WO2021017417A1 - 一种人绒毛膜促性腺激素半定量检测试纸、试剂杯及其制备方法和应用 - Google Patents

一种人绒毛膜促性腺激素半定量检测试纸、试剂杯及其制备方法和应用 Download PDF

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WO2021017417A1
WO2021017417A1 PCT/CN2020/071034 CN2020071034W WO2021017417A1 WO 2021017417 A1 WO2021017417 A1 WO 2021017417A1 CN 2020071034 W CN2020071034 W CN 2020071034W WO 2021017417 A1 WO2021017417 A1 WO 2021017417A1
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WIPO (PCT)
Prior art keywords
chorionic gonadotropin
human chorionic
semi
antibody
colloidal gold
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PCT/CN2020/071034
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English (en)
French (fr)
Inventor
孙一品
邵志燕
欧卫军
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南通伊仕生物技术股份有限公司
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Priority to AU2020213287A priority Critical patent/AU2020213287B2/en
Priority to EP20746837.2A priority patent/EP3792633A4/en
Priority to RU2020126123A priority patent/RU2752742C1/ru
Priority to CA3089441A priority patent/CA3089441C/en
Publication of WO2021017417A1 publication Critical patent/WO2021017417A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Definitions

  • the application relates to the technical field of biological detection, and in particular to a test paper, a reagent cup for the semi-quantitative detection of human chorionic gonadotropin, and a preparation method and application thereof.
  • hCG Human chorionic gonadotropin
  • the reference value of hCG is 5-50mIU/ml at 0.2-1 weeks of pregnancy; pregnancy 1- 2 weeks, the reference value of hCG is 50-500mIU/ml; 2-3 weeks of pregnancy, the reference value of hCG is 100-5000mIU/ml; 3-4 weeks of pregnancy, the reference value of hCG is 500-10000mIU/ml; 4-5 weeks of pregnancy , HCG reference value is 1000-50000mIU/ml; 5-6 weeks of pregnancy, hCG reference value is 10000-100000mIU/ml; 6-8 weeks of pregnancy, hCG reference value is 15000-200000mIU/ml; 8-12 weeks of pregnancy, hCG The reference value is 10000-100000mIU/ml. After that, hCG decreases rapidly after a higher concentration for 1-2 weeks, then gradually decreases and maintains at a peak level of about 1/5-1/10 until delivery. Therefore, the human pregnancy cycle can be judged by detecting the hCG concentration in blood and urine.
  • hCG human chorionic gonadotropin
  • Chinese patent document CN108761099A discloses a hCG cycle test strip, including a first test strip and a second test strip. The color of the two test strips is used to determine whether the pregnancy time is the first 3 weeks or 3 weeks. More than weeks.
  • the document also discloses a hCG cycle detection kit including the test paper.
  • the operation method of the test paper and the kit is simple, suitable for on-site, immediate, and rapid testing, and suitable for the detection of pregnancy weeks at home, but the test paper only includes 2 test strips, the minimum detection limit of the first test strip is 25mIU/ml, and the minimum detection limit of the second test strip is 100mIU/ml, which can only determine whether the pregnancy time is the first 3 weeks or more than 3 weeks.
  • the pregnancy time is longer and the hCG concentration is higher, higher than 100mIU/ml, because the free ⁇ antibody used in the second test strip is a mouse-derived antibody, the quality control line is prone to non-color development and cannot be performed
  • the judgment of the higher concentration range of hCG makes it impossible to diagnose and distinguish various diseases, and the concentration detection range is narrow; and the test paper is compared and judged by the color development degree of the detection lines of the two test strips, which is more subjective , The test result is not accurate enough.
  • the technical problem to be solved by this application is to overcome the shortcomings in the prior art that the hCG detection test paper has a narrow concentration detection range, high subjectivity in the detection results, and insufficient accuracy of the results, thereby providing a wide concentration detection range, accurate detection results, and high sensitivity.
  • this application provides a test paper for the semi-quantitative detection of human chorionic gonadotropin, which includes at least three test strips, each test strip includes a substrate and a sample attached to the substrate in turn A pad, a colloidal gold adsorption pad, an antibody carrying membrane and a water absorbing pad, wherein the loading pad, the colloidal gold adsorption pad, the antibody carrying membrane and the water absorbing pad are sequentially overlapped by partial overlap;
  • a detection line is provided at one end close to the colloidal gold adsorption pad, and a quality control line is provided at one end close to the absorbent pad;
  • the detection line is coated with anti-human chorionic gonadotropin ⁇ - hCG monoclonal antibody, the quality control line is coated with an anti-mouse IgG polyclonal antibody;
  • the colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody conjugate is adsorbed on the colloidal gold adsorption pad of one of the test strips; colloids are adsorbed on the colloidal gold adsorption pads of the other test strips Gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody conjugate and free ⁇ antibody, and colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG adsorbed on the colloidal gold adsorption pad of the remaining test strips
  • the mass ratios of monoclonal antibody conjugates and free ⁇ antibodies are different; wherein, the free ⁇ antibodies are non-mouse antibodies.
  • the free beta antibody is a humanized, equine, rabbit or goat antibody.
  • the semi-quantitative detection test paper for human chorionic gonadotropin includes six test paper strips, namely the first test strip, the second test strip, the third test strip, the fourth test strip, and the fifth test strip. Paper strip and the sixth test strip.
  • the semi-quantitative detection test paper for human chorionic gonadotropin, the second test strip, the third test strip, the fourth test strip, the fifth test strip, and the sixth test strip on the colloidal gold adsorption pad The mass ratios of the adsorbed colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody conjugate and free ⁇ antibody are 1:1, 1:2, 1:40, 1:80, 1:100.
  • the antibody-bearing membrane is a nitrocellulose membrane with a pore size of 3-10 ⁇ m.
  • the distance between the detection line and the quality control line is 0.3-1.0 cm, preferably, the distance between the detection line and the quality control line is 0.5 cm.
  • the sample loading pad and the absorbent pad are both provided with a protective film.
  • This application provides a method for preparing the semi-quantitative detection test paper for human chorionic gonadotropin, including:
  • the free ⁇ antibody is a non-mouse antibody.
  • the number of test paper strips is six, which are the first test strip, the second test strip, the third test strip, and the third test strip respectively.
  • the anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody is 4 ⁇ g/ ml labeled colloidal gold
  • the anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody labeled colloidal gold at 5 ⁇ g/ml.
  • the method for preparing the semi-quantitative detection test paper for human chorionic gonadotropin, in the second, third, fourth, fifth, and sixth test strips, the anti-human chorionic gonadotropin ⁇ -hCG The mass ratios of the monoclonal antibody to the free ⁇ antibody are 1:1, 1:2, 1:40, 1:80, and 1:100, respectively.
  • the preparation method of the semi-quantitative detection test paper for human chorionic gonadotropin the concentration of the anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody is 2-3 mg/ml, and the anti-mouse IgG polyclonal antibody is The concentration is 1-2mg/ml.
  • the sealing treatment solution comprises a 0.08-0.12 Mol buffer, a sugar content of 0.3-0.7% by mass, and a mass percentage of 0.8 -1.2% of occluded protein and a preservative with a mass percentage of 0.03-0.07%; wherein the buffer is phosphate buffer, the sugar is sucrose or trehalose, and the preservative is NaN 3 or Thimerosal, the blocking protein is complex protein or bovine serum albumin; preferably, the blocking treatment solution includes 0.1 Mol buffer, 0.5% by mass sugar content, and 1% by mass blocking protein , Preservative with 0.05% mass percentage.
  • step S2 the pH value is adjusted, and the pH value is adjusted to 6.5-7.0.
  • the preparation method of the semi-quantitative detection test paper for human chorionic gonadotropin, the colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody conjugate compound solution, and the colloidal gold-anti-human chorionic The mixed solution of the gonadotropin ⁇ -hCG monoclonal antibody conjugate and free ⁇ antibody was poured on the colloidal gold adsorption pad in an amount of 50 ⁇ l/cm 2 .
  • This application also provides a reagent cup for the semi-quantitative detection of human chorionic gonadotropin, including the semi-quantitative detection test paper for human chorionic gonadotropin or the semi-quantitative human chorionic gonadotropin prepared by the preparation method Test paper.
  • the reagent cup for the semi-quantitative detection of human chorionic gonadotropin further includes a cup body and a card housing, the card housing is placed inside the reagent cup; the card housing is provided with slots arranged in parallel, the slot One end of the test strip is provided with an opening for the test strip to be inserted therein, and the sample loading pad of the test strip extends from the opening to the outside of the card housing.
  • the reagent cup for the semi-quantitative detection of human chorionic gonadotropin further includes a cup cover, which is detachably covered on the cup body.
  • the card housing In the semi-quantitative detection reagent cup for human chorionic gonadotropin, the card housing is vertically placed inside the cup body, and the open end of the slot is close to the bottom of the cup body.
  • the cup body is provided with a detection cavity, a liquid storage cavity, and a liquid guiding cavity; the upper part of the liquid guiding cavity is provided with a second fluid communicating with the liquid storage cavity A through hole, the lower part is provided with a second through hole communicating with the detection cavity; the liquid guide cavity is provided with a piston that slides along the liquid guide cavity, and the piston has a connection between the liquid guide cavity and the The initial state of communication between the liquid storage cavity and the detection state of connecting the liquid guide cavity with the detection cavity; a third through hole is provided on the cup wall on the side of the cup body away from the detection cavity, and The liquid guiding cavity is communicated with the third through hole; the card housing is vertically placed inside the detection cavity.
  • the piston In the semi-quantitative detection reagent cup for human chorionic gonadotropin, the piston is provided with a reservoir; in the initial state, the reservoir of the piston is aligned with the first through hole, thereby Make the liquid guide cavity communicate with the liquid storage cavity; in the detection state, the liquid storage groove of the piston is aligned with the second through hole, so that the liquid guide cavity communicates with the detection cavity .
  • the cup cover is provided with a booster for boosting the piston, and the booster is detachably fitted on the cup cover.
  • the card housing is fixed in parallel inside the cup cover.
  • the outer edge of the cup cover is provided with a support body for fixing the reagent cup placed upside down, and the cup body is provided with graduation lines.
  • the cartridge In the semi-quantitative detection reagent cup for human chorionic gonadotropin, the cartridge is made of transparent material.
  • the present application provides a method for semi-quantitative detection of human chorionic gonadotropin using the reagent cup, which includes collecting a urine sample in the cup body, starting timing from the adsorption of the sample on the test strip, Observe whether the detection line and quality control line of the test strip are colored within minutes.
  • the application also provides a semi-quantitative detection test paper for human chorionic gonadotropin or a semi-quantitative detection test paper for human chorionic gonadotropin prepared by the preparation method or the semi-quantitative detection test paper for human chorionic gonadotropin
  • the detection reagent cup is used in the detection of human pregnancy cycle, ectopic pregnancy, abortion and trophoblast disease.
