 mL of EDC solution. 4.4.5 After mixing, incubate at 37°C for 30 min; 4.4.6 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.4.7 Add 1 mL of washing buffer and resuspend in an ultrasonic cleaner for 20 min; 4.4.8 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.4.9 Add 2 mL of activation coupling buffer and resuspend in an ultrasonic cleaner for 20 min; 4.4.10 Add 200 μg of mouse anti-genital mucosal antigen factor monoclonal antibody 1, mix manually, place on a mixer, and incubate at 37°C for 4 h; 4.4.11 Centrifuge at 12,000 rpm at 4°C for 20 min and discard the supernatant; 4.4.12 Add 1 mL of blocking buffer and resuspend in an ultrasonic cleaner for 20 min; 4.4.13 Incubate on a mixer for 4 h. 4.4.14 Centrifuge at 12000 rpm, 4°C for 20 min and discard the supernatant. 4.4.15 Add 1 mL of nanosphere storage solution and resuspend in an ultrasonic cleaner for 20 min. Set aside.

[0094] 5. Preparation of Biotinylated Antibody-Conjugate Membrane Cut the bonding pad into 8mm*300mm strips.

[0095] Candida: Spray 60 μL / strip of biotin-labeled rabbit anti-Candida polyclonal antibody 2 (Biotin-Rabbit Pab2 to Candida) onto the conjugate pad. Dry for 3 hours at 25°C and humidity ≤ 30%.

[0096] Trichomonas vaginalis: Spray 60 μL / strip of biotin-labeled mouse anti-Trichomonas monoclonal antibody 2 onto the conjugate pad. Dry for 3 hours at 25°C and humidity ≤ 30%.

[0097] Gardnerella vaginalis: Prepare biotin-labeled mouse anti-Gardnerella vaginalis monoclonal antibody 2 (Biotin-Mouse Mab 2 to Gardnerella). Spray 60 μL of the solution onto the conjugate pad evenly. Dry at 25°C and humidity ≤ 30% for 3 hours.

[0098] 6. Preparation of Nanosphere Particle-labeled Conjugate Film 6.1 Preparation of Nanoparticle Working Solution 6.1.1 Preparation of Candida albicans Nanoparticles Working Solution: Mix the R-COO-Rabbit Mab 1 to Candida solution and the B-COO-Mouse Mab 1 to GMAF solution in a volume ratio of 1:2 and set aside.

[0099] 6.1.2 Preparation of Trichomonas vaginalis Nanoparticles Working Solution: Mix the R-COO-Mouse Mab 1 to Trichomonas solution and the B-COO-Mouse Mab 1 to GMAF solution in a volume ratio of 1:2 and set aside.

[0100] 6.1.3 Preparation of Gardnerella vaginalis Nanoparticles Working Solution: Mix the R-COO-Mouse Mab 1 to Gardnerella solution and the B-COO-Mouse Mab 1 to GMAF solution in a volume ratio of 1:2 and set aside.

[0101] 6.2 Immobilization of Nanosphere-Labeled Complexes Cut the integrated sample pad into 20mm*300mm strips.

[0102] Candida: Spray the Candida nanosphere working solution evenly onto the integrated sample pad at a rate of 2 mL / strip; dry for 6 hours at 37°C and humidity ≤ 30%.

[0103] Trichomonas vaginalis: Spray the Trichomonas vaginalis nanosphere working solution evenly onto the integrated sample pad at a concentration of 2 mL / strip; dry for 6 hours at a temperature of 37°C and a humidity of ≤30%.

[0104] Gardnerella vaginalis: Spray the working solution of Gardnerella vaginalis nanosphere particles evenly onto the integrated sample pad at a concentration of 2 mL / strip; dry for 6 hours at a temperature of 37°C and a humidity of ≤30%.

[0105] 7. Coating membrane preparation 7.1 Back plate-Nitrocellulose Membrane-absorbent: Bp-NC-Abs) Paste the cut nitrocellulose membrane (2.4cm*30cm) to the plastic base (6.6cm*30cm), and then affix 2.0cm*30cm absorbent paper.

[0106] 7.2 Preparation of membrane coating working solution Preparation of membrane coating working solution: Preparation of T-line membrane coating working solution: Take a certain amount of streptavidin and dilute it to 1.5 mg / mL with coating buffer.

[0107] Preparation of C-line membrane coating working solution: Take a certain amount of mouse genital mucosal antigen factor monoclonal antibody 2 (MouseMab 2 to GMAF) and dilute it to 1.5 mg / mL with coating buffer.

