Test strip for detecting human chorionic gonadotropins in saliva and method for manufacturing the same

Abstract

A test strip for detecting human chorionic gonadotropins in saliva and a method for producing the same, wherein the sample pad treatment solution, based on 100 mL, contains Tris 1.0-1.5 g, casein 0.3-0.8 g, Tween-20 50-100 μL, NaCl 0.5-1.5 g, dodecyltrimethylammonium bromide 0.15-0.2 g, 4-nonylphenyl-polyethylene glycol 0.8-1.2 g, borax 3.5-4.0 g, polyvinylpyrrolidone 0.5-1 g, dithiothreitol 3-5 g, and L-cysteine ​​0.8-2 g. By treating the sample pad with the sample pad treatment solution, interference from other impurities in saliva can be reduced, and nonspecific reactions can be completely eliminated. The test strip has many advantages, including high sensitivity, good stability, can be stored at room temperature for a long time, is cleaner and more convenient, can be tested at any time, and is not affected by the time or place of sample collection.

Description

[[Technical Field]]

[0001] The present invention relates to the technical field of detection using test strips, and particularly relates to a test strip for detecting human chorionic gonadotropin in saliva and a method for producing the same. [[Background Art]]

[0002] Human chorionic gonadotropin (HCG) is a glycoprotein secreted by trophoblast cells of the placenta, and is a dimeric glycoprotein composed of an α-subunit and a β-subunit. The α-subunit is common to anterior pituitary hormones, and the β-subunit is specific to HCG. The main function of HCG is to stimulate the corpus luteum, thereby contributing to the sustained secretion of estrogen and progesterone, promoting the formation of the decidua of the uterus, and facilitating the growth and maturation of the placenta. Currently, it is considered that HCG is produced by transitional trophoblast cells and syncytiotrophoblast cells. During the first 8 weeks of early pregnancy, HCG proliferates rapidly to maintain pregnancy. After approximately 8 weeks of pregnancy, HCG gradually decreases and reaches a relatively stable state by approximately 20 weeks of pregnancy. Detecting the HCG content in urine of women of childbearing age using the HCG double antibody method allows obtaining results rapidly in early pregnancy, and is an effective means for auxiliary diagnosis.

[0003] Saliva is a biofluid rich in clinical information, containing a variety of soluble biomarkers, enabling rapid detection or more standardized, centralized clinical laboratory procedures. Saliva is commonly used in in vitro diagnostics, and the quantitative detection of steroid hormones such as cortisol, estriol, and testosterone using saliva is widely applied. Currently, numerous rapid detection test strips for HIV using saliva exist in China. Importantly, saliva is closely related to its corresponding components in blood, and can function as a "window" to blood. Furthermore, relevant experimental results show that the concentration of human chorionic gonadotropin correlates well with its concentration in blood. Therefore, saliva can function as a non-invasive, rapid, and more acceptable biofluid for pregnancy detection. The secretion mechanism of human chorionic gonadotropin in saliva is thought to be related to the fact that saliva is a hypotonic fluid, and capillaries and lymphatic capillaries are tightly connected to the basement membrane of glands. Human chorionic gonadotropin may be transferred from the blood to the saliva through back leakage via the interacinar spaces and tight junctions. In a clinical trial on human chorionic gonadotropin in saliva conducted in a hospital in China, two types of samples, urine and saliva, were tested simultaneously under identical conditions. The results showed that pregnant women had hCG in both their urine and saliva. When the test results of the two types of samples were compared, there was a good correlation and the test results were consistent.

[0004] While saliva-based early pregnancy detection offers many advantages, including being cleaner, more convenient, allowing testing at any time, and unaffected by sample collection time, test strips for saliva-based early pregnancy detection are rarely mentioned. The development challenges include the presence of many non-detection interfering substances in saliva samples, low sensitivity, difficulty in chromatography, and nonspecificity. For example, under normal circumstances, human hCG levels are below 5 mIU / mL, and a level greater than 5 mIU / mL in women of childbearing age may indicate pregnancy. However, most commercially available urine-based early pregnancy detection test strips have a detection limit of 25 mIU / mL, failing to detect pregnancy earlier. They cannot detect hCG levels below 25 mIU / mL in early pregnancy, for example, within the first week. Furthermore, both direct urine application and methods using urine collection cups and droppers make it difficult to avoid "getting your hands dirty." Direct urine application can also affect chromatography results due to urine wetting of the detection window. Urine detection methods are also subject to time and location constraints. Furthermore, when the test strip sample type is saliva, saliva samples are relatively viscous, resulting in a slow flow rate when the saliva is chromatographed upwards from the sample pad. Additionally, latex microspheres can clog the nitrocellulose membrane, causing the liquid chromatography to stop midway and leading to problems such as false positives. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] In view of this, the present invention aims to provide a test strip for detecting human chorionic gonadotropins in saliva and a method for manufacturing the same, wherein the test strip is clean and convenient for detection in early pregnancy, has high detection sensitivity and strong specificity. [Means for solving the problem]

[0006] To achieve the objectives of the above invention, the present invention provides the following technical solutions.

[0007] The present invention provides a test strip for detecting human chorionic gonadotropins in saliva, the test strip comprising a sample pad, a conjugate pad, a water filtration pad, a coating film, and a substrate, wherein the sample pad, the conjugate pad, the water filtration pad, and the coating film are sequentially layered on the substrate. The aforementioned sample pad is a glass fiber film treated with a sample pad treatment solution. Based on 100 mL, the sample pad processing solution is: It contains Tris 1.0-1.5g, casein 0.3-0.8g, Tween-20 50-100μL, NaCl 0.5-1.5g, dodecyltrimethylammonium bromide 0.15-0.2g, 4-nonylphenyl polyethylene glycol 0.8-1.2g, borax 3.5-4.0g, polyvinylpyrrolidone 0.5-1g, dithiothreitol 3-5g, and L-cysteine ​​0.8-2g. The aforementioned coating film is a nitrocellulose film coated with T-lines and C-lines. The T-line is sprayed with a 0.5-1 mg / mL diluted mouse anti-α-HCG monoclonal antibody solution. The C line is sprayed with a 0.4-0.6 mg / mL dilution of goat anti-mouse IgG polyclonal antibody. The spray volume when spraying the T-line or C-line is 0.5 to 1.5 μL / cm. The aforementioned conjugate pad is a glass fiber membrane or polyester fiber membrane treated with a conjugate pad treatment solution. The aforementioned conjugate pad treatment solution contains 1 to 1.5 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.2 to 1 g of casein, 50 to 100 μL of Tween-20, and 75 to 85 mL of water. HCG latex microspheres are sprayed onto the glass fiber membrane or polyester fiber membrane, and the spray volume is 1.5 to 3 μL / cm.