  • test paper for the semi-quantitative detection of human chorionic gonadotropin, which includes at least three test strips, each test strip includes a substrate and a sample loading pad sequentially adhered to the substrate , Colloidal gold adsorption pad, antibody bearing membrane and water absorbing pad, the loading pad, colloidal gold adsorption pad, antibody bearing membrane and water absorbing pad are sequentially overlapped by partial overlap; on the antibody bearing membrane, close to the colloidal gold adsorption One end of the pad is provided with a test line, and one end close to the absorbent pad is provided with a quality control line; the test line is coated with anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody, and the quality control line is coated with Anti-mouse IgG polyclonal antibody; the colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody conjugate is adsorbed on the colloidal gold adsorption pad of one of the test strips; the colloid
  • the detection limit of the test strip is adjusted to a wide range. If the mouse-derived free ⁇ antibody is used, when the hCG concentration in the test sample is high, a large amount of free ⁇ antibody needs to be contained to adjust the detection limit of the test strip to a higher However, since the free ⁇ antibody is a murine antibody, a large amount of free ⁇ antibody will bind to the anti-mouse IgG polyclonal antibody on the quality control line, and only a very small amount of colloidal gold-labeled hCG will bind to the quality control line. The quality control line does not develop color, which makes it difficult to detect the defects of higher concentrations of hCG.
  • the concentration detection range can be wider; in addition, the test strip is equipped with at least three test strips , And the antibody type and concentration in each test strip are different.
  • the color development of each test strip is different. You can judge whether the detection line of each test strip is color-developed, and compare the concentration in a wide range. Semi-quantitative judgment is performed, and subjective judgment of color depth is not required, and the concentration range of judgment is more accurate.
  • a method for preparing a test paper for semi-quantitative detection of human chorionic gonadotropin which uses non-mouse-derived free ⁇ antibody and colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody
  • the colloidal gold adsorption pad mixed with the conjugate can overcome the high concentration of hCG and the high concentration of mouse-derived free ⁇ antibody in the sample to be tested when the mouse-derived free ⁇ antibody is used, which causes the quality control line to not show color, which makes it difficult.
  • the concentration detection range is wider; in addition, by preparing at least three test strips, and the antibody type and concentration in each test strip are different, When detecting the hCG concentration, you can determine whether the detection line of each test strip is colored, so as to perform a semi-quantitative determination of the concentration in a wider range, without subjective judgment of color depth, and the determined concentration range is more accurate.
  • a method for preparing a test paper for semi-quantitative detection of human chorionic gonadotropin provided by the present application.
  • the anti-human chorionic gonadotropin promotes
  • the mass ratios of the gonadal hormone ⁇ -hCG monoclonal antibody to the free ⁇ antibody are 1:1, 1:2, 1:40, 1:80, 1:100, respectively.
  • the lowest detection can be obtained.
  • the limits are 5mIU/ml, 25mIU/ml, 100mIU/ml, 500mIU/ml, 2500mIU/ml, 10000mIU/ml test strips respectively, so as to obtain semi-quantitative test paper with high sensitivity, wide concentration detection range and accurate detection results.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application, comprising the semi-quantitative detection test paper for human chorionic gonadotropin or the semi-quantitative human chorionic gonadotropin prepared by the preparation method Detection test paper, the reagent cup is used to detect hCG concentration, and the detection range is wider; in addition, the reagent cup can be used to determine whether the detection line of each test strip is colored to determine the concentration semi-quantitatively, without the need for color shade Subjective judgment, the judged concentration range is more accurate.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application, further comprising a cup body and a card housing, the card housing is placed inside the reagent cup; the card housing is provided with parallel slots , One end of the slot is provided with an opening for the test strip to be inserted therein, the sample loading pad of the test strip extends from the opening to the outside of the card housing, and the slot is provided with
  • the cartridge can be used to fix the test strip in the reagent cup to facilitate sample absorption; since the sample loading pad of the test strip extends from the opening to the outside of the cartridge, it is convenient for the sample loading pad to absorb samples; when using the reagent cup, you only need to The urine sample is placed in the cup to complete the test, which is convenient and hygienic.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application, further comprising a cup cover, the cup cover is detachably closed on the cup body, and the urine sample is put into After the cup body, cover the cup lid, on the one hand, it can isolate the sample from the outside world, ensure the stability of the detection environment, and prevent the external environment from affecting the detection effect; on the other hand, it can prevent the emission of peculiar smell and make it more hygienic.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application.
  • the cup body is provided with a detection cavity, a liquid storage cavity and a liquid guide cavity; the upper part of the liquid guide cavity is provided with the storage
  • the first through hole communicating with the liquid cavity is provided with a second through hole communicating with the detection cavity at the lower part;
  • the liquid guiding cavity is provided with a piston sliding along the liquid guiding cavity, and the piston has the guide
  • the cup wall on the side of the cup away from the detection cavity is provided with a third A through hole, the liquid guiding cavity is in communication with the third through hole;
  • the card housing is vertically placed inside the detection cavity, and a detection cavity containing the card housing and test paper and a liquid storage cavity for collecting urine are provided,
  • the test paper in the detection cavity can be separated from the urine sample in the initial state,
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application, wherein the piston is provided with a reservoir; in the initial state, the reservoir of the piston and the first The through holes are aligned, so that the liquid guiding cavity is connected to the liquid storage cavity; in the detection state, the liquid storage groove of the piston is aligned with the second through hole, so that the liquid guiding cavity and The detection chamber is connected.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application, the cup cover is provided with a booster for boosting the piston, and the booster is detachably fitted in the cup On the cover, by providing a booster, the piston can be pushed from the initial state to the detection state more conveniently, and when not in use, the booster can be fitted on the lid to facilitate storage and prevent its loss.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application.
  • the card housing is fixed in parallel to the inside of the cup cover. By fixing the card housing on the cup cover, the card housing can be removed before the test is started.
  • the test strip in the middle is separated from the urine sample in the cup.
  • a reagent cup for the semi-quantitative detection of human chorionic gonadotropin provided by the present application.
  • the outer edge of the cup cover is provided with a support body for fixing the reagent cup placed sideways, and the cup body is provided with a scale Line, by setting the support body on the edge of the cup lid, it can be used to fix the reagent cup when the reagent cup is placed on the side for testing, to prevent the reagent cup from rolling; by setting the scale line, it is necessary to ensure that the urine exceeds the scale line when collecting urine , When the reagent cup is placed upside down, it can ensure that there is a sufficient amount of urine sample to make the test strip smoothly perform adsorption detection.
  • the semi-quantitative detection test paper for human chorionic gonadotropin or the semi-quantitative detection test paper for human chorionic gonadotropin prepared by the preparation method provided by this application or the semi-quantitative detection test paper for human chorionic gonadotropin Quantitative detection reagent cup is used in the detection of human pregnancy cycle, ectopic pregnancy, abortion and trophoblastic disease.
  • the above detection test paper or reagent cup can be used for semi-quantitative detection of hCG concentration in a wide range, with a wide detection range. The sensitivity is high and the results are accurate, so that the pregnancy cycle can be judged more accurately, and the detected concentration range can be used to diagnose and distinguish diseases such as ectopic pregnancy, abortion and trophoblastic disease.
  • FIG. 1 is a schematic diagram of the structure of a test paper for semi-quantitative detection of human chorionic gonadotropin in Examples 1-6 of the present application;
  • FIG. 2 is a schematic diagram of the structure of the test paper and the jam in the reagent cup for the semi-quantitative detection of human chorionic gonadotropin in Examples 4-6 of the present application;
  • Example 3 is a schematic diagram of the structure of the reagent cup for the semi-quantitative detection of human chorionic gonadotropin in Example 4 of the present application;
  • Example 4 is a schematic structural diagram of a reagent cup for semi-quantitative detection of human chorionic gonadotropin in Example 5 of the present application;
  • Example 5 is a schematic diagram of the structure of the reagent cup for the semi-quantitative detection of human chorionic gonadotropin in Example 5 of the present application in the initial state;
  • Example 6 is a schematic diagram of the structure of the semi-quantitative detection reagent cup for human chorionic gonadotropin in Example 5 of the present application in the detection state;
  • Example 7 is a schematic diagram of the structure of the reagent cup for the semi-quantitative detection of human chorionic gonadotropin in Example 6 of the present application;
  • test strip for the semi-quantitative detection of human chorionic gonadotropin, which includes six test strips, namely the first test strip 11, the second test strip 12, the third test strip 13, and the fourth test strip.
  • a detection line 6 is provided at one end close to the colloidal gold adsorption pad 3, and a quality control line 7 is provided at one end close to the absorbent pad 5; the detection line 6 is coated with anti-human chorionic gonadotropin ⁇ -hCG mono Cloned antibody (anti- ⁇ -hCG monoclonal antibody), quality control line 7 is coated with anti-mouse IgG polyclonal antibody.
  • the colloidal gold-anti-human chorionic gonadotropin ⁇ -hCG monoclonal antibody conjugate (colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate) is adsorbed on the colloidal gold adsorption pad 3 of the first test strip 11, and the second The colloidal gold adsorption pad 3 of the test strip 12, the third test strip 13, the fourth test strip 14, the fifth test strip 15, and the sixth test strip 16 adsorb colloidal gold-anti- ⁇ -hCG monoclonal Antibody conjugate and free ⁇ antibody, and the mass ratios of the adsorbed colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate and free ⁇ antibody are 1:1, 1:2, 1:40, 1:80, 1: 100, wherein the free ⁇ antibody is a non-mouse antibody, and the free ⁇ antibody in this example is a humanized antibody derived from a hamster.
  • the detection line 6 and the quality control line 7 are arranged in parallel with a distance of 0.3-1.0 cm. In this embodiment, the distance is 0.5 cm.
  • sample loading pad 2 and the water-absorbing pad 3 are both provided with a protective film 8.
  • the antibody-carrying membrane 4 is a nitrocellulose membrane with a pore size of 3-10 ⁇ m.
  • This embodiment also provides a method for preparing the above semi-quantitative detection test paper for human chorionic gonadotropin, including:
  • the antibody concentration is 2-3mg/ml, in this example it is 2.5mg/ml, and the mass percentage is used.
  • a 0.85% sodium chloride buffer was used to prepare an anti-mouse IgG polyclonal antibody for quality control line coating, so that the antibody concentration was 1-2 mg/ml, in this embodiment 1.5 mg/ml.
  • the soaking solution for sealing treatment contains 0.1Mol buffer and 0.5% sugar by mass
  • the occluded protein is 1% by mass, and the preservative is 0.05% by mass.
  • the buffer is phosphate buffer, the sugar is sucrose, the preservative is thimerosal, and the occluded protein is bovine serum albumin. .
  • Drying of the board tear off the white paper in the middle of the cutting line on the double-sided tape of the rubber sheet, use tweezers to place the sealed antibody carrier film on the blank position in the center of the rubber sheet, and the right side of the rubber sheet is flush with the right side of the film , To avoid errors in the production process, to ensure that the color development position is relatively accurate.
  • all the top quality control lines are pasted.
  • the film surface is smoothed with double-sided adhesive paper to avoid air bubbles.
  • the temperature in the control room is 18-28°C, the relative humidity is ⁇ 40%, and the air in the drying room can circulate and the air from the dehumidifier will not be directly blown on the film surface. Drying time ⁇ 4 hours, later use.
  • colloidal gold complex solution add the actual production amount of purified water to the mixing tank; weigh trehalose, bovine serum albumin, trisodium citrate, polyethylene glycol and NaN 3 with an electronic analytical balance, and add directly Stir in the liquid mixing tank, stir until it is completely dissolved, add purified water to the required volume, stir well, and the stirring time is longer than 30 minutes.
