Test strip, kit and method for quantitatively detecting testosterone

Abstract

The embodiment of the invention belongs to the technical field of detection, and relates to a test strip, a kit and a method for quantitatively detecting testosterone, the test strip comprises a PVC base plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped on the PVC base plate; a quality control line and a detection line are arranged on the nitrocellulose membrane, the quality control line is coated with a chicken IgY antibody, the detection line is coated with a testosterone-BSA conjugate, and a fluorescently-labeled testosterone antibody and a fluorescently-labeled goat anti-chicken IgY antibody are fixed on the conjugate pad. The detection speed is effectively improved.

Description

Technical Field

[0001] This application relates to the field of detection technologies, and particularly to test strips, reagent kits and methods for quantitatively detecting testosterone. Background Art

[0002] Testosterone is a very important steroid male hormone in the human body. Testosterone in men is mainly secreted by adult testicular interstitial cells and is regulated by luteinizing hormone (LH). The secretion of testosterone is regulated by the negative feedback mechanism of gonadotropin-releasing hormone from the hypothalamus. The hypothalamus secretes gonadotropin-releasing hormone, which promotes the synthesis and release of LH and follicle-stimulating hormone (FSH) by the anterior pituitary gland. Testosterone promotes the formation of male secondary sexual characteristics and maintains the functions of the prostate and seminal vesicles. Abnormally low levels of testosterone in men indicate hypogonadism, hypopituitarism, hyperprolactinemia, renal failure, cirrhosis or "Klinefelter" syndrome; while adrenal and testicular tumors, congenital adrenal hyperplasia or abnormalities in the hypothalamic-pituitary-testicular axis can lead to elevated testosterone levels in men. Testosterone in women is secreted by theca cells and ovarian interstitial cells, and adrenal androgen metabolism also produces testosterone. Androgens at physiological levels have no special effects on women, and elevated androgen levels in women can lead to masculinization. Persistently elevated total testosterone levels in women indicate polycystic ovary syndrome, theca cell hyperplasia, adrenal and ovarian tumors, congenital adrenal hyperplasia and some other abnormalities in the hypothalamic-pituitary-testicular axis function.

[0003] Testosterone belongs to small molecule steroid hormones with a small molecular weight. Currently, immunological analysis methods (chemiluminescence analysis, enzyme-linked immunosorbent assay, cloned enzyme donor immunoassay, immunochromatography) and chromatography-mass spectrometry coupling methods are mostly used for detection. Specifically as follows:

[0004] Liquid chromatography-tandem mass spectrometry: Liquid chromatography-tandem mass spectrometry is a powerful analytical technique that combines the separation ability of liquid chromatography and the high-sensitivity selective mass analysis ability of mass spectrometry to achieve a more accurate quantitative and qualitative analysis method for complex mixtures. This method has the characteristics of high sensitivity, rapid analysis, good accuracy, etc., and is the gold standard for clinical monitoring of small molecule hormones. However, it has defects such as high cost, strict environmental site requirements, complex sample pretreatment, relatively low detection throughput, and difficulty in meeting the needs of large-scale clinical samples, and the manual operation is relatively complex, and the operators need to undergo relatively professional training, so it is difficult to be promoted.

[0005] Chemiluminescence method: Chemiluminescence analysis method is mainly based on the principle that the concentration of the analyte in the chemiluminescence detection system shows a linear quantitative relationship with the chemiluminescence intensity of the system under certain conditions. It is an analytical method that uses an instrument to detect the chemiluminescence intensity of the system to determine the content of the analyte. The double-antigen competitive antibody analysis mode for testosterone chemiluminescence detection has a narrow analysis range, especially poor accuracy at both ends of the analysis range, and this method has a high cost and requires a specific chemiluminescence instrument, which results in a small application range in the clinical detection of testosterone.

[0006] Enzyme-linked immunosorbent assay and cloned enzyme donor immunoassay: Enzyme immunoassay is an immunoassay method that combines the specific immune reaction of antigens and antibodies with the high catalytic efficiency of enzymes and is applied to the detection of serum testosterone. It has the advantages of easy preparation and stable properties of enzyme-labeled reagents, no radioactive pollution, and high sensitivity. Enzyme-linked immunosorbent assay is less used due to its high requirements for storage temperature, long detection time, poor precision, easy contamination, poor repeatability, easy false positives, and cumbersome operation with many interfering factors. Summary of the Invention

[0007] The purpose of the embodiments of this application is to provide a test strip, a kit and a method for quantitatively detecting testosterone, effectively improving the detection speed.