[0008] Preferably, the pH of the sample pad processing solution is 8.0 to 8.5.

[0009] Preferably, the mouse anti-α-HCG monoclonal antibody diluent or the goat anti-mouse IgG polyclonal antibody diluent is prepared by diluting the mouse anti-α-HCG monoclonal antibody or the goat anti-mouse IgG polyclonal antibody, respectively, with a coating diluent, the coating diluent being a PBS solution containing 50 to 150 mg / mL of trehalose.

[0010] Preferably, the volume ratio of the red latex microspheres to the borax buffer is 0.1 to 0.2:1, and the concentration of the borax buffer is 0.05 to 0.15 M.

[0011] Preferably, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 5 to 15 mg / mL, the concentration of the ethanolamine solution is 0.08 to 0.12 M, and the concentration of the Tris buffer solution is 50 to 100 mM.

[0012] Preferably, the blocking time is 15 to 30 minutes, and the ultrasonic treatment time is 10 to 20 minutes.

[0013] The present invention further provides test strips manufactured based on the above manufacturing method.

[0014] The present invention further provides a method for manufacturing the above-mentioned test strip, (1) Manufacturing of sample pads The steps include uniformly absorbing the sample pad processing solution into a glass fiber film, then drying it to obtain a sample pad, (2) Manufacturing of conjugate pads The process involves uniformly absorbing a conjugate pad treatment solution into a glass fiber membrane or polyester fiber membrane, drying it, and then spraying HCG latex microspheres onto the glass fiber membrane or polyester fiber membrane treated with the conjugate pad treatment solution at a spray rate of 1.5 to 3 μL / cm to obtain a conjugate pad. (3) Manufacturing of coating films The process involves diluting mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody with a coating diluent to obtain diluted mouse anti-α-HCG monoclonal antibody and diluted goat anti-mouse IgG polyclonal antibody solutions, spraying the diluted mouse anti-α-HCG monoclonal antibody solution onto the T-line of a nitrocellulose membrane, spraying the diluted goat anti-mouse IgG polyclonal antibody solution onto the C-line of a nitrocellulose membrane, drying, and obtaining a coated membrane. (4) Manufacturing of test strips The process includes the steps of cutting the sample pad, conjugate pad, and water filtration pad, then sequentially attaching the sample pad, conjugate pad, water filtration pad, and coating film to a substrate, such that one side of the conjugate pad is positioned above the sample pad, the other side of the conjugate pad is positioned below one side of the water filtration pad, the other side of the water filtration pad is positioned below the coating film, and the T-line on the coating film is close to the water filtration pad.

[0015] Preferably, the method for producing the HCG latex microspheres is: The steps include washing red latex microspheres in borax buffer to obtain the red latex microspheres after washing, The process involves mixing β-HCG-labeled antibody with washed red latex microspheres, activating them with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to obtain β-HCG-labeled antibody-modified red latex microspheres, and mixing an ethanolamine solution with β-HCG-labeled antibody-modified red latex microspheres for blocking, obtaining blocked HCG red latex microspheres, removing the supernatant by centrifugation, further adding Tris buffer, uniformly mixing the blocked HCG red latex microspheres by ultrasonic treatment, removing the supernatant by centrifugation, then adding Tris buffer and uniformly mixing by ultrasonic treatment, further rotating and uniformly mixing at room temperature for 4 to 6 hours to obtain HCG latex microspheres.

[0016] Preferably, the addition ratio of said β-HCG-labeled antibody, washed red latex microspheres and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1 mg: 10 mg: 0.05 mL.

[0017] The present invention further provides a kit for detecting early pregnancy, said kit comprising the above test strip and a sample diluent.

[0018] Preferably, said sample diluent is 0.9% physiological saline.

[0019] The present invention further provides use of the above test strip or the above production method,[ (1) use of said test strip or said production method in detection of human chorionic gonadotropin; (2) use of said test strip or said production method in production of products for detecting human chorionic gonadotropin; (3) use of said test strip or said production method in detection of early pregnancy; (4) use of said test strip or said production method in production of products for detecting early pregnancy, comprising at least one of the above.

[0020] Preferably, the criteria for detecting and judging whether pregnancy occurs are: (1) Positive: two red reaction lines, that is, one red reaction line appears in the detection region and one red reaction line appears in the control region respectively; (2) Negative: One red reaction line appears, i.e., one red reaction line appears only in the control region, (3) Invalid: No red reaction line appears in the control region, indicating a test error or an invalid result.

[0021] Preferably, the detection sample is saliva.