[0108] 7.3 Coating 7.3.1 Film Scraping Instrument Debugging Place the fixed nitrocellulose membrane plate (Bp-NC-Abs) on the working surface of the membrane stripper, set the T-line and C-line spray volume to 0.8uL / cm and the speed to 100mm / s, and start the device to start membrane stripping.

[0109] 7.3.2 Drying Dry for 36 hours at a temperature of 35°C and a humidity of ≤30%.

[0110] 8. Assembly Candida: Press the upper edge of the biotin molecular particle-labeled antibody Biotin-Rabbit Mab 2 to Candida conjugate membrane 2 mm against the lower edge of the Candida-coated nitrocellulose membrane (NC membrane) and then press the lower edge of the Candida nanosphere particle-labeled conjugate membrane flush with the plastic base. Press the upper edge of the Candida nanosphere particle-labeled conjugate membrane against the lower edge of the biotin molecular particle-labeled antibody Biotin-Rabbit Mab 2 to Candida conjugate membrane and mark it on absorbent paper: VVC.

[0111] Trichomonas vaginalis: Press the upper edge of the biotin molecular particle-labeled antibody Biotin-Mouse Mab 2 to Trichomonas conjugate membrane 2 mm against the lower edge of the nitrocellulose membrane (NC membrane) coating the Trichomonas vaginalis membrane, then press the lower edge of the Trichomonas vaginalis nanomicrosphere particle-labeled conjugate membrane flush with the plastic base, and mark the upper edge of the Trichomonas vaginalis nanomicrosphere particle-labeled conjugate membrane with the biotin molecular particle-labeled antibody Biotin-Mouse Mab 2 to Trichomonas conjugate membrane, and mark it on absorbent paper: TV.

[0112] Gardnerella vaginalis: Press the upper edge of the biotin molecular particle-labeled antibody Biotin-Mouse Mab 2 toGardnerella conjugate membrane 2mm against the lower edge of the nitrocellulose membrane (NC membrane) coated with the Gardnerella vaginalis membrane, then press the lower edge of the Gardnerella vaginalis nanomicrosphere particle-labeled conjugate membrane flush with the plastic bottom plate, and press the upper edge of the Gardnerella vaginalis nanomicrosphere particle-labeled conjugate membrane with the biotin molecular particle-labeled antibody Biotin-Mouse Mab 2 toGardnerella conjugate membrane, and mark it on the absorbent paper: GV.

[0113] 9. Cut into strips Cut the assembled test strip sheet into strips with a width of 4.0 mm.

[0114] 10. Card installation Place the qualified test strips into the corresponding card shells in turn, cover the card cover, fasten it, and press the shell.

[0115] The detection principle of the kit of this embodiment: Polystyrene carboxyl nanoparticle immunochromatography technology and a biotin-streptavidin amplification system were used. Streptavidin (SA) was coated on the test line (T) of the nitrocellulose membrane, and mouse genital mucosal antigen factor monoclonal antibody 2 (Mouse Mab 2 to GMAF) was coated on the control line (C). Red polystyrene carboxyl nanoparticles-labeled Candida antibody 1 / Trichomonas vaginalis antibody 1 / Gardnerella vaginalis antibody 1 and blue polystyrene nanospheres-coupled mouse anti-genital mucosal antigen factor monoclonal antibody 1 (B-COO-Mouse Mab 1 to GMAF) were immobilized on the right end of the integrated sample pad. Biotin-molecular particle-labeled rabbit anti-Candida polyclonal antibody 2 (abbreviated as: Biotin-Rabbit Pab 2 to Candida) and biotin-molecular particle-labeled mouse anti-Trichomonas vaginalis monoclonal antibody 2 (abbreviated as: Biotin-Mouse Mab 2 to When testing positive samples, the Candida / Trichomonas / Gardnerella vaginalis antigens in the sample can respectively bind to the red polystyrene carboxyl nanoparticles labeled Candida antibody 1 / Trichomonas antibody 1 / Gardnerella vaginalis antibody 1 on the integrated sample pad to form a complex, which then moves to the binding pad under the action of chromatography and then binds to the Biotin-Rabbit Pab 2 toCandida / Biotin-Mouse Mab 2 to Trichomonas / Biotin-Mouse Mab 2 binds to Trichomonas to form a (Biotin-Ab2)-ag-(Ab1-COO-R) sandwich complex, which then moves along the membrane. When passing the detection line (T), it forms a SA-(Biotin-Ab2)-ag-(Ab1-COO-R) complex with the pre-coated streptavidin body and condenses to form a red strip. Regardless of whether the sample is negative or positive, as long as the sample is collected accurately and sufficiently, the reproductive tract mucosal antigen factor in the sample will bind to the mouse anti-genital tract mucosal antigen factor monoclonal antibody 1 (B-COO-Mouse Mab 1 to GMAF) coupled to the blue polystyrene nanosphere particles on the integrated sample pad to form a complex. The complex will move forward under the action of chromatography, pass through the quality control line (C), and bind to the mouse reproductive tract mucosal antigen factor monoclonal antibody 2 (Mouse Mab 2 to GMAF), forming a (B-COO-Ab1)-GMAF-Ab2 sandwich complex and condensing to form a blue strip.