  • the obtained colloidal gold compound solution contains 5% by mass of trehalose, Bovine serum albumin with a mass percentage of 2%, trisodium citrate with a mass percentage of 0.5%, polyethylene glycol with a mass percentage of 0.05%, and NaN 3 with a mass percentage of 0.05%.
  • the temperature of the drying room is controlled at 18-28°C and the relative humidity is ⁇ 40% to ensure unobstructed air flow and airflow cannot directly blow on the colloidal gold adsorption pad.
  • the dried colloidal gold adsorption pad is placed in an aluminum foil bag containing a desiccant, sealed and stored, and the colloidal gold adsorption pad labeled as the second test strip is ready for use.
  • the temperature of the drying room is controlled at 18-28°C and the relative humidity is ⁇ 40% to ensure unobstructed air flow and airflow cannot directly blow on the colloidal gold adsorption pad.
  • the temperature of the drying room is controlled at 18-28°C and the relative humidity is ⁇ 40% to ensure unobstructed air flow and airflow cannot directly blow on the colloidal gold adsorption pad.
  • the temperature of the drying room is controlled at 18-28°C and the relative humidity is ⁇ 40% to ensure unobstructed air flow and airflow cannot directly blow on the colloidal gold adsorption pad.
  • the temperature of the drying room is controlled at 18-28°C and the relative humidity is ⁇ 40% to ensure unobstructed air flow and airflow cannot directly blow on the colloidal gold adsorption pad.
  • the colloidal gold is cast, because the cast colloidal gold can be freely cut to the width, so that it is convenient to adjust the depth and color of the product.
  • the first test strip absorbent pad is laminated on the transparent substrate near the side of the quality control line and overlaps with the antibody carrier film about 1mm, and the first test strip is overlapped with the antibody carrier film by about 1mm.
  • the sample loading pad is compounded on the end of the first test strip colloidal gold adsorption pad far away from the antibody bearing membrane and overlapped with it for about 1mm. Mark it for later use;
  • This embodiment provides a semi-quantitative detection test paper for human chorionic gonadotropin.
  • the difference from embodiment 1 is that the free ⁇ antibody is horse-derived antibody, and the distance between the detection line 6 and the quality control line 7 is 0.3 cm.
  • This example also provides the preparation method of the above semi-quantitative detection test paper for human chorionic gonadotropin.
  • the difference from the preparation method of Example 1 is that the free beta antibody used is horse-derived antibody; preparation of antibody carrier membrane During the process, the concentration of anti- ⁇ -HCG monoclonal antibody is 2mg/ml, the concentration of anti-mouse IgG polyclonal antibody is 1mg/ml, and the distance between the detection line and the quality control line is 0.3cm; the blocking treatment solution used includes, 0.08Mol buffer, 0.3% by mass of sugar, 0.8% by mass of occluded protein, 0.03% by mass of preservative, the buffer in the blocking treatment solution is phosphate buffer , The sugar is trehalose, the preservative is NaN 3 , and the blocking protein is complex protein.
  • This embodiment provides a semi-quantitative detection test paper for human chorionic gonadotropin.
  • the difference from embodiment 1 is that the free ⁇ antibody is rabbit-derived antibody, and the distance between the detection line 6 and the quality control line 7 is 1.0 cm.
  • This example also provides the preparation method of the above semi-quantitative detection test paper for human chorionic gonadotropin.
  • the free beta antibody used is a rabbit-derived antibody; preparation of the antibody carrier membrane During the process, the concentration of anti- ⁇ -HCG monoclonal antibody was 3mg/ml, the concentration of anti-mouse IgG polyclonal antibody was 2mg/ml, and the distance between the detection line and the quality control line was 0.7cm; the blocking treatment solution used included, 0.12Mol buffer, 0.7% by mass of sugar, 1.2% by mass of occluded protein, 0.07% by mass of preservative, the buffer in the blocking treatment solution is phosphate buffer , The sugar is sucrose, the preservative is thimerosal, and the blocking protein is bovine serum albumin.
  • this embodiment provides a reagent cup for the semi-quantitative detection of human chorionic gonadotropin, which includes the semi-quantitative detection test paper for human chorionic gonadotropin in embodiment 1, and also includes a cup body 9, a cup lid 10 and the card housing 17, the card housing 17 is vertically placed inside the cup body 9, and the cup cover 10 is detachably covered on the cup body 9; as shown in Figure 2, there are more than six parallel slots 18 in the card housing 17, The slot 18 has an opening at one end close to the bottom of the cup body 9, the first test strip 11, the second test strip 12, the third test strip 13, the fourth test strip 14, the fifth test strip 15 and the The six test strips 16 are sequentially inserted into the six slots 18 from the opening, and the sample loading pad 2 of the test strip extends from the opening to the outside of the card housing 17.
  • the card housing 17 is made of plastic. Of course, as an alternative embodiment, the card housing 17 can also be made of other transparent materials.
  • the structure is shown in Figure 1, which respectively include a substrate 1 and a sample loading pad 2, a colloidal gold adsorption pad 3, an antibody bearing film 4 and a water-absorbing pad 5 which are sequentially adhered to the substrate, a sample loading pad 2, a colloidal gold adsorption
  • the pad 3, the antibody carrying membrane 4 and the absorbent pad 5 are sequentially overlapped by partial overlap.
  • the antibody carrying membrane 4 is located below the colloidal gold adsorption pad 3 and the absorbent pad 5, and the loading pad 2 is located above the colloidal gold adsorption pad 3.
  • the length of the overlapping part is 1mm.
  • a detection line 6 is provided at the end close to the colloidal gold adsorption pad 3, and a quality control line 7 is provided at the end close to the absorbent pad 5; the detection line 6 is coated with anti- ⁇ -hCG monoclonal antibody, the quality control line 7 is coated with anti-mouse IgG polyclonal antibody.
  • the colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate is adsorbed on the colloidal gold adsorption pad 3 of the first test strip 11, the second test strip 12, the third test strip 13, the fourth test strip 14, and the The colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate and free ⁇ antibody are adsorbed on the colloidal gold adsorption pad 3 of the fifth test strip 15 and the sixth test strip 16, wherein the free ⁇ antibody is a non-mouse antibody.
  • the free ⁇ antibody is a humanized antibody from a hamster.
  • the detection line 6 and the quality control line 7 are arranged in parallel with a distance of 0.3-1.0 cm. In this embodiment, the distance is 0.5 cm. Further, the sample loading pad 2 and the water-absorbing pad 3 are both provided with a protective film 8. Further, the antibody-carrying membrane 4 is a nitrocellulose membrane with a pore size of 3-10 ⁇ m.
  • the cup lid 10 When in use, open the cup lid 10, collect urine samples in the cup body 9, cover the cup lid 10 on the cup body 9, place the cup body 9 upright, and immerse the sample loading pads 2 of the six test strips. In the sample, the sample is sucked into the test strip from the sample loading pad 2 due to capillary action, and then passes through the colloidal gold adsorption pad 3 and the antibody carrier film 4 to reach the absorbent pad 5.
  • the hCG in the sample and the colloid adsorbed in the colloidal gold adsorption pad 3 Gold-anti- ⁇ -hCG monoclonal antibody conjugate or free ⁇ antibody binding.
  • the hCG bound to the colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate follows the flow of the liquid to reach the antibody carrier membrane 4, and is respectively coated with the anti- ⁇ -hCG monoclonal antibody coated in the detection line and the quality control line.
  • Anti-mouse IgG polyclonal antibody binding because it is labeled with colloidal gold, it will be colored at the detection line and quality control line.
  • the hCG bound to free ⁇ antibody does not have a colloidal gold label, so it will not develop color at the detection line, and because the free ⁇ antibody is not a murine antibody, it will not interact with the anti-coated antibody at the quality control line.
  • the hCG concentration in the sample can be determined semi-quantitatively.
  • this embodiment provides a reagent cup for the semi-quantitative detection of human chorionic gonadotropin, which includes the semi-quantitative detection test paper for human chorionic gonadotropin in embodiment 1, and also includes a cup body 9 and Card shell 17.
  • the cup body 9 is provided with a detection cavity 19, a liquid storage cavity 20, and a liquid guiding cavity 21.
  • the liquid guiding cavity 21 is located below the liquid storage cavity 20; the upper portion of the liquid guiding cavity 21 is provided with a first through hole 211 communicating with the liquid storage cavity 20 , The lower part is provided with a second through hole 212 communicating with the detection cavity 19.
  • the liquid guiding cavity 21 is provided with a piston 22 sliding along the liquid guiding cavity.
  • the piston 22 has an initial state that connects the liquid guiding cavity 21 and the liquid storage cavity 20, and a detection state that makes the liquid guiding cavity 21 communicate with the detection cavity 19;
  • a third through hole 213 is provided on the cup wall of the cup body 9 away from the detection cavity 19, and the liquid conducting cavity 21 is in communication with the third through hole 213.
  • the piston 22 is provided with a liquid storage tank 221, as shown in FIG. 5, in the initial state, the liquid storage tank 221 of the piston is aligned with the first through hole 211, so that the liquid guiding cavity 21 is communicated with the liquid storage cavity 20; As shown in FIG. 6, in the detection state, the liquid storage tank 221 of the piston is aligned with the second through hole 212, so that the liquid guiding cavity 21 and the detection cavity 19 are connected.
  • the inner bottom of the cup body 9 is provided with a liquid guiding groove 26, the liquid guiding groove 26 is in communication with the detection cavity 19, and the liquid guiding groove 26 is located below the second through hole 212, so that the liquid guiding groove 26 communicates with the second through hole 212.
  • the structure and number of the card housing 17 and the test strips are the same as those of the fourth embodiment, except that the card housing 17 with the test strips inserted therein is vertically placed inside the detection cavity 19.
  • the reagent cup can fix the test strip in the detection cavity 19 of the reagent cup by setting the card shell 17.
  • the cup body can be kept upright, so that the test strip can also be kept upright, which is convenient for sucking the sample;
  • Set the test strips to six you can semi-quantitatively detect the hCG concentration in urine according to the color development of different test strips, the detectable concentration range is wider, and there is no need to compare the color development Judgment, the test result is more accurate.
  • the test paper in the detection chamber 19 can be separated from the urine sample in the initial state, and then by setting the piston 22 and the upper piston
  • the liquid storage tank 221 can be in the initial state, urine enters the liquid storage tank 221 from the liquid storage chamber 20 through the first through hole 211; by sliding the piston 22 in the liquid guiding chamber 21 to the detection state, the urine
  • the liquid storage tank 221 flows into the liquid guiding tank 26 through the second through hole 212, and then flows into the detection cavity 19, so that the test strip in the cartridge 17 in the detection cavity 19 contacts the urine sample to complete the detection; through the above structure , It is more convenient to control the timing of the start of the test, you can start the test after the sample collection is completed, to prevent the problem of inaccurate test results that is easy to occur while collecting the samples while performing the test, making the results more accurate and easy to operate;
  • the liquid guiding cavity 21 with the third through hole 213, the sliding of the piston 22 can be conveniently controlled; by
  • the reagent cup also includes a cup cover 10, and the cup cover 10 is detachably covered on the cup body 9.
  • the cup cover 10 of the reagent cup is also provided with a booster 23 for boosting the piston 22.