[0022] Preferably, the product comprises a kit or a detection card. Advantageous Effects of Invention

[0023] Compared with the prior art, the present invention has the following beneficial effects. The present invention provides a test strip for detecting human chorionic gonadotropin in saliva and a method for manufacturing the same. In the present invention, by directly collecting saliva and treating the sample pad with a sample pad treatment solution, the water solubility of the saliva sample is improved, the chromatography of the saliva sample is smoothed, the interference from other impurity components in saliva is reduced, the false positive phenomenon in the detection of saliva samples is eliminated, and the effect of completely eliminating non-specific reactions can be achieved. At the same time, the test strip of the present invention has good stability and can be stored for a long time at normal temperature. The detection limit of the test strip of the present invention is 5 mIU / mL, which can detect pregnancy earlier. As a potential, user-friendly and highly acceptable product, it has many advantages such as being cleaner and more convenient, allowing testing at any time, and not being affected by the time and place of sample collection. Brief Description of the Drawings

[0024] [Figure 1] It is a schematic diagram for detecting and judging whether a subject is pregnant by using the test strip of the present invention. [Figure 2] It is the specificity test result of test strips for detecting human chorionic gonadotropin in saliva with different detection cards. In the figure, from left to right in order are the test results using the detection cards of Comparative Example 3, Comparative Example 2, Comparative Example 1 and Example 2. [Figure 3]The figures show the specificity test results after storing test strips for detecting human chorionic gonadotropins in saliva using different detection cards at room temperature for 182 days. From left to right in the figure, the test results are from Comparative Example 2, Comparative Example 1, and Example 2 using the respective detection cards. [Figure 4] These are the test results from 20 negative saliva samples. [Figure 5] These are the test results for 8 positive samples. [Modes for carrying out the invention]

[0025] The present invention provides a test strip for detecting human chorionic gonadotropins in saliva, the test strip comprising a sample pad, a conjugate pad, a water filtration pad, a coating film, and a substrate, wherein the sample pad, the conjugate pad, the water filtration pad, and the coating film are sequentially layered on the substrate. The aforementioned sample pad is a glass fiber film treated with a sample pad treatment solution. Based on 100 mL, the sample pad processing solution is: It contains Tris 1.0-1.5g, casein 0.3-0.8g, Tween-20 50-100μL, NaCl 0.5-1.5g, dodecyltrimethylammonium bromide 0.15-0.2g, 4-nonylphenyl polyethylene glycol 0.8-1.2g, borax 3.5-4.0g, polyvinylpyrrolidone 0.5-1g, dithiothreitol 3-5g, and L-cysteine ​​0.8-2g.

[0026] In the present invention, the width of the sample pad is preferably 2 ± 0.1 cm, and the length is preferably 30 ± 0.2 cm. The water filtration pad is purchased from Whatman, with part number 8151-6621.

[0027] In the present invention, the sample pad processing solution preferably further contains 750 to 850 mL of water, and the pH of the sample pad processing solution preferably is 8.0 to 8.5. The method for preparing the sample pad processing solution is to sequentially weigh 1.0 to 1.5 g of Tris, 0.3 to 0.8 g of casein, 50 to 100 μL of Tween-20, 0.5 to 1.5 g of NaCl, 0.15 to 0.2 g of dodecyltrimethylammonium bromide, 0.8 to 1.2 g of 4-nonylphenyl polyethylene glycol, 3.5 to 4.0 g of borax, and 0.5 to 1 g of polyvinylpyrrolidone, add 750 to 850 mL of water, stir to dissolve, then add 3 to 5 g of dithiothreitol and 0.8 to 2 g of L-cysteine, stir to dissolve, adjust the pH to 8.0 to 8.5, and then dilute to 100 mL to obtain the sample pad processing solution. By using dithiothreitol and L-cysteine ​​in the sample pad processing solution, the objective of removing nonspecific reactions in the chromatographic process can be achieved. At the same time, dithiothreitol has antioxidant properties, preventing L-cysteine ​​from being oxidized to cystine by air and forming precipitates, thus creating a synergistic effect. The two reagents, dithiothreitol and L-cysteine, are usually prepared and used as solutions. These solutions are easily oxidized, making them unsuitable for storage and resulting in a short shelf life. Therefore, they usually need to be used as soon as possible or stored at low temperatures. For products such as rapid detection reagents, refrigeration is clearly not practical. In this invention, by placing the above two reagents and other reagents in the sample pad processing solution into the sample pad, the problem of the short shelf life of the reagents is effectively solved, allowing for long-term storage at room temperature, with a shelf life of 6 months or more. The 4-nonylphenyl-polyethylene glycol is the wetting agent P-40 purchased from Jiangsu Pulesi Biotechnology Co., Ltd.

[0028] The test strip of the present invention employs a novel sample pad processing method, in which saliva is dropped onto the sample pad, reacts with the active ingredients in the sample pad, reduces the viscosity of the saliva, resulting in very smooth chromatography and no membrane clogging. This improves the specificity of the test strip, reduces interference from other components in the saliva, and avoids the likelihood of saliva samples causing false positives.

[0029] In the present invention, the coating film is preferably a nitrocellulose film coated with T-lines and C-lines, wherein the T-lines are preferably sprayed with a mouse anti-α-HCG monoclonal antibody diluent at a concentration of 0.5 to 1 mg / mL, and the C-lines are preferably sprayed with a goat anti-mouse IgG polyclonal antibody diluent at a concentration of 0.4 to 0.6 mg / mL, and the spray volume when spraying the T-lines or C-lines is preferably 0.5 to 1.5 μL / cm. The mouse anti-α-HCG monoclonal antibody diluent or the goat anti-mouse IgG polyclonal antibody diluent is prepared by diluting the mouse anti-α-HCG monoclonal antibody or the goat anti-mouse IgG polyclonal antibody with a coating diluent, and the coating diluent is a PBS solution containing 50 to 150 mg / mL of trehalose. The concentration of the PBS solution is preferably 0.005 to 0.015 M. The distance between the T-lines and C-lines on the coating film is preferably 0.4 to 0.6 cm. The present invention enhances sensitivity and reduces the occurrence of nonspecific reactions by selecting the above antibody raw materials. In the present invention, the mouse anti-α-HCG monoclonal antibody is purchased from Nanjing Jingda Biotechnology Co., Ltd., with catalog number W4003, and the mouse anti-α-HCG monoclonal antibody is purchased from Aochuang Biotechnology (Shandong) Co., Ltd., with catalog number A01-Ab2.