[0116] Therefore, regardless of the presence of Candida / Trichomonas vaginalis / Gardnerella vaginalis antigens in the clinical sample, a blue band will appear at the control line (C). This blue band serves as a criterion for determining whether the sample was collected accurately, whether it was sufficient, whether the sample was female secretions, and whether the chromatography process was normal. It also serves as an internal control standard for the reagent.

[0117] How to use the test card: (1) Sample type: Female vaginal swab sample, female urine sample, male urethral swab sample, male urine, male semen.

[0118] (2) Swab sample testing Vertically drip 0.8-1.0 mL of sample processing solution into the sample processing tube. Immediately insert the sampled swab. Squeeze the sample processing tube and rotate the swab 15 times. Then, let the swab sit in the sample processing tube for 2 minutes. Squeeze the swab along the tube wall, remove it, and discard it. Drip 90 μL of the processed sample solution into each well of the test card. Read the results within 10 minutes and within 20 minutes.

[0119] (3) Urine sample testing Vertically drip 0.4-0.5mL of sample treatment solution into the sample treatment tube. Pipette 0.4-0.5mL of urine sample and mix thoroughly. After 2 minutes, drip 90µL of treated sample solution into each well of the test card. Read the results within 10 minutes and 20 minutes.

[0120] (4) Semen sample testing Vertically drip 1 mL of sample processing solution into the sample processing tube. Pipette 0.05 mL to 0.1 mL of liquefied semen sample and mix thoroughly. After 2 minutes, drip 90 μL of the processed sample solution into each well of the test card. Read the results within 10 minutes and within 20 minutes.

[0121] (5) Result judgment a. Negative: Only a blue band appears in the control area (C), and no red band appears in the test area (T). A negative result indicates that the sample does not contain the microorganism to be tested, or the content is below the detectable range; b. Positive: Two bands appear. One red band is located in the test area (T), and the other blue band is located in the quality control area (C). A positive result indicates that the sample contains the microorganism to be tested; c. Invalid: No blue strip appears in the quality control area (C), indicating that the operation process is incorrect or the test card is damaged.

[0122] Comparative Example 1 This comparative example is similar to Example 1, except that 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (chaps) is not added to the sample treatment solution. Other reagents and experimental methods are the same as those in Example 1.

[0123] Comparative Example 2 This comparative example is similar to Example 1, except that no brown algal oligosaccharide is added to the sample treatment solution, and other reagents and experimental methods are the same as those in Example 1.

[0124] Comparative Example 3 This comparative example is similar to Example 1, except that the brown algal oligosaccharide in the sample treatment solution is replaced with sucrose, and the other reagents and experimental methods are the same as those in Example 1.

[0125] Comparative Example 4 This comparative example is similar to Example 1, except that the brown algae oligosaccharide in the sample treatment solution is replaced with trehalose, and the other reagents and experimental methods are the same as those in Example 1.

[0126] Comparative Example 5 This comparative example is similar to Example 1, except that no lentinan is added to the integrated sample pad / conjugate pad processing solution, and other reagents and experimental methods are the same as those in Example 1.

[0127] Application Example 1 The clinical effectiveness of the kit was evaluated by conducting comparative experiments using the kits of Example 1 and Comparative Examples 1-4 and the gold standard "isolation and culture method".

