Test strip for detecting human chorionic gonadotropin in saliva and preparation method therefor

Abstract

A test strip for detecting human chorionic gonadotropin in saliva and a preparation method therefor. Calculated based on 100 mL, a sample pad treatment liquid comprises the following ingredients: 1.0-1.5 g of Tris, 0.3-0.8 g of casein, 50-100 μL of Tween-20, 0.5-1.5 g of NaCl, 0.15-0.2 g of dodecyltrimethylammonium bromide, 0.8-1.2 g of 4-nonylphenyl-polyethylene glycol, 3.5-4.0 g of borax, 0.5-1 g of polyvinylpyrrolidone, 3-5 g of dithiothreitol and 0.8-2 g of L-cysteine. The sample pad treatment liquid is used to treat a sample pad, so as to reduce the interference of other impurity ingredients in saliva, thereby achieving the effect of completely eliminating non-specific reactions. The test strip has high sensitivity and good stability, can be stored for a long time at room temperature, and has the advantages of being cleaner and more convenient, allowing testing to take place at any time, being unaffected by sample collection time and place, etc.

Description

Test strip for detecting human chorionic gonadotropin in saliva and preparation method thereof Technical Field

[0001] The invention belongs to the technical field of test strip detection, and in particular relates to a test strip for detecting human chorionic gonadotropin in saliva and a preparation method thereof. Background Art

[0002] Human chorionic gonadotropin (HCG) is a glycoprotein secreted by the trophoblast cells of the placenta. It is composed of α and β dimers of glycoproteins. The α-subunit is common to all anterior pituitary hormones; the β-subunit is specific to HCG. The main function of HCG is to stimulate the corpus luteum, which is conducive to the continuous secretion of estrogen and progesterone to promote the formation of the uterine decidua and the growth and maturation of the placenta. Modern thinking is that HCG is produced by trophoblast transitional cells and syncytial cells. It proliferates rapidly in the first 8 weeks of pregnancy to maintain pregnancy. After about 8 weeks of pregnancy, HCG gradually decreases until it reaches relative stability at about 20 weeks. The use of HCG double antibodies to detect the HCG content in the urine of women of pregnant age can quickly obtain results in early pregnancy and is an effective means of auxiliary diagnosis.

[0003] Saliva is a clinically rich biofluid containing a variety of soluble biomarkers, making it amenable to rapid testing or centralized clinical laboratory procedures in a more standardized format. The use of saliva for in vitro testing is not uncommon. Quantitative detection of steroid hormones such as cortisol, estriol, and testosterone using saliva has been widely used, and a large number of rapid saliva-based HIV test strips are currently available in China. Importantly, saliva closely correlates with corresponding components in blood, serving as a window to the blood. Experimental results have shown that human chorionic gonadotropin concentrations correlate well with blood levels. Therefore, saliva could be a non-invasive, rapid, and more acceptable biofluid for pregnancy testing. The secretion mechanism of human chorionic gonadotropin in saliva may be that saliva is a hypotonic fluid, and capillary capillaries and lymph nodes are tightly connected to the basement membrane of the gland. Human chorionic gonadotropin can migrate from the blood into saliva through the intercellular spaces and tight junctions. Domestic hospitals conducted clinical tests on human chorionic gonadotropin in saliva. Urine and saliva samples were tested simultaneously under the same conditions. The results showed that not only HCG is contained in the urine of pregnant women, but also in their saliva. The test results of the two samples were compared and correlated well, and the test results were consistent.

[0004] Saliva pregnancy tests offer numerous advantages, including cleaner, more convenient, and anytime testing, regardless of sample collection time. However, saliva pregnancy test strips are not often discussed. Development challenges stem from the presence of numerous non-detectable interferences in saliva samples, low sensitivity, chromatographic difficulties, and nonspecificity. For example, under normal circumstances, human hCG levels are less than 5mIU / mL. When HCG levels exceed 5mIU / mL in women of gestational age, pregnancy is possible. However, commercially available urine pregnancy test strips often have a detection limit of 25mIU / mL, preventing pregnancy from being detected earlier. In the early stages of pregnancy, such as within the first week of pregnancy, if HCG levels are below 25mIU / mL, a test strip cannot be detected. Furthermore, both urine and urine dropper methods involve the risk of dirty hands. Urine testing can also wet the detection window, potentially affecting chromatographic results. Furthermore, urine testing is also subject to time and location constraints. In addition, when the sample type of the test strip is saliva, the saliva sample is relatively viscous, and the flow rate of saliva is slow when it is chromatographed upward from the sample pad. There are even problems such as latex microspheres being blocked on the nitrate membrane, liquid chromatography being halfway, and false positives.

[0005] Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a test strip for detecting human chorionic gonadotropin in saliva and a preparation method thereof. The test strip is clean and convenient for detecting early pregnancy and has high detection sensitivity and strong specificity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a test strip for detecting human chorionic gonadotropin in saliva, wherein the test strip comprises a sample pad, a conjugation pad, a water filter pad, a coating membrane and a bottom plate, wherein the sample pad, the conjugation pad, the water filter pad and the coating membrane are sequentially overlapped on the bottom plate;

[0009] The sample pad is a glass fiber membrane treated with a sample pad treatment solution;

[0010] The sample pad treatment solution includes the following components in 100 mL:

[0011] Tris 1.0-1.5 g, casein 0.3-0.8 g, Tween-2050-100 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.