[0030] In the present invention, the conjugate pad is preferably a glass fiber membrane or a polyester fiber membrane treated with a conjugate pad treatment solution.

[0031] In the present invention, the conjugate pad treatment solution preferably contains 1 to 1.5 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.2 to 1 g of casein, 50 to 100 μL of Tween-20, and 75 to 85 mL of water. The pH of the conjugate pad treatment solution is preferably 8.0 to 8.5. The method for preparing the conjugate pad treatment solution is to weigh 1 to 1.5 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.2 to 1 g of casein, 50 to 100 μL of Tween-20, and 75 to 85 mL of water, stir to dissolve, adjust the pH to 8.0 to 8.5, and then dilute to 100 mL to obtain the conjugate pad treatment solution. In a preferred embodiment of the present invention, HCG latex microspheres are sprayed onto the glass fiber membrane or polyester fiber membrane, with a spray volume of 1.5 to 3 μL / cm. The 3-(N-morpholino)-2-hydroxypropanesulfonic acid is purchased from Adamas, with catalog number 76496B.

[0032] In the present invention, the width of the conjugate pad is preferably 1 ± 0.2 cm, and the length is preferably 30 ± 0.2 cm.

[0033] The present invention further provides a method for manufacturing the above-mentioned test strip, (1) Manufacturing of sample pads The steps include uniformly absorbing the sample pad processing solution into a glass fiber film, then drying it to obtain a sample pad, (2) Manufacturing of conjugate pads The process involves uniformly absorbing a conjugate pad treatment solution into a glass fiber membrane or polyester fiber membrane, drying it, and then spraying HCG latex microspheres onto the glass fiber membrane or polyester fiber membrane treated with the conjugate pad treatment solution at a spray rate of 1.5 to 3 μL / cm to obtain a conjugate pad. (3) Manufacturing of coating films The process involves diluting mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody with a coating diluent to obtain diluted mouse anti-α-HCG monoclonal antibody and diluted goat anti-mouse IgG polyclonal antibody solutions, spraying the diluted mouse anti-α-HCG monoclonal antibody solution onto the T-line of a nitrocellulose membrane, spraying the diluted goat anti-mouse IgG polyclonal antibody solution onto the C-line of a nitrocellulose membrane, drying, and obtaining a coated membrane. (4) Manufacturing of test strips The process includes the steps of cutting the sample pad, conjugate pad, and water filtration pad, then sequentially attaching the sample pad, conjugate pad, water filtration pad, and coating film to a substrate, such that one side of the conjugate pad is positioned above the sample pad, the other side of the conjugate pad is positioned below one side of the water filtration pad, the other side of the water filtration pad is positioned below the coating film, and the T-line on the coating film is close to the water filtration pad.

[0034] In the present invention, the production of the sample pad involves uniformly absorbing the sample pad processing solution into a glass fiber film, and then drying it to obtain the sample pad. The drying method is preferably drying at 40-45°C for 2 hours or more.

[0035] In the present invention, a glass fiber membrane or polyester fiber membrane is uniformly absorbed with a conjugate pad treatment solution and then dried. The drying method is preferably drying at 40 to 45°C for 8 to 12 hours.

[0036] In the present invention, a conjugate pad is obtained by spraying HCG latex microspheres at a spray rate of 1.5 to 3 μL / cm onto a glass fiber membrane or polyester fiber membrane treated with a conjugate pad treatment solution. The method for producing HCG latex microspheres includes the steps of: washing red latex microspheres with borax buffer to obtain washed red latex microspheres; mixing β-HCG-labeled antibody with the washed red latex microspheres, activating with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to obtain β-HCG-labeled antibody-modified red latex microspheres; mixing ethanolamine solution with β-HCG-labeled antibody-modified red latex microspheres and blocking to obtain blocked HCG red latex microspheres; removing the supernatant by centrifugation; further adding Tris buffer and uniformly mixing with the blocked HCG red latex microspheres by sonication; further rotating at room temperature for 4 to 6 hours to uniformly mix; removing the supernatant by centrifugation; and then adding Tris buffer and uniformly mixing by sonication to obtain HCG latex microspheres.

[0037] In this invention, red latex microspheres are washed with borax buffer to obtain washed red latex microspheres. The particle size of the red latex microspheres is preferably 150 to 250 nm, and more preferably 200 nm. The volume ratio of the red latex microspheres to the borax buffer is preferably 0.1 to 0.2:1. The concentration of the borax buffer is preferably 0.05 to 0.15 M. The washing method is preferably centrifugation at 13500 rpm for 10 to 20 minutes, removal of the supernatant, and then adding 0.5 to 1.5 mL of borax buffer to the red latex microspheres after washing by centrifugation, and mixing uniformly by sonication. The red latex microspheres are purchased from Suzhou Weidu Biotechnology Co., Ltd., and the product number is DR0200CA-1.

[0038] In the present invention, a β-HCG-labeled antibody is mixed with washed red latex microspheres, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution is added to activate the mixture and obtain β-HCG-labeled antibody-modified red latex microspheres. The addition ratio of the β-HCG-labeled antibody, washed red latex microspheres, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1 mg:10 mg:0.05 mL. The 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is preferably 5 to 15 mg / mL. The method for preparing the 5-15 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is preferably to weigh 5-15 mg of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and dissolve it in 1 mL of purified water. The method for activation is preferably to shake to mix uniformly and then mix uniformly at room temperature for 4-8 hours. The β-HCG-labeled antibody was purchased from Aochuang Biotechnology (Shandong) Co., Ltd., and its catalog number is A01-Ab1.

[0039] In this invention, an ethanolamine solution and β-HCG-labeled antibody-modified red latex microspheres are mixed and blocked to obtain HCG-red latex microspheres after blocking. The concentration of the ethanolamine solution is preferably 0.08 to 0.12 M ethanolamine solution. The blocking time is preferably 15 to 30 minutes.