  • the booster 23 is detachably fitted on the cup cover 10. By providing the booster 23, the piston can be moved more conveniently. 22 is pushed from the initial state to the detection state, and when not in use, the booster 23 can be fitted on the lid 10 to facilitate storage and prevent its loss.
  • the sample loading pads 2 of the six test strips in the detection chamber 19 are all immersed in the sample.
  • the sample is sucked into the test strip by the sample loading pad 2 due to capillary action, and then adsorbed by colloidal gold.
  • the pad 3 and the antibody carrying membrane 4 reach the absorbent pad 5, and the hCG in the sample is combined with the colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate or free ⁇ antibody adsorbed on the colloidal gold adsorption pad 3.
  • the hCG bound to the colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate follows the flow of the liquid to reach the antibody carrier membrane 4, and is respectively coated with the anti- ⁇ -hCG monoclonal antibody coated in the detection line and the quality control line.
  • Anti-mouse IgG polyclonal antibody binding because it is labeled with colloidal gold, it will show color at the detection line 6 and quality control line 7.
  • the hCG bound to free ⁇ antibody does not have a colloidal gold label, so it will not develop color at the detection line 6, and because the free ⁇ antibody is not a mouse antibody, it will not be coated with the quality control line 7.
  • Binding of anti-mouse IgG polyclonal antibodies because the content ratios of colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate and free ⁇ antibody in the colloidal gold adsorption pads of the six test strips are different, so the six test strips The detection line 6 will show different color development conditions.
  • the hCG concentration in urine can be semi-quantitatively detected. Compared with the two test strips, the detectable concentration range is wider, and There is no need to compare and judge by the degree of color rendering, and the detection result is more accurate.
  • the reagent cup is used, only the urine sample is directly collected in the cup, and then the piston 22 is pushed to perform detection and result reading, which is convenient and sanitary to use.
  • this embodiment provides a reagent cup for the semi-quantitative detection of human chorionic gonadotropin, including a cup body 9, a card shell 17 and the test paper in embodiment 1.
  • the reagent cup also includes a cup cover 10, and the cup cover 10 is detachably covered on the cup body 9.
  • the structure and number of the card shell 17 and the test strips are the same as those of the fourth embodiment, except that the card shell 17 is fixed in parallel inside the cup cover 10.
  • the reagent cup is provided with a card shell 17, so that the test strip can be fixed in parallel in the lid 10 of the reagent cup.
  • the cup body 9 When in use, the cup body 9 is in a sideways state, which is convenient for the test strip to absorb samples; by setting the test strip There are six. According to the color development of different test strips, the hCG concentration in urine can be semi-quantitatively detected. The detectable concentration range is wider, and there is no need to compare and judge by the degree of color development, and the detection result is more accurate.
  • the test strip in the card housing 17 can be separated from the urine sample in the cup body when the test is not started.
  • the outer edge of the lid 10 of the reagent cup is provided with a support body 24 for fixing the reagent cup placed upside down, and a scale line 25 is provided on the cup body 9.
  • a support body 24 for fixing the reagent cup placed upside down
  • a scale line 25 is provided on the cup body 9.
  • the cup lid 10 When in use, open the cup lid 10, collect the urine sample in the cup body 9, make the urine sample volume exceed the scale line 25 on the cup body 9, cover the cup lid 10 on the cup body 9 and tighten it, the reagent
  • the cup is placed upside down, and the reagent cup is fixed by the support 24.
  • the sample loading pads 2 of the six test strips are all immersed in the sample. Due to capillary action, the sample is sucked into the test strip by the sample loading pad 2 and passes through the colloidal gold.
  • the adsorption pad 3 and the antibody carrying membrane 4 reach the absorbent pad 5, and the hCG in the sample is combined with the colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate or free ⁇ antibody adsorbed in the colloidal gold adsorption pad 3.
  • the hCG bound to the colloidal gold-anti- ⁇ -hCG monoclonal antibody conjugate follows the flow of the liquid to reach the antibody carrier membrane 4, and is respectively coated with the anti- ⁇ -hCG monoclonal antibody coated in the detection line and the quality control line.
  • Anti-mouse IgG polyclonal antibody binding because it is labeled with colloidal gold, it will show color at the detection line 6 and quality control line 7.
  • the hCG bound to free ⁇ antibody does not have a colloidal gold label, so it will not develop color at the detection line 6, and because the free ⁇ antibody is not a mouse antibody, it will not be coated with the quality control line 7.
  • the detection line 6 will show different color development conditions. According to the color development of different test strips, the hCG concentration in urine can be semi-quantitatively detected. Compared with the two test strips, the detectable concentration range is wider, and There is no need to compare and judge by the degree of color rendering, and the detection result is more accurate.
  • the reagent cup it is only necessary to collect the urine sample directly in the cup body, then tighten the lid of the cup, and place it upside down to perform detection and result reading, which is convenient and hygienic to use.
  • This embodiment provides a method of using the reagent cup for the semi-quantitative detection of human chorionic gonadotropin in embodiment 4, which includes balancing the sample and reagent cup at room temperature; then opening the lid 10 to collect the urine sample In the cup body 9, cover the cup lid 10 on the cup body 9, and place the cup body 9 upright.
  • the sample loading pads 2 of the six test strips are all immersed in the sample. Observe the test strips within 5 minutes. Whether the detection line and the quality control line are colored.
  • the test results include the following situations:
  • the test start time is the day when menstruation should come but not. If you test in advance, you need to test again on the expected menstrual day. If the menstruation is delayed, the test should be performed again after 3 days. If the menstrual period is irregular, the longest menstrual period in the most recent month should be used before the test. If you do not know when your menstruation is expected to begin, you should be tested at least 19 days after your last intercourse without protective measures.
  • This embodiment provides the use of the semi-quantitative detection reagent cup for human chorionic gonadotropin in embodiment 4 in detecting human pregnancy cycle, ectopic pregnancy, abortion or trophoblastic disease.
  • the urine sample was detected. According to the description in Table 1, the content range of human chorionic gonadotropin in the urine sample was detected semi-quantitatively.
  • the pregnancy cycle of a person is judged, specifically: 0.2-1 week of pregnancy, hCG reference value 5-50mIU/ml; 1-2 weeks of pregnancy, hCG reference The value is 50-500mIU/ml; 2-3 weeks of pregnancy, the reference value of hCG is 100-5000mIU/ml; 3-4 weeks of pregnancy, the reference value of hCG is 500-10000mIU/ml; 4-5 weeks of pregnancy, the reference value of hCG 1000-50000mIU/ml; 5-6 weeks of pregnancy, hCG reference value is 10000-100000mIU/ml; 6-8 weeks of pregnancy, hCG reference value is 15000-200000mIU/ml; 8-12 weeks of pregnancy, hCG reference value is 10000- 100000mIU/ml.
  • hCG human chorionic gonadotropin content in urine samples is used to diagnose ectopic pregnancy, miscarriage, and trophoblastic disease and observe the course of the disease.
  • hCG is often 312-625mIU/ml during ectopic pregnancy. After 3 days of uterine bleeding, hCG can still be positive, so hCG examination can be used to distinguish it from other acute abdominal diseases.
  • miscarriage In terms of the diagnosis of miscarriage, if an incomplete miscarriage occurs, if there is still placental tissue remaining in the uterus, the hCG test can still be positive, while in the case of a complete miscarriage or stillbirth, hCG turns from positive to negative. Therefore, it can be used as a fetal protection or aspiration Reference basis for palace treatment.
  • hCG In the diagnosis of threatened abortion, if the hCG in the urine has been maintained at a high level, miscarriage will not occur. If the hCG is below 2500mIU/ml and gradually decreases, there is a possibility of miscarriage or stillbirth. When it drops to 600mIU/ml, then Miscarriage is unavoidable.
  • hCG if hCG continues to decline during fetal protection treatment, it means that the fetus is ineffective, and if hCG continues to rise, it means that the fetus is successful.
  • patients with hydatidiform mole, malignant hydatidiform mole, chorioepithelial carcinoma, and other patients will have a significant increase in urine hCG, reaching 100,000 to millions of mIU/ml.
  • urine hCG should be less than 50mIU/ml after 3 weeks after surgery, and it should be negative at 8-12 weeks. If hCG does not decrease or become negative, it may indicate residual disease.
  • This comparative example provides a test paper for the semi-quantitative detection of human chorionic gonadotropin similar to that of Example 1, except that the free beta antibody used is a mouse-derived antibody, and a national standard human chorionic gonadotropin is used. , Prepare a standard solution with a concentration of 10000mIU/ml, use the test paper to detect it, and the result is that among the six test strips, the quality control lines of the fourth, fifth, and sixth test strips are not colored , The display result is invalid.
  • Phosphate buffered saline (PBS) containing protein is 0mIU/ml HCG sample solution.
  • HCG sample solution use the standard human chorionic gonadotropin (HCG) containing Phosphate buffered saline (0.01M PBS) of protein is prepared to the corresponding concentration.
  • 500mIU/ml human luteinizing hormone (hLH) After the hLH standard is reconstituted with protein-containing phosphate buffered saline (PBS), it is formulated into hLH with a concentration of 500mIU/ml with 0mIU/ml HCG sample solution Solution A: Use 5mIU/ml human chorionic gonadotropin sample solution to prepare hLH B solution with a concentration of 500mIU/ml.
  • PBS protein-containing phosphate buffered saline
  • hFSH Human Follicle Stimulating Hormone
  • hTSH Human Thyroid Stimulating Hormone
  • Inspect appearance properties take out the test strip and observe it visually.
  • the strip should be clean and complete, free of burrs, damage, and pollution; the material should be firmly attached.
  • the width of the test paper film 6 kinds of sensitivity test paper, take 2 of each, use a vernier caliper to measure the width of the film on the paper, and calculate the average of the two measurement results.
  • Liquid migration speed 6 kinds of sensitivity test strips, each take 2 strips, operate according to the use method in Example 7, from the test strip immersed in the sample liquid, use a stopwatch (with an accuracy of 0.01s) to time until the liquid reaches Stop timing at the boundary line between the antibody-bearing membrane and the absorbent pad. The time used is recorded as (t).
  • the minimum detection limit of the fifth test strip is 2500mIU/ml; use the national standard human chorionic gonadotropin, and the concentration is 9900mIU/ml, 10000mIU/ml, 10100mIU/ml
  • the standard solution of each concentration is tested 3 times, and the results are observed within 5 minutes.
  • the minimum detection limit of the sixth test strip is 10000mIU/ml;
  • the concentration of 5mIU/ml, 25mIU/ml, 100mIU/ml, 500mIU/ml, 2500mIU/ml, 10000mIU/ml standard solution each concentration of standard solution Tested 20 times, the difference between the test result and the label value of the corresponding standard solution was no more than one order of magnitude, no reverse difference, and no negative result in the positive standard solution.
  • Negative specificity the concentration of 500mIU/ml hLH solution A, 1000mIU/ml hFSH solution A, and 1000 ⁇ IU/ml hTSH solution A were tested separately, each repeated 3 times, and the test lines of each solution 3 times were negative .
  • Test stability Take test strips and reagent cups, test them one month after the expiration date, and perform physical property tests, accuracy, repeatability and specificity tests, and the results meet the requirements.