[0012] Preferably, the pH of the sample pad treatment solution is 8.0-8.5.

[0013] Preferably, the coating membrane is nitrocellulose coated with T lines and C lines;

[0014] The T line is sprayed with 0.5-1 mg / mL of mouse anti-α-HCG monoclonal antibody dilution;

[0015] The C line is sprayed with 0.4-0.6 mg / mL goat anti-mouse IgG polyclonal antibody dilution solution;

[0016] The spray volume during T-line spraying or C-line spraying is 0.5 to 1.5 μL / cm.

[0017] Preferably, the mouse anti-α-HCG monoclonal antibody diluent or the goat anti-mouse IgG polyclonal antibody diluent is prepared by preparing a coating diluent; the coating diluent is a PBS solution containing 50-150 mg / mL trehalose.

[0018] Preferably, the binding pad is a glass fiber membrane or a polyester fiber membrane treated with a binding pad treatment solution;

[0019] The conjugate pad treatment solution includes 1-1.5 g of 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, 0.2-1 g of casein, 50-100 μL of Tween-20, and 75-85 mL of water.

[0020] Preferably, the volume ratio of the red latex microspheres to the borax buffer solution is 0.1-0.2:1; and the concentration of the borax buffer solution is selected to be 0.05-0.15M.

[0021] 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 is 50 to 100 mM.

[0022] Preferably, the sealing time is 15 to 30 minutes; the ultrasonic time is 10 to 20 minutes.

[0023] The present invention also provides a test strip prepared according to the above preparation method.

[0024] Preferably, HCG latex microspheres are sprayed on the glass fiber membrane or polyester fiber membrane, and the spraying volume is 1.5 to 3 μL / cm.

[0025] The present invention also provides a method for preparing the above-mentioned test strip, comprising the following steps:

[0026] (1) Preparation of sample pad

[0027] After the glass fiber membrane absorbs the sample pad treatment liquid evenly, it is dried to obtain the product;

[0028] (2) Preparation of conjugate pad

[0029] After the glass fiber membrane or polyester fiber membrane absorbs the binding pad treatment liquid evenly, it is dried, and HCG latex microspheres are sprayed on the glass fiber membrane or polyester fiber membrane treated with the binding pad treatment liquid at a spray volume of 1.5 to 3 μL / cm;

[0030] (3) Preparation of coating membrane

[0031] Dilute mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody with coating diluent respectively to obtain mouse anti-α-HCG monoclonal antibody dilution solution and goat anti-mouse IgG polyclonal antibody dilution solution, spray the mouse anti-α-HCG monoclonal antibody dilution solution on the T line of the nitrocellulose membrane, and spray the goat anti-mouse IgG polyclonal antibody dilution solution on the C line of the nitrocellulose membrane, and dry them to obtain;

[0032] (4) Preparation of test strips

[0033] After cutting the sample pad, conjugate pad and water filter pad respectively, the sample pad, conjugate pad, water filter pad and coating membrane are sequentially pasted on the bottom plate, one side of the conjugate pad is located above the sample pad, the other side of the conjugate pad is located below the one side of the water filter pad, and the other side of the water filter pad is located below the coating membrane, and the T line on the coating membrane is close to the water filter pad.

[0034] Preferably, the preparation method of the HCG latex microspheres comprises the following steps:

[0035] washing the red latex microspheres with borax buffer to obtain washed red latex microspheres;

[0036] The β-HCG labeled antibody is mixed with the washed red latex microspheres, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution is added for activation to obtain the β-HCG labeled antibody modified red latex microspheres;

[0037] The ethanolamine solution was mixed with the red latex microspheres modified with β-HCG labeled antibodies to obtain blocked HCG red latex microspheres. The supernatant was removed by centrifugation, and Tris buffer was added to the blocked HCG red latex microspheres and ultrasonically mixed. The supernatant was removed by centrifugation, and Tris buffer was added to ultrasonically mix. The mixture was then rotated and mixed at room temperature for 4 to 6 hours to obtain HCG latex microspheres.

[0038] Preferably, 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.

[0039] The present invention also provides a kit for detecting early pregnancy, which comprises the above-mentioned test strip and a sample diluent.

[0040] Preferably, the sample diluent is 0.9% physiological saline.

[0041] The present invention also provides an application of the above-mentioned test strip or the above-mentioned preparation method, comprising at least one of the following:

[0042] (1) The test strip or the preparation method is used to detect human chorionic gonadotropin;

[0043] (2) Use of the test strip or the preparation method in preparing a product for detecting human chorionic gonadotropin;

[0044] (3) The test strip or the preparation method is used to detect early pregnancy;

[0045] (4) Application of the test strip or the preparation method in the preparation of products for detecting early pregnancy.

[0046] Preferably, the criteria for determining whether a woman is pregnant are:

[0047] (1) Positive: Two red reaction lines, i.e. one red reaction line appears in the test area and one red reaction line appears in the control area;

[0048] (2) Negative: a red reaction line, that is, a red reaction line appears only in the control area;

[0049] (3) Invalid: No red reaction line appears in the control area, indicating that the test is wrong or invalid.

[0050] Preferably, the test sample is saliva.