[0040] In the present invention, Tris buffer is added and the blocked HCG red latex microspheres are uniformly mixed by sonication, and further mixed uniformly by rotation at room temperature for 4 to 6 hours. The Tris buffer is preferably 50 to 100 mM Tris buffer.

[0041] In the present invention, HCG latex microspheres are obtained by adding Tris buffer and uniformly mixing it by sonication. The volume of the added Tris buffer is preferably 0.4 to 0.8 mL, and the sonication time is preferably 10 to 20 minutes.

[0042] The present invention further includes the step of spraying HCG latex microspheres and then drying them at 37-55°C for 20-60 minutes.

[0043] The test strip of this invention employs latex microsphere labeling instead of the colloidal gold method, and simultaneously selects red latex microspheres with a particle size of 200 nm to eliminate the influence of viscosity on some saliva samples.

[0044] The criteria for detecting and determining whether or not a woman is pregnant using the test strip of the present invention (shown in Figure 1) are as follows: (1) Positive (Pregnancy): Two red reaction lines appear, one in the detection area (T) and one in the control area (C). If the color of the detection area (T) is weak, it may indicate that the person is recently pregnant. Retest every other day. (2) Negative (not pregnant): One red reaction line appears, i.e., one red reaction line appears only in the control area (C). (3) Invalid: No red reaction line appears in the control area (C), indicating a test error or invalidity.

[0045] If a woman of childbearing age has an hCG level greater than 5 mIU / mL, she may be pregnant. In the early stages of pregnancy, for example within the first 10 days, if the hCG level is greater than 5 mIU / mL but less than 25 mIU / mL, it cannot be detected by common, less sensitive test strips. The detection limit of the detection test strip of the present invention is 5 mIU / mL, allowing for earlier detection of pregnancy. The minimum detection limit of the present invention is the lowest concentration for a detection rate of ≥95%.

[0046] The present invention further provides a kit for detecting early pregnancy, the kit comprising the above-mentioned test strip and sample diluent.

[0047] The sample diluent is preferably 0.9% physiological saline, and the sample diluent enhances the liquefaction effect of the saliva sample. During detection, the volume ratio of saliva sample to sample diluent is 1:1. The kit of the present invention further includes a small funnel, which is used to collect saliva. The lid of the sample processing tube is opened, the attached saliva funnel is placed at the opening of the tube, saliva from the oral cavity is spat into the saliva funnel, the saliva funnel is removed, the lid of the sample processing tube is closed, and the sample can be uniformly mixed and then dropped for detection. When using the kit for detection, the results can be read in 5 minutes, it is hygienic, convenient, can be tested at any time, and is not affected by the time or place of sample collection.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to examples, but these should not be understood as limiting the scope of protection of the present invention.

[0049] Example 1 A method for manufacturing a test strip for detecting human chorionic gonadotropins in saliva includes the following steps:

[0050] (1) Manufacturing of sample pads Preparation of sample pad processing solution: A 100 mL small beaker was prepared, 800 mL of pure water was weighed out, and Tris 1.21 g, casein 0.5 g, Tween-20 80 μL, NaCl 0.9 g, dodecyltrimethylammonium bromide 0.15 g, 4-nonylphenyl-polyethylene glycol 1.0 g, borax 3.811 g, and polyvinylpyrrolidone 0.8 g were weighed out in order and placed into the small beaker one by one, stirring to dissolve each component, and then the next reagent was added. After all the above materials had dissolved, 3 g of dithiothreitol and 1.2 g of L-cysteine ​​were added, stirred to dissolve, the pH was adjusted to 8.5, and finally the volume was reduced to 100 mL for use.

[0051] The treatment solution was prepared according to the sample pad treatment solution formulation, poured onto a single fiberglass membrane, and the fiberglass membrane was gently pulled with tweezers to guide the treatment solution to be uniformly absorbed into the fiberglass membrane. After the fiberglass membrane had absorbed the solution uniformly, the two diagonal corners were grasped with tweezers and the fiberglass membrane was quickly transferred to a draining rack. The mesh of the draining rack was placed in an oven and dried at 45°C for 4 hours until excess moisture evaporated, with the relative humidity of the oven being ≤30%.

[0052] (2) Manufacturing of conjugate pads S1, Washing of red latex microspheres 0.1 mL of 200 nm red latex microspheres were added to a 2 mL centrifuge tube containing 1 mL of 0.1 M borax buffer and thoroughly mixed. The mixture was centrifuged at 14,000 rpm for 15 minutes using a centrifuge, the supernatant was removed using a pipette, and the red latex microsphere particles were further washed. After that, 1 mL of 0.1 M borax buffer was added to the centrifuge tube, and the latex microspheres in the centrifuge tube were homogenized by sonication using an ultrasonic cell disruptor.

[0053] S2. Labeling of red latex microspheres with β-HCG after washing. Using a pipette, 0.5 mg of β-HCG-labeled antibody was transferred, and the amount of labeled antibody transferred was calculated to a final concentration of 1 mg / mL. This was added to 1 mL of washed red latex microspheres, and then 25 μL of latex microsphere activator was added. The latex microsphere activator was an 8 mg / mL solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. After shaking for 1 minute to mix uniformly, the mixture was mixed uniformly at room temperature for 5 hours using a shaker or rotating apparatus to obtain β-HCG-labeled antibody-modified red latex microspheres.

[0054] S3, blocking 50 μL of blocking solution was added to approximately 1 mL of β-HCG-labeled antibody-modified red latex microspheres. The added blocking solution was a 0.1 M ethanolamine solution. The mixture was mixed for 15-30 minutes, and the supernatant was removed by centrifugation. 1 mL of preservation solution was added to the blocked HCG red latex microspheres. The preservation solution was a 100 mM Tris buffer. The mixture was homogenized by sonication for 4-6 hours, and the supernatant was removed by centrifugation. Finally, 0.5 mL of the final volume of preservation solution was added, and the mixture was sonicated in a water bath for 15 minutes to obtain HCG latex microspheres.