Abstract

一种人绒毛膜促性腺激素半定量检测试纸及其制备方法，一种包含该试纸的试剂杯，以及该试纸和试剂杯在检测人怀孕周期、异位妊娠、流产和滋养层病变中的用途。该试纸通过采用非鼠源的游离β抗体，使浓度检测的范围更宽；此外，该试纸设置至少三个试纸条，可以通过判断各试纸条的检测线是否显色，对较宽范围内的浓度进行半定量判断，且不需要进行颜色深浅的主观判断，判断的浓度范围更为准确。该试剂杯使用方便、卫生，适用于现场、即时、快速检测。

Description

一种人绒毛膜促性腺激素半定量检测试纸、试剂杯及其制备方法和应用
交叉引用
本申请要求在2019年7月30日提交中国专利局、申请号为201910693957.1、发明名称为“一种人绒毛膜促性腺激素半定量检测试纸、试剂杯及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及生物检测技术领域，具体涉及一种人绒毛膜促性腺激素半定量检测试纸、试剂杯及其制备方法和应用。
背景技术
人绒毛膜促性腺激素(human chorionic gonadotropin,hCG)是妊娠期期间由胎盘滋养层细胞分泌产生的一种糖蛋白激素，由两个不同的亚基，α、β以非共价键连接组成。在激素的产生、分泌、代谢等过程中，hCG分子会发生断裂、离解等多种变化，从而在血、尿中以多种分子形式存在。hCG是唯一不随胎盘重量增加而分泌增多的胎盘激素,《全国临床检验操作规程》中记载了怀孕妇女的hCG参考值，妊娠0.2-1周，hCG参考值为5-50mIU/ml；妊娠1-2周，hCG参考值为50-500mIU/ml；妊娠2-3周，hCG参考值为100-5000mIU/ml；妊娠3-4周，hCG参考值为500-10000mIU/ml；妊娠4-5周，hCG参考值为1000-50000mIU/ml；妊娠5-6周，hCG参考值为10000-100000mIU/ml；妊娠6-8周，hCG参考值为15000-200000mIU/ml；妊娠8-12周，hCG参考值为10000-100000mIU/ml，此后，hCG在较高浓度持续1-2周后迅速减低，以后逐渐下降并以1/5-1/10左右峰值水平维持至分娩。因此，通过检测血、尿中的hCG浓度可以判断人怀孕的周期。
此外，人绒毛膜促性腺激素(hCG)的检测也已广泛应用于涉及先兆流产、难免流产、稽留流产、异位妊娠、滋养层病变等疾病的诊断及疗效观察，通过检测hCG的浓度，可以对多种疾病进行诊断判别及病程观察，具有重要的临床意义。
目前较为常用的是利用胶体金早早孕检测试纸进行hCG检测，但其仅能作定性判断，应用范围受到限制。
中国专利文献CN108761099A公开了一种hCG周期检测试纸，包括第一试纸条和第二试纸条，通过两条试纸条检测线的显色程度，来判断人怀孕时间是前3周还是3周以上。该文献还公开了包括该试纸的hCG周期检测试剂盒，该检测试纸及试剂盒的操作方法简单，适用于现场、即时、快速检测，适用于家庭进行怀孕周数的检测，但是该试纸仅包括2个试纸条，第一试纸条最低检测限为25mIU/ml，第二试纸条最低检测限为100mIU/ml,其仅可判断人怀孕时间是前3周还是3周以上，当人怀孕的时间更长，hCG浓度更高，高于100mIU/ml时，由于其第二试纸条中所用的游离β抗体为鼠源抗体，使其质控线容易发生不显色，而无法进行hCG更高浓度范围的判断，更无法对各种疾病进行诊断和判别，浓度检测范围较窄；且该试纸是通过两条试纸条检测线的显色程度来进行比较判断，主观性较大，检测结果不够准确。
发明内容
因此，本申请要解决的技术问题在于克服现有技术中hCG检测试纸浓度检测范围窄，检测结果主观性大，结果不够准确的缺陷，从而提供一种浓度检测范围宽、检测结果准确、灵敏度高的人绒毛膜促性腺激素半定量检测试纸、试剂杯及其制备方法和应用。
为此，本申请提供了一种人绒毛膜促性腺激素半定量检测试纸，包括至少三个试纸条，每个试纸条均包括，基片以及依次粘附于所述基片上的上样垫、胶体金吸附垫、抗体承载膜和吸水垫，所述上样垫、胶体金吸附垫、抗体承载膜和吸水垫依次通过部分重叠搭接；
所述抗体承载膜上，靠近所述胶体金吸附垫的一端设置有检测线，靠近所述吸水垫的一端设置有质控线；所述检测线包被有抗人绒毛膜促性腺激素α-hCG单克隆抗体，所述质控线包被有抗鼠IgG多克隆抗体；
所述试纸条中的一个试纸条的胶体金吸附垫上吸附有胶体金－抗人绒毛膜促性腺激素β-hCG 单克隆抗体结合物；其余试纸条的胶体金吸附垫上均吸附有胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物和游离β抗体，且所述其余试纸条的胶体金吸附垫上所吸附的胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的质量比各不相同；其中，所述游离β抗体为非鼠源抗体。
所述的人绒毛膜促性腺激素半定量检测试纸，所述游离β抗体为人源化、马源、兔源或羊源抗体。
所述的人绒毛膜促性腺激素半定量检测试纸，包括六个试纸条，分别为第一试纸条、第二试纸条、第三试纸条、第四试纸条、第五试纸条和第六试纸条。
所述的人绒毛膜促性腺激素半定量检测试纸，所述第二试纸条、第三试纸条、第四试纸条、第五试纸条、第六试纸条的胶体金吸附垫上所吸附的胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的质量比依次为1:1、1:2、1:40、1:80、1:100。
所述的人绒毛膜促性腺激素半定量检测试纸，所述抗体承载膜为孔径为3-10μm的硝酸纤维素膜。
所述的人绒毛膜促性腺激素半定量检测试纸，所述检测线和质控线的距离为0.3-1.0cm，优选的，所述检测线和质控线的距离为0.5cm。
所述的人绒毛膜促性腺激素半定量检测试纸，所述上样垫和所述吸水垫上均设置有保护膜。
本申请提供了一种所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，包括，
S1、将抗体承载膜分别包被抗人绒毛膜促性腺激素α-hCG单克隆抗体和抗鼠IgG多克隆抗体，得到检测线和质控线，并在封闭处理液中进行封闭处理，干燥后备用；
S2、取胶体金，调节pH值后，加入抗人绒毛膜促性腺激素β-hCG单克隆抗体，加入稳定剂搅拌，离心，收集沉淀，用胶体金复溶液将沉淀复溶，得到胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液；
S3、将S2得到的所述胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液浇制在一个试纸条的胶体金吸附垫上，干燥，密封保存备用；
S4、向S2得到的所述胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液中加入游离β抗体混合均匀，得到胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的质量比各不相同的混合物复溶液，分别浇制在其余试纸条的胶体金吸附垫上，干燥，密封保存备用；
S4、分别取上样垫、胶体金吸附垫、抗体承载膜、吸水垫各一个沿基片的长度方向上依次以部分重叠的方式粘贴，得到一个所述的试纸条，共制备至少三个试纸条；
其中，所述游离β抗体为非鼠源抗体。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，所述试纸条的个数为六个，分别为第一试纸条、第二试纸条、第三试纸条、第四试纸条、第五试纸条和第六试纸条。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，所述第一、第二、第三试纸条中，所述抗人绒毛膜促性腺激素β-hCG单克隆抗体按4μg/ml标记胶体金，所述第四、第五、第六试纸条中，所述抗人绒毛膜促性腺激素β-hCG单克隆抗体按5μg/ml标记胶体金。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，所述第二、第三、第四、第五、第六试纸条中，所述抗人绒毛膜促性腺激素β-hCG单克隆抗体与所述游离β抗体的质量比分别为1:1、1:2、1:40、1:80、1:100。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，所述抗人绒毛膜促性腺激素α-hCG单克隆抗体的浓度为2-3mg/ml，所述抗鼠IgG多克隆抗体的浓度为1-2mg/ml。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，所述封闭处理液包括，0.08-0.12Mol缓冲液、质量百分含量为0.3-0.7％的糖份、质量百分含量为0.8-1.2％的封闭蛋白、质量百分含量为0.03-0.07％的防腐剂；其中，所述缓冲液为磷酸盐缓冲液，所述糖份为蔗糖或海藻糖，所述防腐剂为NaN 3或硫柳汞，所述封闭蛋白为络蛋白或牛血清蛋白；优选的，所述封闭处理液包括，0.1Mol缓 冲液、质量百分含量为0.5％的糖份、质量百分含量为1％的封闭蛋白、质量百分含量为0.05％的防腐剂。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，S2步骤中，所述调节pH值为，将pH值调节为6.5-7.0。
所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，所述胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液，及所述胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的混合物复溶液，浇制在胶体金吸附垫上的量均为50μl/cm 2。
本申请还提供了一种人绒毛膜促性腺激素半定量检测试剂杯，包括所述的人绒毛膜促性腺激素半定量检测试纸或所述的制备方法制备得到的人绒毛膜促性腺激素半定量检测试纸。
所述的人绒毛膜促性腺激素半定量检测试剂杯，还包括杯体和卡壳，所述卡壳放置于所述试剂杯的内部；所述卡壳中设有平行设置的插槽，所述插槽的一端设有开口，供所述试纸条插置于其中，所述试纸条的上样垫由所述开口处延伸至所述卡壳外部。