[0008] To solve the above technical problems, the embodiments of this application provide a test strip for quantitatively detecting testosterone, and adopt the following technical solutions:

[0009] A test strip for quantitatively detecting testosterone, the test strip includes a PVC bottom plate and a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad that are sequentially overlapped on the PVC bottom plate;

[0010] The nitrocellulose membrane is provided with a quality control line and a detection line. The quality control line is coated with chicken IgY antibody, and the detection line is coated with testosterone-BSA conjugate. The conjugate pad is fixed with a fluorescence-labeled testosterone antibody and a fluorescence-labeled goat anti-chicken IgY antibody.

[0011] Further, the concentration of the testosterone-BSA conjugate coated on the detection line is 0.5 - 0.8 mg / mL.

[0012] Further, the concentration of the chicken IgY antibody coated on the quality control line is 0.8 - 1.0 mg / mL.

[0013] Further, the fluorescence labels of the fluorescence-labeled testosterone antibody and the fluorescence-labeled goat anti-chicken IgY antibody are Eu 3+ Lanthanide element fluorescent microspheres.

[0014] Further, the Eu 3+The diameter of the lanthanide fluorescent microspheres is 200 nm to 300 nm.

[0015] Further, the sample pad is prepared by the following steps:

[0016] Apply the treatment solution on the preset initial sample pad and dry it overnight to obtain the sample pad. Wherein, by mass concentration, the treatment solution includes 0.1 M PB, 1% sucrose, 1% trehalose, 1% polyvinylpyrrolidone, 0.1 mg / mL anti-human red blood cell antibody, and 0.2 mg / mL antibody blocker.

[0017] Further, the conjugate pad is prepared by the following steps:

[0018] Activate the immunofluorescent microspheres;

[0019] Crosslink the activated immunofluorescent microspheres with testosterone antibody and goat anti-chicken IgY antibody respectively to obtain fluorescently labeled testosterone antibody and fluorescently labeled goat anti-chicken IgY antibody;

[0020] Mix and process the fluorescently labeled testosterone antibody and fluorescently labeled goat anti-chicken IgY antibody, and spray them onto the initial conjugate pad to obtain the conjugate pad.

[0021] Further, the step of activating the immunofluorescent microspheres includes:

[0022] Take immunofluorescent microspheres with a solid content of 1%, add them to the labeling buffer, and vortex and mix. Wherein, the labeling buffer includes morpholineethanesulfonic acid;

[0023] Add EDC and NHS, mix by ultrasound and react. After the reaction is completed, centrifuge to remove the supernatant and retain the precipitate;

[0024] Add HEPES buffer and sonicate to make the immunofluorescent microspheres evenly dispersed to obtain the activated immunofluorescent microspheres.

[0025] To solve the above technical problems, the embodiment of the present application also provides a kit for quantitatively detecting testosterone, and adopts the following technical solution:

[0026] A kit for quantitatively detecting testosterone, including a test card, and the test card includes a card shell and a test strip as described in any one of claims 1 to 8;

[0027] The test strip is arranged in the card shell.

[0028] To solve the above technical problems, the embodiment of the present application also provides a method for quantitatively detecting testosterone, and adopts the following technical solution:

[0029] A method for quantitatively detecting testosterone using the above kit, including the following steps:

[0030] Add the sample to be tested to the sample diluent and then add it to the sample addition hole of the test card;

[0031] Place the test card in a fluorescence analyzer, wherein an ID card chip has been inserted into the fluorescence analyzer;

[0032] Read the testosterone content of the sample to be tested output by the fluorescence analyzer.