[0051] Preferably, the product comprises a kit or a test card.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The present invention provides a test strip for detecting human chorionic gonadotropin in saliva and a method for preparing the same. The present invention directly collects saliva and treats the sample pad with a sample pad treatment solution, thereby increasing the water solubility of the saliva sample, smoothing the chromatography of the saliva sample, reducing interference from other impurities in the saliva, eliminating false positives in saliva sample testing, and completely eliminating nonspecific reactions. The test strip of the present invention also has good stability and can be stored for long periods of time at room temperature. The test strip of the present invention has a detection limit of 5mIU / mL, can detect pregnancy earlier, and can be used as a potential, user-friendly, and highly acceptable product with numerous advantages, such as being cleaner, more convenient, allowing for testing at any time, and being unaffected by the time and location of sample collection. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 is a schematic diagram of using the test strip of the present invention to detect whether a woman is pregnant;

[0055] FIG2 shows the specificity test results of different test cards for detecting human chorionic gonadotropin in saliva. From left to right in the figure, the test results using the test cards of Comparative Example 3, Comparative Example 2, Comparative Example 1, and Example 2 are shown;

[0056] FIG3 shows the specificity test results of different test cards for detecting human chorionic gonadotropin in saliva after storage at room temperature for 182 days. From left to right in the figure, the test results using the test cards of Comparative Example 2, Comparative Example 1, and Example 2 are shown;

[0057] Figure 4 shows the test results of 20 negative saliva samples;

[0058] Figure 5 shows the test results of 8 positive samples. DETAILED DESCRIPTION

[0059] The present invention provides a test strip for detecting human chorionic gonadotropin in saliva, wherein the test strip comprises a sample pad, a conjugation pad, a water filter pad, a coating membrane and a bottom plate, wherein the sample pad, the conjugation pad, the water filter pad and the coating membrane are sequentially overlapped on the bottom plate;

[0060] The sample pad is a glass fiber membrane treated with a sample pad treatment solution;

[0061] The sample pad treatment solution includes the following components in 100 mL:

[0062] Tris 1.0-1.5 g, casein 0.3-0.8 g, Tween-2050-100 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.

[0063] 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 filter pad is purchased from Waterman, item number 8151-6621.

[0064] In the present invention, the sample pad treatment solution preferably further comprises 750-850 mL of water, and the pH of the sample pad treatment solution is preferably 8.0-8.5. The sample pad treatment solution is preferably prepared by weighing 1.0-1.5 g of Tris, 0.3-0.8 g of casein, 50-100 μL of Tween-20, 0.5-1.5 g of NaCl, 0.15-0.2 g of dodecyltrimethylammonium bromide, 0.8-1.2 g of 4-nonylphenyl-polyethylene glycol, 3.5-4.0 g of borax, and 0.5-1 g of polyvinylpyrrolidone in sequence, adding 750-850 mL of water, stirring to dissolve, then adding 3-5 g of dithiothreitol and 0.8-2 g of L-cysteine, stirring to dissolve, adjusting the pH to 8.0-8.5, and finalizing the volume to 100 mL to obtain the sample pad treatment solution. The combined use of dithiothreitol and L-cysteine ​​in the sample pad treatment liquid can achieve the purpose of eliminating nonspecific reactions in the chromatography process. At the same time, dithiothreitol has an antioxidant effect that can prevent L-cysteine ​​from being oxidized by air to form cystine and form a precipitate, which has a synergistic effect. The two reagents of dithiothreitol and L-cysteine ​​are usually formulated into solutions for use. The solutions are easily oxidized, which is not conducive to storage and has a short shelf life. They usually need to be used as soon as possible or placed at a low temperature. For products of this type of rapid detection reagent, frozen storage conditions are obviously impractical. Therefore, the present invention places the above two reagents and other sample pad treatment liquid reagent formulas in the sample pad, which can effectively solve the problem of short shelf life of the reagents, and can be stored for a long time at room temperature, with a shelf life of more than 6 months. The 4-nonylphenyl-polyethylene glycol was purchased from the wetting agent P-40 of Jiangsu Pulesi Biotechnology Co., Ltd.

[0065] The test strip of the present invention uses a novel sample pad processing method. Saliva drips into the sample pad and reacts with the active ingredients in the sample pad, reducing the viscosity of the saliva and making the chromatography very smooth without membrane clogging. This increases the specificity of the test strip, reduces interference from other components in saliva, and avoids the false positive phenomenon caused by saliva samples.

[0066] In the present invention, the coating membrane is preferably nitrocellulose coated with T-lines and C-lines; the T-lines are preferably sprayed with 0.5-1 mg / mL of mouse anti-α-HCG monoclonal antibody dilution; the C-lines are preferably sprayed with 0.4-0.6 mg / mL of goat anti-mouse IgG polyclonal antibody dilution; the spray volume when spraying the T-line or C-line is preferably 0.5-1.5 μL / cm. The mouse anti-α-HCG monoclonal antibody dilution or the goat anti-mouse IgG polyclonal antibody dilution is prepared by coating diluent; the coating diluent is a PBS solution containing 50-150 mg / mL trehalose. The concentration of the PBS solution is preferably 0.005-0.015 M. The interval between the T-line and the C-line on the coating membrane is preferably 0.4-0.6 cm. The present invention increases sensitivity while reducing the occurrence of nonspecific reactions by screening the above-mentioned antibody raw materials. In the present invention, the mouse anti-α-HCG monoclonal antibody was purchased from Nanjing Jingda Biotechnology Co., Ltd. with the item number W4003; the mouse anti-α-HCG monoclonal antibody was purchased from AoChuang Biotechnology (Shandong) Co., Ltd. with the item number A01-Ab2.