所述的人绒毛膜促性腺激素半定量检测试剂杯，还包括杯盖，所述杯盖可拆卸地盖合在所述杯体上。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述卡壳竖直放置于所述杯体内部，所述插槽的开口端靠近所述杯体底部。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述杯体内部设有检测腔、储液腔和导液腔；所述导液腔上部设有与所述储液腔连通的第一通孔，下部设有与所述检测腔连通的第二通孔；所述导液腔内设有沿所述导液腔滑动的活塞，所述活塞具有使所述导液腔与所述储液腔连通的起始状态，以及使所述导液腔与所述检测腔连通的检测状态；所述杯体远离所述检测腔一侧的杯壁上设有第三通孔，所述导液腔与所述第三通孔连通；所述卡壳竖直放置于所述检测腔内部。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述活塞上设有储液槽；在所述起始状态下，所述活塞的储液槽与所述第一通孔对齐，从而使所述导液腔与所述储液腔连通；在所述检测状态下，所述活塞的储液槽与所述第二通孔对齐，从而使所述导液腔与所述检测腔连通。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述杯盖上设有用于助推活塞的助推件，所述助推件可拆卸地嵌合在所述杯盖上。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述卡壳平行固定于所述杯盖内部。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述杯盖的外部边缘设有支撑体，用于固定侧倒放置的试剂杯，所述杯体上设有刻度线。
所述的人绒毛膜促性腺激素半定量检测试剂杯，所述卡壳为透明材料制成。
本申请提供了一种利用所述的试剂杯半定量检测人绒毛膜促性腺激素的方法，包括，将尿液样本收集于所述杯体中，从所述试纸条吸附样本开始计时，5分钟内观察所述试纸条的检测线和质控线是否显色。
本申请还提供了一种所述的人绒毛膜促性腺激素半定量检测试纸或所述的制备方法制备的人绒毛膜促性腺激素半定量检测试纸或所述的人绒毛膜促性腺激素半定量检测试剂杯，在检测人怀孕周期、异位妊娠、流产和滋养层病变中的用途。
本申请技术方案，具有如下优点：
1.本申请提供的一种人绒毛膜促性腺激素半定量检测试纸，包括至少三个试纸条，每个试纸条均包括，基片以及依次粘附于所述基片上的上样垫、胶体金吸附垫、抗体承载膜和吸水垫，所述上样垫、胶体金吸附垫、抗体承载膜和吸水垫依次通过部分重叠搭接；所述抗体承载膜上，靠近所述胶体金吸附垫的一端设置有检测线，靠近所述吸水垫的一端设置有质控线；所述检测线包被有抗人绒毛膜促性腺激素α-hCG单克隆抗体，所述质控线包被有抗鼠IgG多克隆抗体；所述试纸条中的一个试纸条的胶体金吸附垫上吸附有胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物；其余试纸条的胶体金吸附垫上均吸附有胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物和游离β抗体，且所述其余试纸条的胶体金吸附垫上所吸附的胶体金－抗人绒毛膜促性腺激素β-hCG单 克隆抗体结合物与游离β抗体的质量比各不相同；其中，所述游离β抗体为非鼠源抗体，该试纸通过采用非鼠源的游离β抗体，可以将试纸的检测限调节至较宽的范围，若采用鼠源游离β抗体，当待测样品中hCG浓度较高时，需要含有大量的游离β抗体来将试纸条的检测限调至较高的数值，但由于该游离β抗体为鼠源抗体，大量游离β抗体会与质控线上的抗鼠IgG多克隆抗体结合，只有极少量的胶体金标记的hCG与质控线结合，因此会使质控线不显色，从而导致其难以检测较高浓度的hCG的缺陷，通过采用非鼠源的游离β抗体，可以使浓度检测的范围更宽；此外，该试纸设置至少三个试纸条，且各试纸条中抗体种类和浓度不同，在检测hCG浓度时，各试纸条的显色情况不同，可以通过判断各试纸条的检测线是否显色，对较宽范围内的浓度进行半定量判断，且不需要进行颜色深浅的主观判断，判断的浓度范围更为准确。
2.本申请提供的一种人绒毛膜促性腺激素半定量检测试纸的制备方法，该方法中采用非鼠源的游离β抗体与胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物混合浇制胶体金吸附垫，可以克服采用鼠源游离β抗体时，待测样品中hCG浓度较高、鼠源游离β抗体浓度较高，导致质控线不显色，从而导致其难以检测较高浓度的hCG的缺陷，通过采用非鼠源游离β抗体，使浓度检测的范围更宽；此外，通过制备至少三个试纸条，且各试纸条中抗体种类和浓度不同，在检测hCG浓度时时，可以通过判断各试纸条的检测线是否显色，从而在较宽范围内进行浓度的半定量判断，不需要进行颜色深浅的主观判断，判断的浓度范围更为准确。
3.本申请提供的一种人绒毛膜促性腺激素半定量检测试纸的制备方法，所述第二、第三、第四、第五、第六试纸条中，所述抗人绒毛膜促性腺激素β-hCG单克隆抗体与所述游离β抗体的质量比分别为1:1、1:2、1:40、1:80、1:100，通过设置以上比例梯度，可以制备得到最低检测限分别为5mIU/ml、25mIU/ml、100mIU/ml、500mIU/ml、2500mIU/ml、10000mIU/ml的试纸条，从而得到灵敏度高、浓度检测范围宽、检测结果准确的半定量检测试纸。
4.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，包括所述的人绒毛膜促性腺激素半定量检测试纸或所述的制备方法制备的人绒毛膜促性腺激素半定量检测试纸，采用该试剂杯检测hCG浓度，检测的范围更宽；此外，采用该试剂杯可以通过判断各试纸条的检测线是否显色来进行浓度的半定量判断，不需要进行颜色深浅的主观判断，判断的浓度范围更为准确。
5.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，还包括杯体和卡壳，所述卡壳放置于所述试剂杯的内部；所述卡壳中设有平行设置的插槽，所述插槽的一端设有开口，供所述试纸条插置于其中，所述试纸条的上样垫由所述开口处延伸至所述卡壳外部，通过设置带有插槽的卡壳，可以将试纸条固定在试剂杯中，方便吸取样品；由于试纸条的上样垫由开口处延伸至卡壳外部，可以方便上样垫吸取样品；使用该试剂杯时，只需将尿液样品置于杯体内，即可完成检测，使用方便、卫生。
6.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，还包括杯盖，所述杯盖可拆卸地盖合在所述杯体上，检测时，将尿液样品放入杯体后，盖上杯盖，一方面可以使样品与外界隔绝，保证检测环境的稳定，防止外界环境影响检测效果；另一方面可防止异味散发，更加卫生。
7.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，所述杯体内部设有检测腔、储液腔和导液腔；所述导液腔上部设有与所述储液腔连通的第一通孔，下部设有与所述检测腔连通的第二通孔；所述导液腔内设有沿所述导液腔滑动的活塞，所述活塞具有使所述导液腔与所述储液腔连通的起始状态，以及使所述导液腔与所述检测腔连通的检测状态；所述杯体远离所述检测腔一侧的杯壁上设有第三通孔，所述导液腔与所述第三通孔连通；所述卡壳竖直放置于所述检测腔内部，通过设置包含卡壳和试纸的检测腔以及用于收集尿液的储液腔，可以在起始状态下使检测腔中的试纸与尿液样品隔开，然后通过设置活塞，可以在起始状态下，储液腔与导液腔连通，尿液由储液腔进入导液腔中；通过使活塞在导液腔中滑动至检测状态，尿液由导液腔进入检测腔中，从而使检测腔中卡壳内的试纸条与尿液样品接触，完成检测；通过以上结构，可以更加方便的控制检测开始的时机，可以在样品收集完成后，再开始检测，防止边收集样品，边进行检测容易出现的检测结果不够准确的问题，使得结果更为准确，且操作简便；通过将导液腔与第三通孔连通，可以方便控制活塞 的滑动。
8.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，所述活塞上设有储液槽；在所述起始状态下，所述活塞的储液槽与所述第一通孔对齐，从而使所述导液腔与所述储液腔连通；在所述检测状态下，所述活塞的储液槽与所述第二通孔对齐，从而使所述导液腔与所述检测腔连通，通过设置储液槽，可以在起始状态下，尿液样品由储液腔流入储液槽内，通过推动活塞，在检测状态下，尿液样品由储液槽流入检测腔内，从而使处于检测腔内的试纸条吸取样品开始检测，通过设置储液槽，可以保证导入检测腔的尿液样品量是固定的，从而防止进入检测腔的尿液样品过多而影响检测结果，保证检测结果的准确性。
9.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，所述杯盖上设有用于助推活塞的助推件，所述助推件可拆卸地嵌合在所述杯盖上，通过设置助推件，可以更加方便地将活塞由起始状态推至检测状态，且在不使用时，可以将助推件嵌合在杯盖上，方便收纳，防止其丢失。
10.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，所述卡壳平行固定于所述杯盖内部，通过将卡壳固定在杯盖上，可以在检测未开始时，将卡壳中的试纸条与杯体中的尿液样品分开，开始检测时，只需拧紧杯盖，将试剂杯侧倒放置，即可开始检测，防止边收集样品，边进行检测容易出现检测结果不够准确的问题，使得结果更为准确，且操作简便。
11.本申请提供的一种人绒毛膜促性腺激素半定量检测试剂杯，所述杯盖的外部边缘设有支撑体，用于固定侧倒放置的试剂杯，所述杯体上设有刻度线，通过在杯盖边缘设置支撑体，可以在将试剂杯侧倒放置检测时，用于固定试剂杯，防止试剂杯的滚动；通过设置刻度线，收集尿液时需保证尿液超过刻度线，可以在试剂杯侧倒放置时，保证有足够量的尿液样品，使试纸条顺利进行吸附检测。
12.本申请提供的一种所述的人绒毛膜促性腺激素半定量检测试纸或所述的制备方法制备的人绒毛膜促性腺激素半定量检测试纸或所述的人绒毛膜促性腺激素半定量检测试剂杯，在检测人怀孕周期、异位妊娠、流产和滋养层病变中的用途，采用以上检测试纸或试剂杯可以在较宽范围内对hCG浓度进行半定量检测，检测的范围宽，灵敏度高，结果准确，从而可以较为准确地判断人怀孕的周期，并通过检测出的浓度范围来诊断和判别异位妊娠、流产和滋养层病变等疾病。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本申请实施例1-6中人绒毛膜促性腺激素半定量检测试纸的结构示意图；
图2是本申请实施例4-6中人绒毛膜促性腺激素半定量检测试剂杯中试纸和卡壳的结构示意图；
图3是本申请实施例4中人绒毛膜促性腺激素半定量检测试剂杯的结构示意图；
图4是本申请实施例5中人绒毛膜促性腺激素半定量检测试剂杯的结构示意图；
图5是本申请实施例5中人绒毛膜促性腺激素半定量检测试剂杯起始状态下的结构示意图；
图6是本申请实施例5中人绒毛膜促性腺激素半定量检测试剂杯检测状态下的结构示意图；
图7是本申请实施例6中人绒毛膜促性腺激素半定量检测试剂杯的结构示意图；