[0128] The sample to be tested is female vaginal secretions. The test kits of Example 1 and Comparative Examples 1-4 all refer to the method of use of the test card mentioned in Example 1: vertically drip 0.8 mL of sample treatment liquid into the sample treatment tube, immediately put in the sampled swab, squeeze the sample treatment tube, and rotate the swab 15 times. Then, after the swab is allowed to stand in the sample treatment tube for 2 minutes, the swab is squeezed along the tube wall and then taken out and discarded. 90 uL of sample liquid treated with the sample treatment liquid is dripped into each well of the test card sample well (the Candida in the test kits of Example 1 and Comparative Examples 1-4 is tested simultaneously using the same sample, and the same applies to Trichomonas vaginalis and Gardnerella vaginalis), and the results are interpreted at 15 minutes. The results of the separation and culture of Candida, Trichomonas vaginalis, Gardnerella vaginalis and vaginal secretions detected by the test kits obtained in Example 1 and Comparative Examples 1-4 are shown in Table 1-5; the sensitivity, specificity and efficacy of Candida, Trichomonas vaginalis and Gardnerella vaginalis detected by Example 1 and Comparative Examples 1-4 are shown in Table 1-5. Figure 3-5 As shown, where: sensitivity = (true positive / N1) × 100%; Specificity = (true negative / N2) × 100%; Efficacy = [(true positives + true negatives) / N] × 100%.

[0129] Table 1 Results of the kit of Example 1 Table 2 Results of the kit of Comparative Example 1 Table 3 Results of the Comparative Example 2 Kit Table 4 Results of the Comparative Example 3 Kit Table 5 Results of the kit of Comparative Example 4 Application Example 2 Take the Candida vaginalis Trichomonas vaginalis Gardnerella vaginalis three-in-one antigen diagnostic kit prepared in Example 1, Comparative Example 2, and Comparative Example 5, place the kit on a horizontal surface, take Candida antigen standard, Trichomonas vaginalis antigen standard, and Gardnerella vaginalis antigen standard, all of which are prepared using the corresponding sample treatment liquids in Example 1, Comparative Example 2, and Comparative Example 5, and prepare 0.02 ng / ml, 0.06 ng / ml, 0.1 ng / ml, 0.2 ng / ml, 0.4 ng / ml, and 0.8 ng / ml of Candida antigen standard (Leinco Technologies, Inc.), 0.02 ng / ml, 0.06 ng / ml, 0.1 ng / ml, 0.2 ng / ml, 0.4 ng / ml, and 0.8 ng / ml of Trichomonas vaginalis antigen standard (Leinco Technologies, Inc.), respectively. Inc.) and Gardnerella vaginalis antigen standards (Bioss Inc.) at concentrations of 0.06 ng / ml, 0.1 ng / ml, 0.2 ng / ml, 0.4 ng / ml, 0.8 ng / ml, and 1.6 ng / ml. 90 μl of the sample solution was pipetted and added dropwise to the sample well. Results were read 15 minutes after sample addition. Each concentration was repeated five times (any negative result in any of the five tests was considered negative). The results are shown in Tables 6-8.

[0130] Table 6 Candida antigen concentration (ng / ml) Example 1 Comparative Example 2 Comparative Example 5 0.02 1 -1 -1 0.06 1 -1 -1 0.1 1 -1 -1 0.2 1 -1 -1 0.4 1 -1 1 0.8 1 1 1 In the table, 1 indicates positive; -1 indicates negative.

[0131] Table 7 Trichomonas vaginalis antigen concentration (ng / ml) Example 1 Comparative Example 2 Comparative Example 5 0.02 1 -1 -1 0.06 1 -1 -1 0.1 1 -1 -1 0.2 1 -1 -1 0.4 1 -1 -1 0.8 1 1 1 In the table, 1 indicates positive; -1 indicates negative.

[0132] Table 8 Gardnerella vaginalis antigen concentration (ng / ml) Example 1 Comparative Example 2 Comparative Example 5 0.06 1 -1 -1 0.1 1 -1 -1 0.2 1 -1 -1 0.4 1 -1 -1 0.8 1 -1 1 1.6 1 1 1 In the table, 1 indicates positive; -1 indicates negative.

[0133] From Table 6-8 and Figure 6 The results show that the three-in-one Candida vaginalis and Trichomonas vaginalis antigen diagnostic kit prepared by the present invention has a detection concentration of Candida albicans antigen and Trichomonas vaginalis antigen ≤ 0.02 ng / ml, and a concentration of Gardnerella vaginalis antigen ≤ 0.06 ng / ml, which has high sensitivity; in addition, the detection concentrations of Candida albicans antigen, Trichomonas vaginalis antigen and Gardnerella vaginalis antigen in Comparative Examples 2 and 5 are significantly higher than those in Example 1, indicating that the brown algae oligosaccharides in the sample processing solution of the present invention and the lentinan in the binding pad processing solution synergistically improve the sensitivity of the kit.