[0067] 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;

[0068] In the present invention, the conjugate pad treatment solution preferably includes 1-1.5 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.2-1 g of casein, 50-100 μL of Tween-20, and 75-85 mL of water. The pH of the conjugate pad treatment solution is preferably 8.0-8.5. The conjugate pad treatment solution is prepared by weighing 1-1.5 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.2-1 g of casein, 50-100 μL of Tween-20, and 75-85 mL of water, stirring to dissolve, adjusting the pH to 8.0-8.5, and diluting to 100 mL. As a preferred embodiment of the present invention, HCG latex microspheres are sprayed on the glass fiber membrane or polyester fiber membrane, and the spraying volume is 1.5-3 μL / cm. The 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid was purchased from Adamas, product number 76496B.

[0069] In the present invention, the width of the binding pad is preferably 1±0.2 cm, and the length is preferably 30±0.2 cm.

[0070] The present invention also provides a method for preparing the above-mentioned test strip, comprising the following steps:

[0071] (1) Preparation of sample pad

[0072] After the glass fiber membrane absorbs the sample pad treatment liquid evenly, it is dried to obtain the product;

[0073] (2) Preparation of conjugate pad

[0074] After the glass fiber membrane or polyester fiber membrane absorbs the binding pad treatment liquid evenly, it is dried, and HCG latex microspheres are sprayed on the glass fiber membrane or polyester fiber membrane treated with the binding pad treatment liquid at a spray volume of 1.5 to 3 μL / cm;

[0075] (3) Preparation of coating membrane

[0076] Dilute mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody with coating diluent respectively to obtain mouse anti-α-HCG monoclonal antibody dilution solution and goat anti-mouse IgG polyclonal antibody dilution solution, spray the mouse anti-α-HCG monoclonal antibody dilution solution on the T line of the nitrocellulose membrane, and spray the goat anti-mouse IgG polyclonal antibody dilution solution on the C line of the nitrocellulose membrane, and dry them to obtain;

[0077] (4) Preparation of test strips

[0078] After cutting the sample pad, conjugate pad, and water filter pad respectively, the sample pad, conjugate pad, water filter pad, and coating membrane are sequentially pasted on the bottom plate, with one side of the conjugate pad located above the sample pad, the other side of the conjugate pad located below one side of the water filter pad, and the other side of the water filter pad located below the coating membrane, with the T line on the coating membrane close to the water filter pad.

[0079] In the present invention, the sample pad is prepared by uniformly absorbing the sample pad treatment solution on the glass fiber membrane and then drying the sample pad. The drying method is preferably drying at 40-45° C. for more than 2 hours.

[0080] In the present invention, the glass fiber membrane or polyester fiber membrane absorbs the bonding pad treatment liquid uniformly and is then dried, preferably at 40-45° C. for 8-12 hours.

[0081] In the present invention, HCG latex microspheres are sprayed on a glass fiber membrane or polyester fiber membrane treated with a conjugated pad treatment liquid at a spray rate of 1.5 to 3 μL / cm. The preparation method of the HCG latex microspheres comprises the following steps: washing red latex microspheres with a borax buffer to obtain washed red latex microspheres; mixing β-HCG labeled antibodies with the washed red latex microspheres, adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution for activation, to obtain β-HCG labeled antibody modified red latex microspheres; mixing ethanolamine solution with the β-HCG labeled antibody modified red latex microspheres for blocking, to obtain blocked HCG red latex microspheres, centrifuging to remove the supernatant, then adding Tris buffer and ultrasonically mixing the blocked HCG red latex microspheres, then rotating and mixing at room temperature for 4 to 6 hours, centrifuging to remove the supernatant, and then adding Tris buffer and ultrasonically mixing to obtain HCG latex microspheres.

[0082] In the present 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 cleaning method is preferably centrifugation at 13500 rpm for 10 to 20 minutes, removing the supernatant, and then adding 0.5 to 1.5 mL of borax buffer to the red latex microspheres after centrifugal washing, and ultrasonically mixing. The red latex microspheres are purchased from Suzhou Weidu Biotechnology Co., Ltd., item number DR0200CA-1.

[0083] In the present invention, β-HCG labeled antibody is mixed with cleaned red latex microspheres, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) solution is added for activation to obtain β-HCG labeled antibody modified red latex microspheres. The addition ratio of the β-HCG labeled antibody, cleaned 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 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution. The 5-15 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is preferably prepared by dissolving 5-15 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution in 1 mL of purified water. The activation method is preferably vortex mixing followed by mixing at room temperature for 4-8 hours. The β-HCG-labeled antibody was purchased from Chuang Biotechnology (Shandong) Co., Ltd., catalog number A01-Ab1.

[0084] In the present invention, an ethanolamine solution is mixed with red latex microspheres modified with β-HCG labeled antibodies to obtain blocked HCG red latex microspheres. 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.

[0085] In the present invention, Tris buffer is added to the blocked HCG red latex microspheres, ultrasonically mixed, and then rotated and mixed at room temperature for 4 to 6 hours. The Tris buffer is preferably 50 to 100 mM Tris buffer.