[0033] Compared with the prior art, the embodiments of the present application mainly have the following beneficial effects:

[0034] The present application adopts the competitive method and utilizes the technical principle of fluorescence immunoassay chromatography. During the test, the sample to be tested is mixed with the sample diluent and added to the sample addition hole, and chromatography is carried out under the capillary effect. The testosterone antigen in the sample binds to the fluorescence-labeled testosterone antibody to form a reaction complex. At this time, the analyte-fluorescence-labeled antibody complex and the unbound fluorescence-labeled antibody coexist in the liquid and diffuse to the test area. The fluorescence-labeled testosterone antibody that is not bound to the analyte is captured by the testosterone-BSA conjugate coated on the detection line of the nitrocellulose membrane; the testosterone concentration in the sample is inversely proportional to the fluorescence intensity of the complex on the detection line. According to the set standard curve, the fluorescence analyzer converts the fluorescence signal value into the testosterone concentration in the sample. The detection of the present application is fast, the operation is simple, and the sensitivity is high. Description of the Drawings

[0035] In order to more clearly illustrate the solutions in the present application, the following will briefly introduce the drawings required for the description of the embodiments of the present application. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0036] Figure 1 is a schematic structural diagram of an embodiment of a test strip for quantitatively detecting testosterone according to the present application;

[0037] Figure 2 is a flowchart of an embodiment of a method for quantitatively detecting testosterone according to the present application;

[0038] Figure 3 is a clinical correlation test result diagram of a test strip for quantitatively detecting testosterone according to the present application.

[0039] Reference numerals: 1, sample pad; 2, conjugate pad; 3, nitrocellulose membrane; 4, absorbent pad; 5, PVC bottom plate; 6, detection line; 7, control line. Detailed Embodiments

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0041] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in connection with the embodiments can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0042] The following embodiments facilitate a better understanding of this application, but do not limit this application. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from regular biochemical reagent stores unless otherwise specified.

[0043] To enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0044] Testosterone belongs to small molecule steroid hormones with a small molecular weight. Currently, immunological analysis methods (chemiluminescence analysis, enzyme-linked immunosorbent assay, cloned enzyme donor immunoassay, immunochromatography) and chromatography-mass spectrometry are mostly used for detection. Since it only contains one antigenic determinant and belongs to a hapten, it cannot meet the requirements of the sandwich mode. Therefore, when this application uses immunological analysis for detection, it is mainly achieved through the competitive method, specifically as follows:

[0045] This application provides a test strip for quantitatively detecting testosterone, as Figure 1 shown, Figure 1 is a schematic structural diagram of an embodiment of the test strip for quantitatively detecting testosterone according to this application; the test strip includes a PVC bottom plate 5, and a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 3, and an absorbent pad 4 that are sequentially lapped on the PVC bottom plate 5; a quality control line 7 (C line) and a detection line 6 (T line) are provided on the nitrocellulose membrane 3. The quality control line 7 is coated with chicken IgY antibody, the detection line 6 is coated with testosterone-BSA conjugate, and a fluorescently labeled testosterone antibody and a fluorescently labeled goat anti-chicken IgY antibody are fixed on the conjugate pad 2.

[0046] In this embodiment, both ends of the nitrocellulose membrane are lapped with the absorbent pad and the conjugate pad respectively, and the other end of the conjugate pad is lapped with the sample pad.

[0047] As an option of the present application, the concentration of the testosterone-BSA conjugate coated on the test line is 0.5-0.8 mg / mL, and the concentration of the chicken IgY antibody coated on the control line is 0.8-1.0 mg / mL. The scribing amounts of both the testosterone-BSA conjugate and the chicken IgY antibody are 1 μL / cm.

[0048] In this embodiment, the concentration of the testosterone-BSA conjugate coated on the test line is 0.5-0.8 mg / mL, optionally including 0.5-0.7 mg / ml, 0.6-0.8 mg / ml. For example, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml; the concentration of the chicken IgY antibody coated on the control line is 0.8-1.0 mg / mL, optionally including 0.8-0.9 mg / ml, 0.9-1 mg / ml. For example, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml.

[0049] As an option of the present application, the fluorescent labels of the fluorescently labeled testosterone antibody and the fluorescently labeled goat anti-chicken IgY antibody are Eu 3+ lanthanide fluorescent microspheres. The Eu 3+ lanthanide fluorescent microspheres have a diameter of 200 nm to 300 nm. Preferably, the Eu 3+ lanthanide fluorescent microspheres of the present application have a diameter of 200 nm or 300 nm.