[0134] The above-described embodiments merely represent specific implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A three-in-one antigen diagnostic reagent for Candida vaginalis and Trichomonas vaginalis, characterized in that: The invention comprises a sample processing solution and an integrated sample pad / conjugate pad processing solution. The sample processing solution comprises: 3.36-16.81 g / L citric acid, 10-100 g / L sodium citrate, 0.1-1 g / L 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt, 0.5-1.5 g / L brown algal oligosaccharide, 5-20 g / L lauryl alcohol polyoxyethylene ether, 0.5-2 mL / L Proclin-300, and the pH is adjusted to 4.5-7.

8.

2. A Candida vaginalis Trichomonas vaginalis Gardnerella vaginalis three-in-one antigen diagnostic reagent according to claim 1, characterized in that: The integrated sample pad / conjugate pad treatment solution contains 4.32-43.2 g / L disodium hydrogen phosphate dodecahydrate, 1.12-11.2 g / L sodium dihydrogen phosphate dihydrate, 1.00-10.0 g / L bovine serum albumin, 1.00-5.00 g / L lentinan, and 1.00-10.0 mL / L sodium lauryl ether sulfate, and the pH is adjusted to 7.5-8.

5.

3. A kit, characterized in that The test kit box body is provided with a test card and a reagent bottle containing the sample processing solution according to claim 2; the test card includes a card cover and a card body, and the card body is provided with a Candida detection test strip, a Trichomonas vaginalis detection test strip, and a Gardnerella vaginalis detection test strip. The Candida detection test strip, the Trichomonas vaginalis detection strip, and the Gardnerella vaginalis detection strip are all provided with an integrated sample pad treated with the integrated sample pad / conjugation pad processing solution according to claim 2, a conjugation pad treated with the integrated sample pad / conjugation pad processing solution according to claim 2, a nitrocellulose membrane, and absorbent paper along the chromatography direction.

4. The kit according to claim 1, wherein In the Candida detection test strip, the connection between the integrated sample pad and the conjugate pad is fixed with: rabbit anti-Candida polyclonal antibody 1 coupled with red polystyrene nanoparticles and mouse anti-genital mucosal antigen factor monoclonal antibody 1 coupled with blue polystyrene nanoparticles; In the described vaginal trichomonas detection test strip, the connection between the integrated sample pad and the binding pad is fixed with: red polystyrene nanoparticles coupled to mouse anti-vaginal trichomonas monoclonal antibody 1 and blue polystyrene nanoparticles coupled to mouse anti-genital mucosal antigen factor monoclonal antibody 1; In the vaginal Gardnerella detection test strip, the connection between the integrated sample pad and the binding pad is fixed with: red polystyrene nanoparticles coupled to mouse anti-vaginal Gardnerella monoclonal antibody 1 and blue polystyrene nanoparticles coupled to mouse anti-genital mucosal antigen factor monoclonal antibody 1.

5. The kit according to claim 1, wherein In the Candida detection test strip, the conjugate pad is fixed with: rabbit anti-Candida polyclonal antibody 2 labeled with biotin molecular particles; In the Trichomonas vaginalis test strip, the binding pad is fixed with: mouse anti-Trichomonas vaginalis monoclonal antibody 2 labeled with biotin molecular particles; In the Gardnerella vaginalis test strip, the binding pad is fixed with: mouse anti-Gardnerella vaginalis monoclonal antibody 2 labeled with biotin molecular particles.

6. The kit according to claim 1, wherein The nitrocellulose membranes of the Candida detection test strip, the Trichomonas vaginalis detection test strip, and the Gardnerella vaginalis detection test strip are all provided with a detection line T line and a quality control line C line. The T line area of the nitrocellulose membrane is fixedly coated with streptavidin, and the C line area is fixedly coated with mouse anti-genital mucosal antigen factor monoclonal antibody 2.