[0086] In the present invention, Tris buffer is then added and ultrasonically homogenized to obtain HCG latex microspheres. The volume of the added Tris buffer is preferably 0.4 to 0.8 mL, and the ultrasonic time is preferably 10 to 20 minutes.

[0087] In the present invention, after spraying the HCG latex microspheres, a drying step at 37-55° C. for 20-60 minutes is also included.

[0088] The test strip of the present invention does not adopt the colloidal gold method but selects latex microspheres for labeling, and simultaneously selects red latex microspheres with a particle size of 200 nm to eliminate the influence caused by the viscosity of some saliva samples.

[0089] The present invention uses the above test strips to detect whether the standard for determining whether a woman is pregnant is (as shown in FIG1 ):

[0090] (1) Positive (pregnancy): Two red reaction lines, one in the test area (T) and one in the control area (C). If the color of the test area (T) is very weak, it means you may have just gotten pregnant. Please test again the next day.

[0091] (2) Negative (not pregnant): one red reaction line, that is, only one red reaction line appears in the control area (C).

[0092] (3) Invalid: No red reaction line appears in the control area (C), indicating that the test is wrong or invalid.

[0093] When HCG levels exceed 5mIU / mL in women of gestational age, pregnancy is possible. In the early stages of pregnancy, such as within 10 days, HCG levels above 5mIU / mL but below 25mIU / mL cannot be detected by commonly used, less sensitive test strips. The test strips of the present invention have a minimum detection limit of 5mIU / mL, enabling earlier pregnancy detection. The minimum detection limit of the present invention refers to the lowest concentration with a detection rate of ≥95%.

[0094] The present invention also provides a kit for detecting early pregnancy, which comprises the above-mentioned test strip and a sample diluent.

[0095] The sample diluent is preferably 0.9% normal saline, which increases the liquefaction effect of the saliva sample. During testing, the added volume ratio of the saliva sample to the sample diluent is 1:1. The kit of the present invention also includes a small funnel for collecting saliva. The cap of the sample processing tube is opened, and the matching saliva funnel is placed at the tube mouth to spit saliva from the mouth into the saliva funnel. The saliva funnel is removed, and the cap of the sample processing tube is replaced. After the sample is evenly mixed, the sample can be dripped for testing. When the kit of the present invention is used for testing, the result can be read in 5 minutes. It is hygienic, convenient, and can be tested at any time, regardless of the time and place of sample collection.

[0096] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0097] Example 1

[0098] A method for preparing a test strip for detecting human chorionic gonadotropin in saliva comprises the following steps:

[0099] (1) Preparation of sample pad

[0100] Preparation of sample pad treatment solution: prepare a 100mL small beaker, measure 800mL of purified water, weigh 1.21g of Tris, 0.5g of casein, 80μL of Tween-20, 0.9g of NaCl, 0.15g of dodecyltrimethylammonium bromide, 1.0g of 4-nonylphenyl-polyethylene glycol, 3.811g of borax, and 0.8g of polyvinylpyrrolidone, put them into the small beaker in sequence, stir and dissolve them one by one, and then add the next reagent. After all the above materials are dissolved, add 3g of dithiothreitol and 1.2g of L-cysteine, stir and dissolve, adjust the pH to 8.5, and finally make up to 100mL for use.

[0101] Prepare the treatment liquid according to the sample pad treatment liquid formula, pour it on a piece of glass fiber, gently scrape the glass fiber with tweezers to guide the treatment liquid to be evenly absorbed by the glass fiber membrane. After the glass fiber membrane is evenly absorbed, clamp the two diagonal corners with tweezers and quickly move the glass fiber to the drain rack; place the drain rack mesh in an oven at 45℃ and dry for 4 hours until the excess water evaporates. The relative humidity of the oven is ≤30%.

[0102] (2) Preparation of conjugate pad

[0103] S1. Cleaning red latex microspheres

[0104] Add 0.1 mL of 200 nm red latex microspheres to a 2 mL centrifuge tube containing 1 mL of 0.1 M borax buffer and mix thoroughly. Centrifuge at 14,000 rpm for 15 minutes. Remove the supernatant with a pipette. Rinse the red latex microsphere pellet again and add 1 mL of 0.1 M borax buffer to the centrifuge tube. Use an ultrasonic cell disruptor to sonicate the microspheres thoroughly.

[0105] S2. Label β-HCG into the washed red latex microspheres

[0106] Use a pipette to transfer 0.5 mg of β-HCG labeled antibody. The amount of labeled antibody transferred is calculated based on a final concentration of 1 mg / mL and added to 1 mL of washed red latex microspheres. Then add 25 μL of latex microsphere activator, which is an 8 mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution. After shaking and mixing for 1 minute, mix on a shaker or rotator at room temperature for 5 hours to obtain red latex microspheres modified with β-HCG labeled antibodies.

[0107] S3, closed

[0108] Add 50 μL of blocking solution to about 1 mL of β-HCG labeled antibody modified red latex microspheres. The added blocking solution is 0.1 M ethanolamine solution. Mix for 15 to 30 minutes. Centrifuge to remove the supernatant. Add 1 mL of preservation solution to the blocked HCG red latex microspheres. The preservation solution is 100 mM Tris buffer. Ultrasonicate, mix for 4 to 6 hours, centrifuge to remove the supernatant, and finally add 0.5 mL of final volume of preservation solution. Ultrasonicate in a water bath for 15 minutes to obtain HCG latex microspheres.