[0050] In this embodiment, the Eu 3+ lanthanide fluorescent microspheres have a diameter of 200 nm to 300 nm, optionally including 200 nm to 260 nm, 260 nm to 300 nm. For example, 200 nm, 240 nm, 260 nm, and 300 nm.

[0051] As an option of the present application, the width of the sample pad is between 22 and 24 mm, and the length is between 290 and 310 mm. The width of the conjugate pad is between 10 and 12 mm, and the length is between 290 and 310 mm. The width of the nitrocellulose membrane is 25 mm, and the length is between 290 and 310 mm. The width of the absorbent pad is between 23 and 25 mm, and the length is between 290 and 310 mm. The width of the test strip is between 3.83 and 3.85 mm.

[0052] Optionally in the present application, the sample pad is prepared through the following steps: applying a treatment solution onto a preset initial sample pad, and drying overnight to obtain the sample pad; by mass concentration (i.e., mass - volume percentage), the treatment solution is 0.1M PB buffer (phosphate buffer), including 1% sucrose, 1% trehalose, 1% PVPK30 (polyvinylpyrrolidone), 0.1 mg / mL RBC (anti - human red blood cell antibody), and 0.2 mg / mL antibody blocker.

[0053] In this embodiment, the treatment solution is uniformly applied onto the initial sample pad at a rate of 45 mL per sheet (the initial sample pad is 25 cm wide and 30 cm long), and placed under the conditions of a temperature of 50 °C and a relative humidity of ≤30% to dry overnight for 16 - 24 h. Under the conditions of a temperature of 18 - 28 °C and a relative humidity of ≤30%, cut off 5 mm from each of the upper and lower long sides of the dried initial sample pad, and then cut it into pieces of size (23 ± 1) mm × (300 ± 10) mm for standby, thus obtaining the sample pad. The antibody blocker is added to the treatment solution of the sample pad, which can prevent non - specific binding of HAMA IgG, rheumatoid factor, etc. in the sample, and improve the specificity of the product.

[0054] Optionally in the present application, the conjugate pad is prepared through the following steps: activating immunofluorescent microspheres; respectively cross - linking the activated immunofluorescent microspheres with testosterone antibody and goat anti - chicken IgY antibody to obtain fluorescently labeled testosterone antibody and fluorescently labeled goat anti - chicken IgY antibody respectively; mixing and treating the fluorescently labeled testosterone antibody and the fluorescently labeled goat anti - chicken IgY antibody, and spraying them onto the initial conjugate pad to obtain the conjugate pad.

[0055] Optionally in the present application, the step of activating immunofluorescent microspheres includes: taking immunofluorescent microspheres with a solid content of 1%, adding them to a labeling buffer, vortex - mixing, adding EDC and NHS, ultrasonic - mixing, centrifuging and removing the supernatant, retaining the precipitate, and adding HEPES buffer. The labeling buffer includes morpholineethanesulfonic acid.

[0056] Optionally in the present application, the step of respectively cross - linking the activated immunofluorescent microspheres with testosterone antibody and goat anti - chicken IgY antibody to obtain fluorescently labeled testosterone antibody and fluorescently labeled goat anti - chicken IgY antibody respectively includes: adding testosterone antibody or goat anti - chicken IgY antibody to the activated immunofluorescent microspheres, and performing rotary reaction treatment; adding 10% bovine serum albumin for blocking treatment; centrifuging, retaining the precipitate, adding a labeling preservation solution, and ultrasonic - mixing to obtain the fluorescently labeled testosterone antibody or the fluorescently labeled goat anti - chicken IgY antibody.

[0057] The specific steps are as follows:

[0058] Activation of immunofluorescent microspheres: Take 100 μL of immunofluorescent microspheres with a solid content of 1% and add 1 mL of labeling buffer MES (morpholineethanesulfonic acid) with a pH of 6.0 and a concentration of 0.05 M; Prepare two reagents with concentrations of 10 mg / mL and 50 mg / mL of EDC (1-ethyl-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) respectively using reaction buffer (i.e., HEPES buffer), then take 100 μL of each of the two reagents and add them to a vortex mixer, ultrasonicate until evenly dispersed, mix well and rotate for reaction for 20 - 40 minutes. After the reaction is completed, centrifuge at 15000 rpm and 4 °C for 15 min. After centrifugation, remove the supernatant, retain the precipitate, add 1 mL of HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer with a pH of 7.0 and a concentration of 0.02 M, and then ultrasonically disperse evenly with an ultrasonic wave to obtain a suspension containing activated fluorescent microspheres; among them, EDC and NHS are used as activators.