[0055] S4, HCG latex microsphere spray Preparation of conjugate pad treatment solution: 1.13 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.5 g of casein, and 50 μL of Tween-20 were weighed out, placed in a small beaker containing 80 mL of pure water, stirred to dissolve, the pH was adjusted to 8.5, and finally the volume was reduced to 100 mL for use.

[0056] Processing the conjugate pad: Pour the prepared conjugate pad processing solution onto a polyester film, gently pull the polyester film with tweezers to guide the processing solution to be evenly absorbed into the polyester film, and once the polyester film has been evenly absorbed, grasp the two diagonal corners with tweezers and quickly transfer the polyester film to a draining rack. The mesh of the draining rack was placed in an oven and dried at 45°C for 8 hours, with the relative humidity in the oven being ≤30%.

[0057] 20 mg of trehalose was weighed and placed in a 1.5 mL centrifuge tube, with a weighing range of ±0.001 g of the calculated amount. 80 μL of latex diluent and 20 μL of HCG latex microspheres were weighed out and added to the 1.5 mL centrifuge tube containing 20 mg of trehalose, and shaken to ensure uniformity. The latex diluent was Tris-Casein buffer prepared with 100 mM Tris buffer and 2.5 mM Casein. A processed conjugate pad was prepared, and HCG latex microspheres were sprayed onto it using a striping sputtering apparatus at a spray rate of 3 μL / cm. The conjugate pad sprayed with HCG latex microspheres was dried in a 55°C oven for 20 minutes, with the oven relative humidity being ≤30%.

[0058] (3) Manufacturing of coating films Using an antibody coating diluent, which was 0.01 M PBS with trehalose added to a final concentration of 100 mg / mL, mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody were diluted to obtain a goat anti-mouse IgG polyclonal antibody diluent (i.e., C-line coating solution) of 0.5 mg / mL and a mouse anti-α-HCG monoclonal antibody diluent (i.e., T-line coating solution) of 0.5 mg / mL. These were then striped onto a substrate with a nitrocellulose film attached using a striping sputtering apparatus at a spray rate of 1 μL / cm, forming two parallel lines, a uniform and continuous C-line and a T-line, on the film. After striping, the coated film was dried in an oven.

[0059] (4) Manufacturing of test strips S1, Test strip assembly: 1) Cutting: The sample pad was cut to a width of 2 ± 0.1 cm and a length of 30 cm ± 0.2 mm. The HCG latex conjugate pad was cut to a width of 1 ± 0.2 cm and a length of 30 cm ± 0.2 mm. The water filtration pad was cut to a length of 30 cm ± 0.1 mm and a width of 4 mm.

[0060] 2) The substrate was placed flat on the surface of the table, and one side of the water filtration pad was attached to the underside of the nitrocellulose membrane, making contact with the lower edge of the nitrocellulose membrane by 2 mm.

[0061] 3) One side of a conjugate pad (also called a latex conjugate pad) sprayed with HCG latex microspheres was attached to the underside of the other side of the water filter pad, making 2mm contact with the lower edge of the latex conjugate pad.

[0062] 4) One side of the sample pad was attached to the underside of the other side of the latex conjugate pad, making contact with the lower edge of the latex conjugate pad by 2 mm.

[0063] 5) The bonding tape was applied. The bonding tape was used to cover the joint between the sample pad, conjugate pad, and water filtration pad, and the upper edge of the water filtration pad was bonded to the lower edge of the nitrocellulose membrane. The tape was then pressed along one edge using the smooth side of the peeled-off sticker.

[0064] 6) The large circuit boards, once assembled, proceeded to the cutting process.

[0065] S2, Cutting (Clean zone with relative humidity ≤ 30%) The slitter was powered on, the cutting program was set, the cutting width was set to 3.05 mm, the front and end of the large circuit board were connected, and it was slowly placed in the slitter. The large circuit board was placed flat on the slitter's platform rails, with the front facing upwards, the screws on the slitter's platform were adjusted to make the slot width the same as the width of the large circuit board, and cutting was started. Each component of the test strip was complete, undamaged, and unfallen, and the test strip was obtained.

[0066] Example 2 Method for manufacturing a detection card for detecting human chorionic gonadotropins in saliva: The test strips manufactured in Example 1 were assembled into a card (in a clean zone with relative humidity ≤ 30%), i.e., each test strip was assembled into a card case, the top lid was closed, and after the assembly of the detection card was complete and it passed the test, the detection card was obtained.

[0067] Example 3 A kit for detecting early pregnancy, the kit comprising a detection card, a small funnel, and a sample diluent, i.e., 0.9% physiological saline, manufactured in Example 2.

[0068] Example 4 Detection method using the kit of Example 3: 1. Sample collection For 10 minutes before the test, participants were required to keep their mouths clean and refrain from eating or drinking (including beverages, coffee, and food), smoking, or using mouth sprays. The cap of the sample processing tube was opened, the attached saliva funnel was placed over the tube opening, and saliva from the oral cavity (saliva is easily secreted when the tongue coating is placed against the roof of the mouth) was spat into the saliva funnel. The liquid level in the sample processing tube was then observed, and the volume of saliva should have matched the volume of the sample diluent as closely as possible.

[0069] 2. Sample Processing Remove the saliva funnel, close the lid of the sample processing tube, pinch the tube body, and invert it for at least 10 seconds to mix the samples as uniformly as possible.

[0070] 3. Detection The aluminum packaging bag was torn along the perforated line, the detection card was removed and placed flat on a table, and three drops of the processed sample were added to the sampling well of the detection card. Results were judged within 5-10 minutes of the start of timing, and results after 15 minutes were considered invalid.

[0071] 4. Explanation of detection results (1) Positive (Pregnancy): Two red reaction lines appeared, one in the detection area (T) and one in the control area (C). If the color in the detection area (T) was weak, it indicated that the person may have recently become pregnant. The test was re-detected every other day. (2) Negative (not pregnant): One red reaction line appeared, i.e., one red reaction line appeared only in the control area (C). (3) Invalid: No red reaction line appeared in the control area (C), indicating a test error or invalidity.