[0109] S4, spraying HCG latex microspheres

[0110] Preparation of conjugate pad treatment solution: Weigh 1.13 g of 3-(N-morpholino)-2-hydroxypropanesulfonic acid, 0.5 g of casein, and 50 μL of Tween-20 into a small beaker filled with 80 mL of purified water, stir to dissolve, adjust the pH to 8.5, and finally dilute to 100 mL for use.

[0111] Treatment of the conjugate pad: Prepare the conjugate pad treatment solution and pour it onto a polyester film. Use tweezers to gently scrape the polyester film to guide the treatment solution to be evenly absorbed by the polyester film. After the polyester film has absorbed the solution evenly, use tweezers to pinch the two diagonal corners and quickly move the polyester film to a drain rack.

[0112] Place the drain rack mesh in an oven at 45°C for 8 hours and dry it with the relative humidity of the oven ≤ 30%.

[0113] Weigh 20 mg of trehalose into a 1.5 mL centrifuge tube within ±0.001 g of the calculated amount. Add 80 μL of latex diluent (Tris-Casein buffer, 100 mM Tris buffer and 2.5 mM Casein) and 20 μL of HCG latex microspheres to the 1.5 mL centrifuge tube containing 20 mg of trehalose and shake thoroughly. Spray the treated conjugate pad with HCG latex microspheres using a gold sprayer at a rate of 3 μL / cm. After spraying, dry the conjugate pad in a 55°C oven for 20 minutes with a relative humidity of ≤30%.

[0114] (3) Preparation of coating membrane

[0115] Use antibody coating diluent, the coating diluent is 0.01M PBS and trehalose with a final concentration of 100 mg / mL, dilute mouse anti-α-HCG monoclonal antibody and goat anti-mouse IgG polyclonal antibody respectively, and obtain goat anti-mouse IgG polyclonal antibody diluent, i.e. C-line coating solution 0.5 mg / mL, and mouse anti-α-HCG monoclonal antibody diluent, i.e. T-line coating solution 0.5 mg / mL. Use the film spray marking device to mark the bottom plate with nitrocellulose membrane at a spray volume of 1 μL / cm. Two uniform and continuous C and T parallel lines are on the membrane. After marking, place the coating membrane in a drying oven to dry.

[0116] (4) Preparation of test strips

[0117] S1. Test strip assembly:

[0118] 1) Cutting: Cut the sample pad to 2±0.1cm in width and 30cm±0.2 in length; cut the HCG latex binding pad to 1±0.2cm in length and 30cm±0.2 in length; and cut the water filter pad to 30cm±0.1 in length and 4mm in width.

[0119] 2) Place the bottom plate flat on the table and place one side of the filter pad under the nitrocellulose membrane, with a 2mm contact with the bottom edge of the nitrocellulose membrane;

[0120] 3) Place one side of the HCG latex microsphere-sprayed bonding pad (also called latex bonding pad) under the other side of the filter pad, with a 2mm contact with the bottom edge of the latex bonding pad;

[0121] 4) Place one side of the sample pad under the other side of the latex binding pad, with a 2mm contact with the bottom edge of the latex binding pad;

[0122] 5) Apply the connecting tape. Cover the joints between the sample pad, conjugate pad, and filter pad with the connecting tape. Connect the upper edge of the filter pad to the lower edge of the nitrocellulose membrane. Use the smooth side of the torn sticker to press along one side of the tape.

[0123] 6) The assembled large board enters the strip cutting process.

[0124] S2, Strip Cutting (clean area with relative humidity ≤ 30%)

[0125] Turn on the strip cutter, set the cutting program, and set the cutting width to 3.05mm. Place the large board end to end and slowly place it on the film cutter. Place the large card flat on the track of the film cutter platform, facing up. Adjust the screws on the strip cutter platform to adjust the card slot width to the same as the large board width, and begin cutting. All components of the test strip should be intact and not broken or falling off. This is the test strip.

[0126] Example 2

[0127] A method for preparing a test card for detecting human chorionic gonadotropin in saliva: the test strips prepared in Example 1 are loaded into the card (in a clean area with a relative humidity of ≤30%), that is, the test strips are loaded into the card housing, the upper cover is closed, and after the test card is assembled and passed the inspection, the test card is obtained.

[0128] Example 3

[0129] A kit for detecting early pregnancy, comprising the test card prepared in Example 2, a small funnel, and a sample diluent, namely 0.9% physiological saline.

[0130] Example 4

[0131] Detection method using the kit of Example 3:

[0132] 1. Sample Collection

[0133] Keep your mouth clean 10 minutes before the test and limit eating (including drinks, coffee, food, etc.), smoking, or mouth sprays.

[0134] Open the cap of the sample processing tube, place the matching saliva funnel at the tube mouth, spit the saliva in the mouth (the tongue coating is against the palate to facilitate saliva secretion) into the saliva funnel, observe the liquid level in the sample processing tube, and the saliva volume should be as consistent as possible with the sample diluent.