[0059] Crosslinking testosterone antibody to immunofluorescent microspheres: Add 50 - 80 μg of testosterone antibody to the above microsphere suspension, rotate for reaction for 2 hours. After the reaction is completed, add 200 μL of 10% bovine serum albumin (BSA) for blocking for 30 min. Centrifuge the blocked microspheres at 4 °C at a speed of 12000 r / min for 15 min. After centrifugation, discard the supernatant, retain the precipitate, add 5 mL of labeling preservation solution, ultrasonically mix well, and store at 2 - 8 °C.

[0060] Crosslinking goat anti-chicken IgY antibody to immunofluorescent microspheres: Add 100 μg of goat anti-chicken IgY antibody to the above microsphere suspension, rotate for reaction for 2 hours. After the reaction is completed, add 200 μL of 10% bovine serum albumin (BSA) for blocking for 30 min. Centrifuge the blocked microspheres at 4 °C at a speed of 12000 r / min for 15 min. After centrifugation, discard the supernatant, retain the precipitate, add 5 mL of labeling preservation solution, ultrasonically mix well, and store at 2 - 8 °C.

[0061] Mixing fluorescently labeled testosterone antibody or fluorescently labeled goat anti-chicken IgY antibody: Mix the fluorescently labeled testosterone antibody and the fluorescently labeled goat anti-chicken IgY antibody evenly according to a volume ratio of 1:1 (V:V), ultrasonicate at 90 W, work for 2 s, pause for 5 s, and repeat 3 times to obtain a labeled mixed solution.

[0062] Spraying the pad: Cut the glass fiber 8964 into (12 ± 0.5) mm × (300 ± 10) mm. Load the fluorescent microsphere-labeled antibody solution into a gold-spraying and scribing machine, and spray the labeled mixed solution onto the cut initial binding pad at a spraying volume of 4.0 μL / cm; Place the sprayed binding pad in an environment at (50 ± 2) °C and a humidity ≤ 30% and dry overnight (16 - 24) h to obtain a binding pad.

[0063] As an option of the present application, the nitrocellulose membrane is prepared by the following steps: diluting testosterone-BSA and chicken IgY antibody with a coating diluent to obtain a detection solution and a quality control solution, respectively; using a gold spray film streaking instrument to draw a detection line and a quality control line on the initial nitrocellulose membrane with the detection solution and the quality control solution, respectively, and drying to obtain a nitrocellulose membrane. Wherein, in terms of mass concentration (i.e., mass volume percentage), the coating diluent includes 10mmol / L PBS (phosphate buffer) and 1% sucrose.

[0064] In this embodiment, the initial nitrocellulose membrane is attached to a PVC plate, and the concentration of testosterone-BSA is diluted to 0.8 mg / mL with a coating diluent to obtain a test solution; the concentration of chicken IgY antibody is diluted to 1 mg / mL with a coating diluent to obtain a quality control solution. Using a gold spray film scribing instrument, the test liquid is drawn at a predetermined position on the initial nitrocellulose membrane to draw a test line (T line), and the quality control liquid is drawn at a predetermined position on the initial nitrocellulose membrane to draw a quality control line (C line). Preferably, the film scribing amount is 1 μL / cm, the film scribing length is 300 mm, the distance between the C line and the T line is (5 ± 0.5) mm, the distance between the C line and one side of the nitrocellulose membrane is (10 ± 0.5) mm, and the distance between the T line and the other side of the nitrocellulose membrane is (10 ± 0.5) mm. After the film is scribing, the nitrocellulose membrane is placed in a drying oven at a temperature of (50 ± 2) ° C and a humidity of ≤ 30% for drying (24 to 48) h.

[0065] As an option of the present application, the absorbent pad is prepared by the following steps: H-1 absorbent paper, cutting the absorbent paper into a size of (25±1) mm×(300±10) mm to obtain an absorbent pad.