[0072] Example 5 Sensitivity test of test strips for detecting human chorionic gonadotropins in saliva Human chorionic gonadotropin (HCG) national standards were prepared at four concentrations: 2.5 mIU / mL, 5 mIU / mL, 10 mIU / mL, and 20 mIU / mL. The HCG at these four concentrations was detected using the detection method from Example 4, and the test was repeated 10 times. The results are shown in Table 1.

[0073] Table 1: Results of the test strip sensitivity test [Table 1]

[0074] In Table 1, "+" indicates detection, and "-" indicates non-detection. The results in Table 1 show that the detection rate for concentrations of 5–20 mIU / mL was 100%, and the detection rate for 2.5 mIU / mL was 70%. Therefore, this product has high sensitivity, and the detection limit was 5 mIU / mL.

[0075] Comparative Example 1 This comparative example differs from Example 2 in that 3 g of dithiothreitol was not added during the preparation of the sample pad processing solution; the other steps were the same as in Example 2.

[0076] Comparative Example 2 This comparative example differs from Example 2 in that 1.2 g of L-cysteine ​​was not added during the preparation of the sample pad processing solution; the other steps were the same as in Example 2.

[0077] Comparative Example 3 This comparative example differs from Example 2 in that 3 g of dithiothreitol and 1.2 g of L-cysteine ​​were not added during the preparation of the sample pad processing solution; the other steps were the same as in Example 2.

[0078] Example 6 Specificity test of test strips for detecting human chorionic gonadotropins in saliva Using the detection cards manufactured in Example 2 and Comparative Examples 1-3, the same saliva samples were tested according to the detection method in Example 4, and the results are shown in Figure 2 and Table 2.

[0079] Table 2 Test results for removing nonspecific reactions [Table 2]

[0080] Note: "+++" indicates clearly visible, "++" indicates visible, "+" indicates faintly visible, and "-" indicates not visible.

[0081] The results are shown in Figure 2 and Table 2. When L-cysteine ​​and dithiothreitol are used alone, the problem of specificity due to the viscosity of the saliva sample is improved in both cases, but the objective of completely eliminating nonspecific reactions is not achieved. Furthermore, there is a problem in that the background of the detection area of ​​the test strip is red, indicating the presence of residual microspheres. Satisfactory results were obtained by using both L-cysteine ​​and dithiothreitol reagents in combination. From the comparison in Figure 2, it can be seen that using both L-cysteine ​​and dithiothreitol reagents in combination completely eliminates nonspecific reactions and clearly cleans the background.

[0082] Furthermore, the detection cards produced in Example 2 and Comparative Examples 1-3 were stored at room temperature for 182 days, and the same saliva samples were tested according to the detection method in Example 4. The results are shown in Figure 3 and Table 3.

[0083] Table 3 Test results after storage at room temperature for 182 days [Table 3]

[0084] Note: "+++" indicates clearly visible, "++" indicates visible, "+" indicates faintly visible, and "-" indicates not visible.

[0085] Figure 2 and Table 3 show that when L-cysteine ​​and dithiothreitol were used alone, their effect in eliminating specific reactions was reduced, but when used in combination, they were still able to completely eliminate specific reactions.

[0086] Furthermore, using the detection cards manufactured in Example 2 and Comparative Example 3, 120 negative saliva samples were tested referring to the detection method in Example 4, and the results are shown in Table 4.

[0087] Table 4 Test results of 120 negative saliva samples using different detection cards. [Table 4]

[0088] Table 4 shows that the sample pad processing method for test strips according to the present invention can effectively remove nonspecific reactions and has a very high negative agreement rate.

[0089] Example 7 Actual sample testing of test strips for detecting human chorionic gonadotropins in saliva Saliva samples were collected from 20 non-pregnant women and 8 women diagnosed with pregnancy. The saliva samples were tested using the detection card from Example 2, referring to the detection method from Example 4, and the chromatographic results of the detection card were observed after 5 minutes.

[0090] From the test results in Figures 4 and 5, the detection card detected negative results for saliva samples from 20 non-pregnant women and positive results for saliva samples from 8 pregnant women. After 5 minutes, the chromatographic background was clear, the lines were distinct, and the chromatographic process was smooth. Therefore, the test strip of the present invention has high detection accuracy and reached 100% accuracy.

[0091] The above are merely preferred embodiments of the present invention, and those skilled in the art should point out that further improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A test strip for detecting human chorionic gonadotropins in saliva, wherein the test strip comprises a sample pad, a conjugate pad, a water filtration pad, a coating film, and a substrate, the sample pad, the conjugate pad, the water filtration pad, and the coating film being sequentially layered on the substrate. The aforementioned sample pad is a glass fiber film treated with a sample pad treatment solution. Based on 100 mL, the sample pad processing solution is: A test strip for detecting human chorionic gonadotropins in saliva, characterized by containing Tris 1.0-1.5 g, casein 0.3-0.8 g, Tween-20 50-100 μL, NaCl 0.5-1.5 g, dodecyltrimethylammonium bromide 0.15-0.2 g, 4-nonylphenyl-polyethylene glycol 0.8-1.2 g, borax 3.5-4.0 g, polyvinylpyrrolidone 0.5-1 g, dithiothreitol 3-5 g, and L-cysteine ​​0.8-2 g.

2. The test strip according to claim 1, characterized in that the pH of the sample pad processing solution is 8.0 to 8.

5.

3. The aforementioned coating film is nitrocellulose coated with T-lines and C-lines. A 0.5-1 mg / mL diluted mouse anti-α-HCG monoclonal antibody solution is sprayed onto the T-line. The C line is sprayed with a 0.4-0.6 mg / mL dilution of goat anti-mouse IgG polyclonal antibody. The test strip according to claim 1, characterized in that the spray volume when spraying the T-line or C-line is 0.5 to 1.5 μL / cm.