[0135] 2. Sample Processing

[0136] Remove the saliva funnel, cover the sample processing tube with the cap, pinch the tube body, and mix it upside down for at least 10 seconds to mix the sample as evenly as possible.

[0137] 3. Detection

[0138] Tear open the aluminum bag along the cutout, remove the test card, and place it flat on a table. Add three drops of the processed sample to the sample well on the card. Start the timer and determine the result within 5-10 minutes. Results after 15 minutes will be invalid.

[0139] 4. Interpretation of test results

[0140] (1) Positive (pregnancy): Two red reaction lines, one in the test area (T) and one in the control area (C). If the color of the test area (T) is very weak, it means you may have just gotten pregnant. Please test again the next day.

[0141] (2) Negative (not pregnant): one red reaction line, that is, only one red reaction line appears in the control area (C).

[0142] (3) Invalid: No red reaction line appears in the control area (C), indicating that the test is wrong or invalid.

[0143] Example 5

[0144] Sensitivity test of test strips for detecting human chorionic gonadotropin in saliva

[0145] The national standard of human chorionic gonadotropin (HCG) was prepared into four concentrations: 2.5mIU / mL, 5mIU / mL, 10mIU / mL, and 20mIU / mL. The HCG at these four concentrations was detected using the detection method of Example 4. The test was repeated 10 times. The results are shown in Table 1.

[0146] Table 1 Sensitivity test results of test strips

[0147] In Table 1, “+” indicates detection, and “-” indicates non-detection.

[0148] The results in Table 1 show that the detection rate for concentrations of 5-20mIU / mL is 100%, and the detection rate for 2.5mIU / mL is 70%. Therefore, this product has high sensitivity, with a detection limit of 5mIU / mL.

[0149] Comparative Example 1

[0150] The difference between this comparative example and Example 2 is that 3 g of dithiothreitol is not added when preparing the sample pad treatment solution in this comparative example, and the remaining steps are the same as those in Example 2.

[0151] Comparative Example 2

[0152] The difference between this comparative example and Example 2 is that 1.2 g of L-cysteine ​​is not added when preparing the sample pad treatment solution in this comparative example, and the remaining steps are the same as those in Example 2.

[0153] Comparative Example 3

[0154] The difference between this comparative example and Example 2 is that 3 g of dithiothreitol and 1.2 g of L-cysteine ​​are not added when preparing the sample pad treatment solution in this comparative example, and the remaining steps are the same as those in Example 2.

[0155] Example 6

[0156] Specificity test of test strips for detecting human chorionic gonadotropin in saliva

[0157] The test cards prepared in Example 2 and Comparative Examples 1 to 3 were used to test the same saliva sample with reference to the test method of Example 4. The results are shown in FIG2 and Table 2.

[0158] Table 2 Elimination of nonspecific reaction test results

[0159] Note: “+++” means clearly visible, “++” means visible, “+” means faintly visible, and “-” means invisible.

[0160] The results, as shown in Figure 2 and Table 2, showed that the use of L-cysteine ​​and dithiothreitol alone improved the specificity issues caused by saliva sample viscosity, but neither completely eliminated nonspecific reactions. Furthermore, there were issues such as a red background in the test strip and noticeable microsphere residue. However, the combined use of L-cysteine ​​and dithiothreitol achieved satisfactory results. As shown in Figure 2, the combined use of L-cysteine ​​and dithiothreitol completely eliminated nonspecific reactions and produced a significantly clearer background.

[0161] Furthermore, the test cards prepared in Example 2 and Comparative Examples 1 to 3 were stored at room temperature for 182 days and tested with the same saliva sample according to the detection method of Example 4. The results are shown in FIG3 and Table 3.

[0162] Table 3 Test after 182 days of storage at room temperature

[0163] Note: “+++” means clearly visible, “++” means visible, “+” means faintly visible, and “-” means invisible.

[0164] As can be seen from Figure 2 and Table 3, the effect of eliminating specific reactions when L-cysteine ​​and dithiothreitol are used alone is reduced, but their combined use can still completely eliminate specific reactions.

[0165] In addition, the test cards prepared in Example 2 and Comparative Example 3 were tested on 120 negative saliva samples according to the detection method of Example 4. The results are shown in Table 4.

[0166] Table 4 Test results of 120 negative saliva samples using different test cards

[0167] As can be seen from Table 4, the sample pad treatment method of the test strip of the present invention can effectively eliminate nonspecific reactions, and the negative coincidence rate is very good.

[0168] Example 7

[0169] Real sample test of test strips for detecting human chorionic gonadotropin in saliva

[0170] Saliva samples were collected from 20 non-pregnant women and 8 women diagnosed as pregnant. The saliva samples were tested using the test card of Example 2 and the detection method of Example 4. The test was timed for 5 minutes and the chromatography results of the test card were observed.

[0171] The test results of Figures 4 and 5 show that the saliva sample test cards of 20 non-pregnant women tested negative, and the saliva sample test cards of 8 pregnant women tested positive. After 5 minutes, the chromatographic background was clean, the lines were clear, and the chromatographic process was smooth. Therefore, the test strip of the present invention has high detection accuracy, with an accuracy rate of 100%.

[0172] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A test strip for detecting human chorionic gonadotropin in saliva, characterized in that: The test strip comprises a sample pad, a conjugation pad, a water filter pad, a coating membrane and a bottom plate, wherein the sample pad, the conjugation pad, the water filter pad and the coating membrane are overlapped on the bottom plate in sequence; The sample pad is a glass fiber membrane treated with a sample pad treatment solution; The sample pad treatment solution includes the following components in 100 mL: 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 treatment solution is 8.0-8.