[0066] The present application also provides an assembly process for the test strip: lay the PVC base plate flat on the work surface, peel off the protective film where the absorbent pad is attached to the PVC base plate, adhere the absorbent pad thereon, ensure that the top of the absorbent pad is aligned with one end of the PVC base plate, and the other side is partially covered on the nitrocellulose membrane by 2 to 3 mm. Peel off the protective film where the binding pad and the sample pad are attached to the PVC base plate, adhere the binding pad thereon, preferably with the front of the binding pad facing upward and one end of the binding pad covering the nitrocellulose membrane by 1 to 2 mm. Adhere the sample pad thereon, align one end of the sample pad with the place where the sample pad is attached to the PVC base plate, and the other end of the sample pad covers the binding pad. Obtain the assembled large plate, and cut the large plate into test strips with a width of (3.83±0.05) mm.

[0067] The present application also provides a quantitative testosterone detection kit, the kit comprising a detection card, the detection card comprising a card housing and the above-mentioned test strip, the test strip being arranged in the card housing.

[0068] As an option in the present application, the above-mentioned kit also includes a sample diluent.

[0069] In this embodiment, the sample diluent is used to dilute the sample. The sample diluent is prepared by the following steps: Add 0.01 - 0.10% of PC300 to 1 L of 10 mmol / L PBS buffer and stir evenly to obtain the sample diluent.

[0070] Optionally, the kit further includes an ID card chip, and the above ID card chip contains the standard curve information of this batch of reagents.

[0071] In this embodiment, on the dry fluorescence immunoassay management tool, fill the prepared curve data into a table, use the MMF Model four-parameter growth curve fitting to complete the preparation of the curve, obtain the chip data, and burn the chip data into the chip in a programmer to obtain the ID card chip.

[0072] Optionally, the reagent card is assembled by the following steps: Load the prepared test strip into the first housing of the card shell. After loading, cover the second housing of the card shell, place it on a card pressing machine, adjust the height of the card pressing machine to 3.5 mm to tightly combine the first housing and the second housing of the card shell to obtain the test card. Each test card is loaded into an aluminum foil bag, and 1 pack of desiccant is added to each bag and sealed.

[0073] The specific components of the kit are shown in Table 1

[0074] Table 1 Kit

[0075]

[0076] In this embodiment, the kit of the present application belongs to the on-site testing or point-of-care testing (POCT) type of testing products. Through the time-resolved fluorescence immunoassay technology, it can quickly and quantitatively detect the content of testosterone in human blood. The present application has the advantages of high sensitivity, strong specificity, good stability of the reagents used, wide detection limit, and especially can eliminate the high background in conventional fluorescence measurements. The present application can determine the content of testosterone in human serum and plasma samples.

[0077] The present application also provides a method for quantitatively detecting testosterone using the above kit. Continuing to refer to Figure 2 , a flowchart showing an embodiment of the method for quantitatively detecting testosterone according to the present application is shown. This method includes the following steps:

[0078] S1: Add the sample to be tested into the sample diluent and add it to the sample addition hole of the test card;

[0079] S2: Place the test card in a fluorescence analyzer, where the ID card chip has been inserted into the fluorescence analyzer;

[0080] S3: Read the testosterone content of the sample to be tested output by the fluorescence analyzer.

[0081] In this embodiment, the present application uses the time-resolved fluorescence immunoassay chromatography method to quantitatively detect testosterone. Fluorescent microspheres are used as markers and a fluorescence immunoassay analyzer is used in combination. The present application can quickly and conveniently quantitatively detect the testosterone content in serum, plasma or whole blood samples.

[0082] The specific steps are as follows:

[0083] 1. Turn on the fluorescence analyzer, insert the ID card chip with the same reagent batch number, and read the chip data in the ID card chip in the fluorescence analyzer;

[0084] 2. Use a pipette to aspirate 100 μL of the sample (the sample is serum, plasma or whole blood), add it to 300 μL of the sample diluent, and mix well;

[0085] 3. Open the aluminum foil bag, take out the test card, and place it horizontally on the table;

[0086] 4. Use a pipette to aspirate 100 μL of the diluted and mixed sample, add it to the sample loading hole of the test card, and react at room temperature for 15 min;

[0087] 5. Select the sample type "plasma, serum or whole blood" on the supporting instrument;

[0088] 6. Test: Place the test card into the card slot of the fluorescence analyzer, click "Test", display the fluorescence signal value of the test line, and according to the set standard curve, the fluorescence analyzer converts the fluorescence signal value into the testosterone concentration in the sample;

[0089] 7. Click "Print" to print the test result report.