4. The test strip according to claim 3, characterized in that the mouse anti-α-HCG monoclonal antibody diluent or the goat anti-mouse IgG polyclonal antibody diluent is prepared with a coating diluent, and the coating diluent is a PBS solution containing 50 to 150 mg / mL of trehalose.

5. The aforementioned conjugate pad is a glass fiber membrane or polyester fiber membrane treated with a conjugate pad treatment solution. The test strip according to claim 1, characterized in that the conjugate pad treatment solution contains 1 to 1.5 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.2 to 1 g of casein, 50 to 100 μL of Tween-20, and 75 to 85 mL of water.

6. The test strip according to claim 5, characterized in that HCG latex microspheres are sprayed onto the glass fiber membrane or polyester fiber membrane, and the spray volume is 1.5 to 3 μL / cm.

7. The method for manufacturing the HCG latex microspheres is as follows: The steps include washing red latex microspheres in borax buffer to obtain the red latex microspheres after washing, The process involves mixing β-HCG-labeled antibody with washed red latex microspheres, activating them with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to obtain β-HCG-labeled antibody-modified red latex microspheres, and The test strip according to claim 6, comprising the steps of: mixing an ethanolamine solution with β-HCG-labeled antibody-modified red latex microspheres and blocking them to obtain the blocked HCG red latex microspheres; removing the supernatant by centrifugation; further adding Tris buffer and mixing it uniformly with the blocked HCG red latex microspheres by sonication; removing the supernatant by centrifugation; then adding Tris buffer and mixing it uniformly by sonication; and further rotating it at room temperature for 4 to 6 hours to obtain HCG latex microspheres.

8. (1) Manufacturing of sample pads The process involves uniformly absorbing the sample pad processing solution into a glass fiber membrane, then drying it to obtain a sample pad, and (2) Manufacturing of conjugate pads The process involves uniformly absorbing a conjugate pad treatment solution into a glass fiber membrane or polyester fiber membrane, drying it, and then spraying HCG latex microspheres onto the glass fiber membrane or polyester fiber membrane treated with the conjugate pad treatment solution at a spray rate of 1.5 to 3 μL / cm to obtain a conjugate pad. (3) Manufacturing of coating films The process involves diluting mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody with a coating diluent to obtain diluted mouse anti-α-HCG monoclonal antibody solution and diluted goat anti-mouse IgG polyclonal antibody solution, spraying the diluted mouse anti-α-HCG monoclonal antibody solution onto the T-line of a nitrocellulose membrane, spraying the diluted goat anti-mouse IgG polyclonal antibody solution onto the C-line of a nitrocellulose membrane, drying, and obtaining a coated membrane. (4) Manufacturing of test strips A method for manufacturing a test strip according to any one of claims 1 to 7, comprising the steps of cutting a sample pad, a conjugate pad, and a water filtration pad, sequentially attaching the sample pad, conjugate pad, water filtration pad, and coating film to a substrate, wherein one side of the conjugate pad is positioned above the sample pad, the other side of the conjugate pad is positioned below one side of the water filtration pad, the other side of the water filtration pad is positioned below the coating film, and the T-line on the coating film is close to the water filtration pad.

9. The method for manufacturing the HCG latex microspheres is as follows: The steps include washing red latex microspheres in borax buffer to obtain the red latex microspheres after washing, The process involves mixing β-HCG-labeled antibody with washed red latex microspheres, activating them with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to obtain β-HCG-labeled antibody-modified red latex microspheres, and The manufacturing method according to claim 7, characterized by comprising the steps of: mixing an ethanolamine solution with β-HCG-labeled antibody-modified red latex microspheres and blocking the mixture to obtain the blocked HCG red latex microspheres; removing the supernatant by centrifugation; further adding Tris buffer and mixing it uniformly with the blocked HCG red latex microspheres by sonication; removing the supernatant by centrifugation; then adding Tris buffer and mixing it uniformly by sonication; and further rotating the mixture at room temperature for 4 to 6 hours to obtain HCG latex microspheres.

10. The manufacturing method according to claim 7, characterized in that the addition ratio of the β-HCG-labeled antibody, the washed red latex microspheres, and the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1 mg:10 mg:0.05 mL.

11. The manufacturing method according to claim 7, characterized in that the volume ratio of the red latex microspheres to the borax buffer is 0.1 to 0.2:1, and the concentration of the borax buffer is 0.05 to 0.15 M.

12. The manufacturing method according to claim 7, characterized in that the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 5 to 15 mg / mL, the concentration of the ethanolamine solution is 0.08 to 0.12 M, and the concentration of the Tris buffer is 50 to 100 mM.

13. The manufacturing method according to claim 7, characterized in that the blocking time is 15 to 30 minutes and the ultrasonic treatment time is 10 to 20 minutes.

14. A kit for detecting early pregnancy, characterized in that the kit comprises a test strip and a sample diluent as described in any one of claims 1 to 7.

15. The kit according to claim 14, characterized in that the sample diluent is 0.9% physiological saline.

16. (1) Use of the test strip or the manufacturing method for the detection of human chorionic gonadotropins, (2) Use of the test strip or the manufacturing method in the manufacture of a product for detecting human chorionic gonadotropins, (3) Use of the test strip or the manufacturing method for the detection of early pregnancy, (4) Using the test strip or the manufacturing method described in any one of claims 1 to 7 in the manufacture of a product for detecting early pregnancy, the use of the test strip or the manufacturing method described in any one of claims 8 to 13, comprising at least one of these.

17. The criteria for detecting whether or not a woman is pregnant are: (1) Positive: Two red reaction lines appear, namely one red reaction line in the detection area and one in the control area. (2) Negative: A single red reaction line appears, that is, a single red reaction line appears only in the control area, (3) Invalid: The use according to claim 16, characterized in that no red reaction line appears in the control area, indicating a test error or invalidity.

18. The use according to claim 16, characterized in that the detection sample is saliva.

19. The use according to claim 16, characterized in that the product includes a kit or a detection card.

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