附图标记：
1-基片；2-上样垫；3-胶体金吸附垫；4-抗体承载膜；5-吸水垫；6-检测线；7-质控线；8-保护膜；9-杯体；10-杯盖；11-第一试纸条；12-第二试纸条；13-第三试纸条；14-第四试纸条；15-第五试纸条；16-第六试纸条；17-卡壳；18-插槽；19-检测腔；20-储液腔；21-导液腔；211-第一通孔；212-第二通孔；213-第三通孔；22-活塞；221-储液槽；23-助推件；24-支撑体；25-刻度线；26-导液槽。
具体实施方式
下述实施例中涉及到的来自仓鼠的人源化游离β抗体、马源游离β抗体、兔源游离β抗体， 抗人绒毛膜促性腺激素α-hCG单克隆抗体，抗鼠IgG多克隆抗体，抗人绒毛膜促性腺激素β-hCG单克隆抗体，均购自于杭州正知生物技术有限公司。
实施例1
本实施例提供了一种人绒毛膜促性腺激素半定量检测试纸，包括六个试纸条，分别为第一试纸条11、第二试纸条12、第三试纸条13、第四试纸条14、第五试纸条15和第六试纸条16，每个试纸条的结构如图1所示，分别包括，基片1以及依次粘附于基片上的上样垫2、胶体金吸附垫3、抗体承载膜4和吸水垫5，上样垫2、胶体金吸附垫3、抗体承载膜4和吸水垫5依次通过部分重叠搭接，具体为抗体承载膜4位于胶体金吸附垫3和吸水垫5的下方，上样垫2位于胶体金吸附垫3的上方，搭接部分长度为1mm。
抗体承载膜4上，靠近胶体金吸附垫3的一端设置有检测线6，靠近吸水垫5的一端设置有质控线7；检测线6包被有抗人绒毛膜促性腺激素α-hCG单克隆抗体(抗α-hCG单克隆抗体)，质控线7包被有抗鼠IgG多克隆抗体。
第一试纸条11的胶体金吸附垫3上吸附有胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物(胶体金-抗β-hCG单克隆抗体结合物)，第二试纸条12、第三试纸条13、第四试纸条14、第五试纸条15、第六试纸条16的胶体金吸附垫3上吸附有胶体金－抗β-hCG单克隆抗体结合物和游离β抗体，且吸附的胶体金－抗β-hCG单克隆抗体结合物与游离β抗体的质量比依次为1:1、1:2、1:40、1:80、1:100，其中游离β抗体为非鼠源抗体，本实施例中该游离β抗体为来自仓鼠的人源化抗体。
进一步的，检测线6和质控线7平行设置，间距为0.3-1.0cm，在本实施例中，间距为0.5cm。
进一步的，上样垫2和吸水垫3上均设置有保护膜8。
进一步的，抗体承载膜4为孔径为3-10μm的硝酸纤维素膜。
本实施例还提供了以上人绒毛膜促性腺激素半定量检测试纸的制备方法，包括，
1.抗体承载膜的制备：
(1)选择3μm～10μm孔径的硝酸纤维素膜，根据需要将膜分切成宽度为2.0cm,长度为30.5cm的规格，备用。
(2)用0.1M磷酸盐缓冲液配制供检测线包被使用的抗α-HCG单克隆抗体，抗体浓度为2-3mg/ml，本实施例中为2.5mg/ml，用质量百分含量0.85％的氯化钠缓冲液配制供质控线包被使用的抗鼠IgG多克隆抗体，使抗体浓度为1-2mg/ml，本实施例中为1.5mg/ml。
(3)选择硝酸纤维素膜的抗体包被面并作标记，将需包被的检测线和质控线的抗体溶液平行均匀的包被在膜片上，且检测线与质控线的间距控制在距离0.3-0.7cm，本实施例中为0.5cm，硝酸纤维素膜在2℃～30℃的恒温条件下干燥备用。
(4)配制封闭处理浸泡液，向配液罐加入实际生产量的纯化水；分别称量缓冲液、糖份、封闭蛋白和防腐剂，将以上组分直接加入到配液罐中搅拌，直至完全溶解，加纯化水定容至所需体积，充分搅拌均匀，搅拌时间不少于10分钟，备用；其中配制的封闭处理浸泡液中含有0.1Mol缓冲液、质量百分含量为0.5％的糖份、质量百分含量为1％的封闭蛋白、质量百分含量为0.05％的防腐剂，其中，缓冲液为磷酸盐缓冲液，糖份为蔗糖，防腐剂为硫柳汞，封闭蛋白为牛血清蛋白。
(5)将包被了检测线和质控线的膜放于处理槽中，加入配好的上述(4)的封闭处理浸泡液，确保每条膜完全浸没在上述(4)的封闭处理浸泡液中30分钟，并保证膜不移动、不重叠，30分钟后将膜从处理槽中取出，将上述(4)的封闭处理浸泡液倒掉；用镊子将膜放在纱布上晾稍干，得到抗体承载膜。
(6)贴板干燥：撕去胶板双面胶上切割线中间的白纸，用镊子将封闭处理后的抗体承载膜正好放在胶板中央空白位置，且胶板右边与膜右边平齐，避免在生产过程中的误差，确保显色位置相对准确，贴板时全部顶质控线一头贴，膜贴在胶板上后，隔着双面胶纸抹平膜面，避免有气泡，控制室内的温度18～28℃、相对湿度≤40％，并保证干燥间空气能循环流通且除湿机风不会直接吹到膜面上。干燥时间≥4小时，后备用。
2.第一试纸条胶体金吸附垫的制备
(1)胶体金复溶液的配制：向配液罐加入实际生产量的纯化水；用电子分析天平称量海藻糖、牛血清白蛋白、柠檬酸三钠、聚乙二醇和NaN 3，直接加入到配液罐中搅拌，搅拌直至完全溶解，加纯化水定容至所需体积，充分搅拌均匀，搅拌时间大于30分钟，得到的胶体金复溶液中含有质量百分含量5％的海藻糖、质量百分含量2％的牛血清白蛋白、质量百分含量0.5％的柠檬酸三钠、质量百分含量0.05％的聚乙二醇、质量百分含量0.05％的NaN 3。
(2)用量筒量取需要标记量的胶体金，按pH6.5-7.0进行调节，即按体积百分含量为0.53％加入0.2mol/L的碳酸钾溶液，于磁力搅拌器上搅拌15分钟后，取抗β-hCG单克隆抗体，用双蒸水将抗β-hCG单克隆抗体进行稀释，并按4ug/ml标记胶体金，将抗β-hCG单克隆抗体加入胶体金中，于磁力搅拌器上搅拌30分钟，加入体积百分含量0.5‰的稳定剂聚乙二醇，搅拌30分钟后离心，收集沉淀，用胶体金复溶液按体积百分含量3％复溶，于磁力搅拌器上搅拌至混合均匀，得到体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，备用。
(3)取上述(2)中体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，用胶体金复溶液按体积百分含量50％进行复溶，于磁力搅拌器上混合均匀，按照50μl/cm 2浇制在准备好的胶体金吸附垫上，置于干燥室晾干≥4个小时，干燥室控制温度18-28℃，相对湿度≤40％，保证空气畅通且气流不能直接吹于胶体金吸附垫上，将干燥好的胶体金吸附垫放入到装有干燥剂的铝箔袋中，密封保存，做好标记为第一试纸条胶体金吸附垫，备用。
3.第二试纸条胶体金吸附垫的制备
(1)用量筒量取需要标记量的胶体金，按pH6.5-7.0进行调节，即按体积百分含量为0.53％加入0.2mol/L的碳酸钾溶液，于磁力搅拌器上搅拌15分钟后，取抗β-hCG单克隆抗体，用双蒸水将抗β-hCG单克隆抗体进行稀释，并按4ug/ml标记胶体金，将抗β-hCG单克隆抗体加入胶体金中，于磁力搅拌器上搅拌30分钟，加入体积百分含量0.5‰稳定剂聚乙二醇，搅拌30分钟后离心，收集沉淀，用胶体金复溶液按体积百分含量3％进行复溶，于磁力搅拌器上搅拌至混合均匀，得到体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，备用。
(2)取上述(1)中体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，用胶体金复溶液按体积百分含量40％进行复溶，再按抗β-hCG单克隆抗体与游离β抗体质量比为1:1加入游离β抗体，该游离β抗体为来源于仓鼠的人源化抗体，于磁力搅拌器上混合均匀，按照50μl/cm 2浇制在准备好的胶体金吸附垫上，置于干燥室晾干≥4个小时，干燥室控制温度18-28℃，相对湿度≤40％，保证空气畅通且气流不能直接吹于胶体金吸附垫上，将干燥好的胶体金吸附垫放入到装有干燥剂的铝箔袋中，密封保存，做好标记为第二试纸条胶体金吸附垫，备用。
4.第三试纸条胶体金吸附垫的制备
(1)用量筒量取需要标记量的胶体金，按pH6.5-7.0进行调节，即按体积百分含量为0.53％加入0.2mol/L的碳酸钾溶液，于磁力搅拌器上搅拌15分钟后，取抗β-hCG单克隆抗体，用双蒸水将抗β-hCG单克隆抗体进行稀释，并按4ug/ml标记胶体金，将抗β-hCG单克隆抗体加入胶体金中，于磁力搅拌器上搅拌30分钟，加入体积百分含量0.5‰稳定剂聚乙二醇，搅拌30分钟后离心，收集沉淀，用胶体金复溶液按体积百分含量3％进行复溶，于磁力搅拌器上搅拌至混合均匀，得到体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，备用。
(2)取上述(1)中体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，用胶体金复溶液按体积百分含量40％进行复溶，再按抗β-hCG单克隆抗体与游离β抗体质量比为1:2加入游离β抗体，该游离β抗体为来源于仓鼠的人源化抗体，于磁力搅拌器上混合均匀，按照50μl/cm 2浇制在准备好的胶体金吸附垫上，置于干燥室晾干≥4个小时，干燥室控制温度18-28℃，相对湿度≤40％，保证空气畅通且气流不能直接吹于胶体金吸附垫上，将干燥好的标记金放入到装有干燥剂的铝箔袋中，密封保存，做好标记为第三试纸条胶体金吸附垫，备用。
5.第四试纸条胶体金吸附垫的制备
(1)用量筒量取需要标记量的胶体金，按pH6.5-7.0进行调节，即按体积百分含量为0.53％加 入0.2mol/L的碳酸钾溶液，于磁力搅拌器上搅拌15分钟后，取抗β-hCG单克隆抗体，用双蒸水将抗β-hCG单克隆抗体进行稀释，并按5ug/ml标记胶体金，将抗β-hCG单克隆抗体加入胶体金中，于磁力搅拌器上搅拌30分钟后加入体积百分含量0.5‰稳定剂聚乙二醇，搅拌30分钟后离心，收集沉淀，用胶体金复溶液按体积百分含量3％进行复溶，于磁力搅拌器上搅拌至混合均匀，得到体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，备用；
(2)取上述(1)中体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，用胶体金复溶液按体积百分含量40％进行复溶，再按抗β-hCG单克隆抗体与游离β抗体质量比为1:40加入游离β抗体，该游离β抗体为来源于仓鼠的人源化抗体，于磁力搅拌器上混合均匀，按照50μl/cm 2浇制在准备好的胶体金吸附垫上，置于干燥室晾干≥4个小时，干燥室控制温度18-28℃，相对湿度≤40％，保证空气畅通且气流不能直接吹于胶体金吸附垫上，将干燥好的标记金放入到装有干燥剂的铝箔袋中，密封保存，做好标记为第四试纸条胶体金吸附垫，备用。
6.第五试纸条胶体金吸附垫的制备