[0090] The present application adopts the competitive method and uses the technical principle of fluorescence immunoassay chromatography. The test line is coated with testosterone-BSA conjugate to distinguish it from testosterone antigen. During the test, the sample to be tested is mixed with the sample diluent and added to the sample loading hole, and chromatography is carried out under capillary action. The testosterone antigen in the sample to be tested binds to the fluorescently labeled testosterone antibody to form a test substance-fluorescently labeled antibody complex; at this time, the test substance-fluorescently labeled antibody complex and the unbound fluorescently labeled testosterone antibody coexist in the liquid and diffuse to the test area. The unbound fluorescently labeled testosterone antibody is captured by the testosterone-BSA conjugate (testosterone conjugated BSA antigen) coated on the test line of the nitrocellulose membrane; the testosterone concentration in the sample is inversely proportional to the fluorescence intensity of the complex on the test line. According to the set standard curve, the fluorescence analyzer converts the fluorescence signal value into the testosterone concentration in the sample. The concentration of testosterone in the sample can be detected by an immunofluorescence detector, and the default measurement result of the instrument is in μmol / L as the unit.

[0091] At present, there are various research methods for detecting testosterone. The more commonly used ones are liquid chromatography method and ELISA method. The ELISA method is cumbersome to operate, time-consuming, and has low sensitivity. The detection results of liquid chromatography are relatively accurate, but the instruments and equipment for such methods are expensive, require professional operators, have high costs, are time-consuming, and are not easy to promote. The fluorescence quantitative immunochromatography method used in this study is a quantitative detection method based on immunological principles, which has the characteristics of rapidity, simple operation, and low cost, and has broad application prospects.

[0092] This application has the following advantages:

[0093] 1) Time-resolved fluorescence immunoassay is a sensitive high-end quantitative immunoassay technology that uses lanthanide rare earth ions as markers to label antigens or antibodies. Due to the unique fluorescence characteristics of lanthanide rare earth ions, this technology can obtain extremely high signal-to-noise ratios, thus having very high sensitivity. It also has the advantages of simple preparation of markers, long storage time, no radioactive pollution, good detection repeatability, short operation process, wide standard curve range, no interference from natural fluorescence of samples, and very wide application range.

[0094] 2) Time-resolved fluorescence immunoassay has higher application value compared to enzyme immunoassay and radioimmunoassay. This technology has the advantages of low cost, simple operation, rapid detection, strong portability, high sensitivity, and accurate quantification, overcoming the deficiencies such as narrow linear range, complex operation, and high instrument requirements in the above-mentioned traditional rapid detection methods for testosterone.

[0095] 3) An antibody blocker is added to the treatment solution of the sample pad, which can prevent non-specific binding of HAMA IgG, rheumatoid factor, etc. in the sample, and improve the specificity of the product.

[0096] This application also provides specific detection data and precision detection data, as follows:

[0097] Example 1: Specificity detection of the kit of this application

[0098] Using this method to determine various steroid hormones, the cross-reaction results are shown in Table 2.

[0099] Table 2 Cross-reaction results

[0100]

[0101]

[0102] Example 2: Precision detection of the kit of this application

[0103] Prepare testosterone standard solutions with low, medium, and high values of 5 μmol / L, 30 μmol / L, and 80 μmol / L. Each sample is detected 10 times, and its coefficient of variation is calculated. The results show that the detection precision of the present invention is good. The coefficient of variation (CV) at level 1 (5 μmol / L) is 6.60%, the CV value at level 2 (30 μmol / L) is 3.98%, and the CV value at level 3 (80 μmol / L) is 2.50%. The detection result of the precision of the kit does not exceed ±10%, meeting the detection requirements. The detection results of the precision of this kit are shown in Table 3 as follows:

[0104] Table 3 Detection Results of Precision

[0105]

[0106]

[0107] Example 3: Clinical Relevance of the Kit of the Present Application

[0108] Detect a certain number of clinical samples. Compared with well-known foreign manufacturers, the correlation coefficient R 2 is 0.9865. The specific results are as Figure 3 shown. Figure 3 is the detection result graph of the clinical relevance of the test strip for quantitatively detecting testosterone according to the present application.