7. The kit according to claim 4, characterized in that The preparation method of the rabbit anti-Candida polyclonal antibody 1 coupled with red polystyrene nanoparticles, the mouse anti-Trichomonas vaginalis monoclonal antibody 1 coupled with red polystyrene nanoparticles, or the mouse anti-Gardnerella vaginalis monoclonal antibody 1 coupled with red polystyrene nanoparticles is as follows: (1) Weigh 20 mg of carbodiimide solid; (2) Add 4 mL of activation coupling buffer to fully dissolve the mixture to a concentration of 5 mg / mL. (3) Take 2 mg of red polystyrene nanosphere particles and add 0.89 mL to 0.95 mL of activation coupling buffer; (4) Add 0.01 mL to 0.06 mL of EDC solution; (5) After mixing, incubate at 25-37°C for 10-50 minutes; (6) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant; (7) Add 1 mL of washing buffer and resuspend in an ultrasonic cleaner for 5–20 min; (8) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant; (9) Add 2 mL of activation coupling buffer and resuspend in an ultrasonic cleaner for 5–20 min; (10) Add 50 μg to 400 μg of rabbit anti-Candida polyclonal antibody 1 or mouse anti-Trichomonas vaginalis monoclonal antibody 1 or mouse anti-Gardnerella vaginalis monoclonal antibody 1, mix manually, place on a mixer, and mix and react at 25-37°C for 1 h to 6 h; (11) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant; (12) Add 1 mL of blocking buffer and resuspend in an ultrasonic cleaner for 5–20 min; (13) Mix the mixture on a mixer for 1–6 h; (14) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. (15) Add 1 mL of nanosphere preservation solution and resuspend in an ultrasonic cleaner for 5 to 20 minutes.

8. The kit according to claim 4, wherein The preparation method of the mouse anti-genital mucosal antigen factor monoclonal antibody 1 coupled with the blue polystyrene nanosphere particles is as follows: (1) Weigh 20 mg of carbodiimide solid; (2) Add 4 mL of activation coupling buffer to fully dissolve the mixture to a concentration of 5 mg / mL. (3) Take 2 mg of blue polystyrene nanosphere particles and add 0.89 mL to 0.95 mL of activation coupling buffer; (4) Add 0.01 mL to 0.06 mL of EDC solution; (5) After mixing, incubate at 25-37°C for 10-50 minutes; (6) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant; (7) Add 1 mL of washing buffer and resuspend in an ultrasonic cleaner for 5–20 min; (8) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant; (9) Add 2 mL of activation coupling buffer and resuspend in an ultrasonic cleaner for 5–20 min; (10) Add mouse anti-genital mucosal antigen factor monoclonal antibody 1, 50 μg~400 μg, mix manually, place on a mixer, and mix and react at 25~37°C for 1 h~6 h; (11) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant; (12) Add 1 mL of blocking buffer and resuspend in an ultrasonic cleaner for 5–20 min; (13) Mix the mixture on a mixer for 1–6 h; (14) Centrifuge at 8000-12000 rpm, 2-8°C for 15-30 min and discard the supernatant. (15) Add 1 mL of nanosphere preservation solution and resuspend in an ultrasonic cleaner for 5 to 20 minutes.

9. The kit according to claim 5, characterized in that The preparation method of the biotin molecular particle-labeled rabbit anti-Candida polyclonal antibody 2 or the biotin molecular particle-labeled mouse anti-Trichomonas vaginalis monoclonal antibody 2 or the biotin molecular particle-labeled mouse anti-Gardnerella vaginalis monoclonal antibody 2 is as follows: (1) Pipette 1 mg of rabbit anti-Candida polyclonal antibody 2 or mouse anti-Trichomonas vaginalis monoclonal antibody 2 or mouse anti-Gardnerella vaginalis monoclonal antibody 2 and dilute it with 0.5 mL to 5 mL of phosphate buffer; (2) Weigh 1 mg of NHS-biotin into a clean centrifuge tube and dissolve it in 1 mL of phosphate buffer. (3) Add 0.05 mL to 0.2 mL of biotin solution to the antibody solution, mix thoroughly, and incubate at 30 to 37 °C for 1 to 4 hours. (4) Add the reaction solution to an ultrafiltration centrifuge tube and centrifuge in a low-temperature centrifuge at 2-8°C and a centrifugal force of 4000-10000g for 10-25 minutes; (5) Measure the volume of the liquid after centrifugation and dilute it to 0.1~0.5mg / ml with phosphate buffer.

10. The kit according to claim 1, wherein The card cover and the areas corresponding to the Candida detection test strip, the Trichomonas vaginalis detection test strip and the Gardnerella vaginalis detection test strip are respectively provided with a sample addition hole and an observation hole.

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