5.

3. The test strip according to claim 1, characterized in that: The coating membrane is nitrocellulose coated with T lines and C lines; The T line is sprayed with 0.5-1 mg / mL of mouse anti-α-HCG monoclonal antibody dilution; The C line is sprayed with 0.4-0.6 mg / mL of goat anti-mouse IgG polyclonal antibody dilution; The spray volume during T-line spraying or C-line spraying 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 by preparing a coating diluent; the coating diluent is a PBS solution containing 50-150 mg / mL trehalose.

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

6. The test strip according to claim 5, characterized in that: The glass fiber membrane or the polyester fiber membrane is sprayed with HCG latex microspheres, and the spraying amount is 1.5-3 μL / cm.

7. The test strip according to claim 6, characterized in that: The preparation method of the HCG latex microspheres comprises the following steps: washing the red latex microspheres with borax buffer to obtain washed red latex microspheres; The β-HCG labeled antibody was mixed with the washed red latex microspheres, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution was added for activation to obtain the β-HCG labeled antibody modified Red latex microspheres; The ethanolamine solution was mixed with the red latex microspheres modified with β-HCG labeled antibody to obtain blocked HCG red latex microspheres, and the supernatant was removed by centrifugation. Tris buffer was then added to the blocked HCG red latex microspheres and ultrasonically mixed. The supernatant was removed by centrifugation, and Tris buffer was then added to ultrasonically mix. The mixture was then rotated and mixed at room temperature for 4 to 6 hours to obtain HCG latex microspheres.

8. A method for preparing the test strip according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Preparation of sample pad After the glass fiber membrane absorbs the sample pad treatment liquid evenly, it is dried to obtain the result; (2) Preparation of conjugate pad After the glass fiber membrane or polyester fiber membrane absorbs the binding pad treatment liquid uniformly, it is dried, and HCG latex microspheres are sprayed on the glass fiber membrane or polyester fiber membrane treated with the binding pad treatment liquid, with a spray volume of 1.5 to 3 μL / cm; (3) Preparation of coating membrane Dilute the mouse anti-α-HCG monoclonal antibody and the goat anti-mouse IgG polyclonal antibody respectively with the coating diluent to obtain the mouse anti-α-HCG monoclonal antibody diluent and the goat anti-mouse IgG polyclonal antibody diluent, spray the mouse anti-α-HCG monoclonal antibody diluent on the T line of the nitrocellulose membrane, spray the goat anti-mouse IgG polyclonal antibody diluent on the C line of the nitrocellulose membrane, and dry to obtain; (4) Preparation of test strips After cutting the sample pad, conjugate pad and water filter pad respectively, the sample pad, conjugate pad, water filter pad and coating membrane are sequentially pasted on the bottom plate, one side of the conjugate pad is located above the sample pad, the other side of the conjugate pad is located below one side of the water filter pad, the other side of the water filter pad is located below the coating membrane, and the T line on the coating membrane is close to the water filter pad.

9. The preparation method according to claim 7, characterized in that: The preparation method of the HCG latex microspheres comprises the following steps: washing the red latex microspheres with borax buffer to obtain washed red latex microspheres; The β-HCG labeled antibody is mixed with the washed red latex microspheres, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solution is added for activation to obtain the β-HCG labeled antibody modified red latex microspheres; The ethanolamine solution was mixed with the red latex microspheres modified with β-HCG labeled antibody to obtain blocked HCG red latex microspheres, and the supernatant was removed by centrifugation. Tris buffer was then added to the blocked HCG red latex microspheres and ultrasonically mixed. The supernatant was removed by centrifugation, and Tris buffer was then added to ultrasonically mix. The mixture was then rotated and mixed at room temperature for 4 to 6 hours to obtain HCG latex microspheres.

10. The preparation 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 preparation method according to claim 7, characterized in that: The volume ratio of the red latex microspheres to the borax buffer solution is 0.1-0.2:1; the concentration of the borax buffer solution is selected to be 0.05-0.15M.

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

13. The preparation method according to claim 7, characterized in that: The sealing time is 15 to 30 minutes; the ultrasonic time is 10 to 20 minutes.

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

15. The test strip box according to claim 14, characterized in that: The sample diluent is 0.9% physiological saline.

16. A use of the test strip according to any one of claims 1 to 7 or the preparation method according to any one of claims 8 to 13, characterized in that: Include at least one of the following: (1) The test strip or the preparation method is used to detect human chorionic gonadotropin; (2) Application of the test strip or the preparation method in the preparation of a product for detecting human chorionic gonadotropin; (3) The test strip or the preparation method is used to detect early pregnancy; (4) Use of the test strip or the preparation method in preparing products for detecting early pregnancy.

17. The use according to claim 16, characterized in that The criteria for determining whether a pregnancy is present are: (1) Positive: Two red reaction lines, i.e. one red reaction line appears in the test area and one red reaction line appears in the control area. Wire; (2) Negative: one red reaction line, that is, only one red reaction line appears in the control area; (3) Invalid: No red reaction line appears in the control area, indicating that the test is wrong or invalid.

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

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

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