[0109] It should be understood that although each step in the flowchart of the accompanying drawings is shown in sequence according to the indication of the arrows, these steps do not necessarily have to be executed in the order indicated by the arrows. Unless otherwise clearly stated in this article, the execution of these steps has no strict order limit, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily have to be executed at the same moment, but can be executed at different moments. Their execution order does not necessarily have to be sequential, but can be executed alternately or in turn with at least a part of other steps or sub-steps or stages of other steps.

[0110] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all of them. The preferred embodiments of the present application are shown in the drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing specific embodiments, or perform equivalent replacements on some of the technical features. Any equivalent structure that makes use of the content of the specification and drawings of the present application, directly or indirectly applied in other related technical fields, is similarly within the scope of the patent protection of the present application.

Claims

1. A test strip for quantitatively detecting testosterone, characterized in that, the test strip comprises a PVC bottom plate, and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad which are sequentially lapped on the PVC bottom plate; a quality control line and a detection line are arranged on the nitrocellulose membrane, the quality control line is coated with chicken IgY antibody, the detection line is coated with testosterone-BSA conjugate, and a fluorescently labeled testosterone antibody and a fluorescently labeled goat anti-chicken IgY antibody are fixed on the conjugate pad.

2. The test strip according to claim 1, characterized in that, the concentration of the testosterone-BSA conjugate coated on the detection line is 0.5-0.8 mg / mL.

3. The test strip according to claim 1, characterized in that, the concentration of the chicken IgY antibody coated on the quality control line is 0.8-1.0 mg / mL.

4. The test strip according to claim 1, characterized in that, The fluorescent labels of the fluorescently labeled testosterone antibody and the fluorescently labeled goat anti-chicken IgY antibody are Eu 3+ lanthanide element fluorescent microspheres.

5. The test strip according to claim 4, characterized in that, The Eu 3+ lanthanide element fluorescent microspheres have a diameter of 200 nm to 300 nm.

6. The test strip according to claim 1, characterized in that, the sample pad is prepared by the following steps: applying a treatment solution on a preset initial sample pad, and drying overnight to obtain the sample pad, wherein, by mass concentration, the treatment solution comprises 0.1 M PB, 1% sucrose, 1% trehalose, 1% polyvinylpyrrolidone, 0.1 mg / mL anti-human red blood cell antibody and 0.2 mg / mL antibody blocker.

7. The test strip according to claim 1, characterized in that, the conjugate pad is prepared by the following steps: activating immunofluorescent microspheres; crosslinking the activated immunofluorescent microspheres with testosterone antibody and goat anti-chicken IgY antibody respectively to obtain a fluorescently labeled testosterone antibody and a fluorescently labeled goat anti-chicken IgY antibody; mixing and treating the fluorescently labeled testosterone antibody and the fluorescently labeled goat anti-chicken IgY antibody, and spraying them onto an initial conjugate pad to obtain the conjugate pad.

8. The test strip according to claim 7, characterized in that, the step of activating immunofluorescent microspheres comprises: taking immunofluorescent microspheres with a solid content of 1%, adding them into a labeling buffer solution, and vortex mixing, wherein the labeling buffer solution comprises morpholineethanesulfonic acid; adding EDC and NHS, and ultrasonically mixing for reaction, after the reaction is completed, centrifuging to remove the supernatant, and retaining the precipitate; adding HEPES buffer solution and ultrasonically mixing to make the immunofluorescent microspheres disperse uniformly to obtain the activated immunofluorescent microspheres.

9. A kit for quantitatively detecting testosterone, characterized in that, it comprises a test card, and the test card comprises a card shell and the test strip according to any one of claims 1 to 8; the test strip is arranged in the card shell.

10. A method for quantitatively detecting testosterone using the kit according to claim 9, characterized in that, it comprises the following steps: adding a sample to be tested into a sample diluent, and adding it into the sample adding hole of the test card; placing the test card in a fluorescence analyzer, wherein an ID card chip has been inserted into the fluorescence analyzer; reading the testosterone content of the sample to be tested output by the fluorescence analyzer.

Images