(1)用量筒量取需要标记量的胶体金，按pH6.5-7.0进行调节，即按体积百分含量为0.53％加入0.2mol/L的碳酸钾溶液，于磁力搅拌器上搅拌15分钟后，取抗β-hCG单克隆抗体，用双蒸水将抗β-hCG单克隆抗体进行稀释，并按5ug/ml标记胶体金，将抗β-hCG单克隆抗体加入胶体金中，于磁力搅拌器上搅拌30分钟，加入体积百分含量0.5‰稳定剂聚乙二醇，搅拌30分钟后离心，收集沉淀，用胶体金复溶液按体积百分含量3％进行复溶，于磁力搅拌器上搅拌至混合均匀，得到体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，备用。
(2)取上述(1)中体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，用胶体金复溶液按体积百分含量40％进行复溶，再按抗β-hCG单克隆抗体与游离β抗体质量比为1:80加入游离β抗体，该游离β抗体为来源于仓鼠的人源化抗体，于磁力搅拌器上混合均匀，按照50μl/cm 2浇制在准备好的胶体金吸附垫上，置于干燥室晾干≥4个小时，干燥室控制温度18-28℃，相对湿度≤40％，保证空气畅通且气流不能直接吹于胶体金吸附垫上，将干燥好的标记金放入到装有干燥剂的铝箔袋中，密封保存，做好标记为第五试纸条胶体金吸附垫，备用。
7.第六试纸条胶体金吸附垫的制备
(1)用量筒量取需要标记量的胶体金，按pH6.5-7.0进行调节，即按体积百分含量为0.53％加入0.2mol/L的碳酸钾溶液，于磁力搅拌器上搅拌15分钟后，取抗β-hCG单克隆抗体，用双蒸水将抗β-hCG单克隆抗体进行稀释，并按5ug/ml标记胶体金，将抗β-hCG单克隆抗体加入胶体金中，于磁力搅拌器上搅拌30分钟，加入体积百分含量0.5‰的稳定剂聚乙二醇，搅拌30分钟后离心，收集沉淀，用胶体金复溶液按体积百分含量3％进行复溶，于磁力搅拌器上搅拌至混合均匀，得到体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，备用；
(2)取上述(1)中体积百分含量3％的胶体金－抗β-hCG单克隆抗体结合物复溶液，用胶体金复溶液按体积百分含量40％进行复溶，再按抗β-hCG单克隆抗体与游离β抗体质量比为1:100加入游离β抗体，该游离β抗体为来源于仓鼠的人源化抗体，于磁力搅拌器上混合均匀，按照50μl/cm 2浇制在准备好的胶体金吸附垫上，置于干燥室晾干≥4个小时，干燥室控制温度18-28℃，相对湿度≤40％，保证空气畅通且气流不能直接吹于胶体金吸附垫上，将干燥好的标记金放入到装有干燥剂的铝箔袋中，密封保存，做好标记为第六试纸条胶体金吸附垫，备用。
注：胶体金为浇制，是因为浇制的胶体金可以自由的裁切宽度，从而方便调节产品的深浅显色。
8.组装及裁切：
(1)取已粘贴抗体承载膜的透明基片半成品，将第一试纸条胶体金吸附垫按0.5cm宽度裁切成0.5cm×30cm的条状后粘贴在靠近检测线一侧的透明基片上，并保持与抗体承载膜呈搭接约1mm，将第一试纸条吸水垫复合在靠近质控线一侧的透明基片上并与抗体承载膜搭接约1mm，将第一试纸条上样垫复合在第一试纸条胶体金吸附垫远离抗体承载膜的一端并与其搭接约1mm，做好标记，备用；
(2)将已组装备用的基片，切割成条状试纸，标记为第一试纸条备用。
(3)取已粘贴抗体承载膜的透明基片半成品，将第二试纸条胶体金吸附垫按0.5cm宽度裁切成 0.5cm×30cm的条状后粘贴在靠近检测线一侧的透明基片，并保持与抗体承载膜呈搭接约1mm，将第二试纸条吸水垫复合在靠近质控线一侧的透明基片上并与抗体承载膜搭接约1mm，将第二试纸条上样垫复合在第二试纸条胶体金吸附垫远离抗体承载膜的一端并与其搭接约1mm，做好标记，备用；
(4)将已组装备用的基片，切割成条状试纸，做好标记第二试纸条备用。
(5)取已粘贴抗体承载膜的透明基片半成品，将第三试纸条胶体金吸附垫按0.5cm宽度裁切成0.5cm×30cm的条状后粘贴在靠近检测线一侧的透明基片上，并保持与抗体承载膜呈搭接约1mm，将第三试纸条吸水垫复合在靠近质控线一侧的透明基片上并与膜搭接约1mm，将第三试纸条上样垫复合在第三试纸条胶体金吸附垫远离抗体承载膜的一端并与其搭接约1mm，做好标记，备用；
(6)将已组装备用的基片，切割成条状试纸，做好标记第三试纸条备用。
(7)取已粘贴抗体承载膜的透明基片半成品，将第四试纸条胶体金吸附垫按0.5cm宽度裁切成0.5cm×30cm的条状后粘贴在靠近检测线一侧的透明基片上，并保持与抗体承载膜呈搭接约1mm，将第四试纸条吸水垫复合在靠近质控线一侧的透明基片上并与抗体承载膜搭接约1mm，将第四试纸条上样垫复合在第四试纸条胶体金吸附垫远离抗体承载膜的一端并与其搭接约1mm，做好标记，备用；
(8)将已组装备用的基片，切割成条状试纸，做好标记第四试纸条备用。
(9)取已粘贴抗体承载膜的透明基片半成品，将第五试纸条胶体金吸附垫按0.5cm宽度裁切成0.5cm×30cm的条状后粘贴在靠近检测线一侧的透明基片上，并保持与抗体承载膜呈搭接约1mm，将第五试纸条吸水垫复合在靠近质控线一侧的透明基片上并与抗体承载膜搭接约1mm，将第五试纸条上样垫复合在第五试纸条胶体金吸附垫远离抗体承载膜的一端并与其搭接约1mm，做好标记，备用；
(10)将已组装备用的基片，切割成条状试纸，做好标记第五试纸条备用。
(11)取已粘贴抗体承载膜的透明基片半成品，将第六试纸条胶体金吸附垫按0.5cm宽度裁切成0.5cm×30cm的条状后粘贴在靠近检测线一侧的透明基片上，并保持与抗体承载膜呈搭接约1mm，将第六试纸条吸水垫复合在靠近质控线一侧的透明基片上并与抗体承载膜搭接约1mm，将第六试纸条上样垫复合在第六试纸条胶体金吸附垫远离抗体承载膜的一端并与其搭接约1mm，做好标记，备用；
(12)将已组装备用的基片，切割成条状试纸，做好标记第六试纸条备用。
实施例2
本实施例提供了一种人绒毛膜促性腺激素半定量检测试纸，与实施例1不同之处在于，其中的游离β抗体为马源抗体，且检测线6和质控线7的间距为0.3cm。
本实施例还提供了以上人绒毛膜促性腺激素半定量检测试纸的制备方法，与实施例1的制备方法的不同之处在于，所使用的游离β抗体为马源抗体；抗体承载膜的制备过程中，抗α-HCG单克隆抗体的浓度为2mg/ml，抗鼠IgG多克隆抗体的浓度为1mg/ml，检测线与质控线的间距为0.3cm；所使用的封闭处理液包括，0.08Mol缓冲液、质量百分含量为0.3％的糖份、质量百分含量为0.8％的封闭蛋白、质量百分含量为0.03％的防腐剂，封闭处理液中的缓冲液为磷酸盐缓冲液，糖份为海藻糖，防腐剂为NaN 3，封闭蛋白为络蛋白。
实施例3
本实施例提供了一种人绒毛膜促性腺激素半定量检测试纸，与实施例1不同之处在于，其中的游离β抗体为兔源抗体，且检测线6和质控线7的间距为1.0cm。
本实施例还提供了以上人绒毛膜促性腺激素半定量检测试纸的制备方法，与实施例1的制备方法的不同之处在于，所使用的游离β抗体为兔源抗体；抗体承载膜的制备过程中，抗α-HCG单克隆抗体的浓度为3mg/ml，抗鼠IgG多克隆抗体的浓度为2mg/ml，检测线与质控线的间距为0.7cm；所使用的封闭处理液包括，0.12Mol缓冲液、质量百分含量为0.7％的糖份、质量百分含量为1.2％的封闭蛋白、质量百分含量为0.07％的防腐剂，封闭处理液中的缓冲液为磷酸盐缓冲液，糖份为蔗糖，防腐剂为硫柳汞，封闭蛋白为牛血清蛋白。
实施例4
如图3所示，本实施例提供了一种人绒毛膜促性腺激素半定量检测试剂杯，包括实施例1中的 人绒毛膜促性腺激素半定量检测试纸，还包括杯体9、杯盖10和卡壳17，卡壳17竖直放置于杯体9内部，杯盖10可拆卸地盖合在杯体9上；如图2所示，卡壳17中有六个以上平行设置的插槽18，插槽18靠近杯体9底部的一端设有开口,第一试纸条11、第二试纸条12、第三试纸条13、第四试纸条14、第五试纸条15和第六试纸条16依次从开口处插置在六个插槽18内，试纸条的上样垫2由开口处延伸至卡壳17的外部。卡壳17为塑料制成，当然，作为可替换的实施方式，卡壳17还可以为其他透明材料。
具体的，第一试纸条11、第二试纸条12、第三试纸条13、第四试纸条14、第五试纸条15和第六试纸条16，每个试纸条的结构如图1所示，分别包括，基片1以及依次粘附于基片上的上样垫2、胶体金吸附垫3、抗体承载膜4和吸水垫5，上样垫2、胶体金吸附垫3、抗体承载膜4和吸水垫5依次通过部分重叠搭接，具体为抗体承载膜4位于胶体金吸附垫3和吸水垫5的下方，上样垫2位于胶体金吸附垫3的上方，搭接部分长度为1mm。
抗体承载膜4上，靠近胶体金吸附垫3的一端设置有检测线6，靠近吸水垫5的一端设置有质控线7；检测线6包被有抗α-hCG单克隆抗体，质控线7包被有抗鼠IgG多克隆抗体。
第一试纸条11的胶体金吸附垫3上吸附有胶体金－抗β-hCG单克隆抗体结合物，第二试纸条12、第三试纸条13、第四试纸条14、第五试纸条15、第六试纸条16的胶体金吸附垫3上吸附有胶体金－抗β-hCG单克隆抗体结合物和游离β抗体，其中游离β抗体为非鼠源抗体，本实施例中该游离β抗体为来自仓鼠的人源化抗体。
进一步的，检测线6和质控线7平行设置，间距为0.3-1.0cm，在本实施例中，间距为0.5cm。进一步的，上样垫2和吸水垫3上均设置有保护膜8。进一步的，抗体承载膜4为孔径为3-10μm的硝酸纤维素膜。
使用时，打开杯盖10，将尿液样本收集于杯体9中，将杯盖10盖合在杯体9上，将杯体9直立放置，六个试纸条的上样垫2均浸入样品中，样品由于毛细作用，由上样垫2吸入试纸条内，依次通过胶体金吸附垫3、抗体承载膜4达到吸水垫5，样品中的hCG与胶体金吸附垫3中吸附的胶体金－抗β-hCG单克隆抗体结合物或游离β抗体结合。与胶体金－抗β-hCG单克隆抗体结合物结合的hCG随着液体的流动，到达抗体承载膜4，分别与检测线中包被的抗α-hCG单克隆抗体和质控线包被的抗鼠IgG多克隆抗体结合，由于其带有胶体金标记，因此会在检测线和质控线处显色。而与游离β抗体结合的hCG，由于其不带有胶体金标记，因此不会在检测线处显色，且由于该游离β抗体不是鼠源抗体，不会与质控线处包被的抗鼠IgG多克隆抗体结合，即使样品中hCG浓度较高，有大量的hCG与游离β抗体结合，也不会造成质控线大部分抗体与hCG-游离β抗体结合物结合，而引起的质控线不显色，从而可以测量hCG浓度较高的样品，检测限可以达到10000mIU/ml，使浓度检测的范围更宽。由于六个试纸条的胶体金吸附垫中胶体金－抗β-hCG单克隆抗体结合物和游离β抗体的含量比例不同，因此，六个试纸条的检测线和质控线会呈现不同的显色情况，根据显色情况，可以来半定量测定样品中的hCG浓度。
实施例5
如图4-6所示，本实施例提供了一种人绒毛膜促性腺激素半定量检测试剂杯，包括实施例1中的人绒毛膜促性腺激素半定量检测试纸，还包括杯体9和卡壳17。
杯体9内部设有检测腔19、储液腔20和导液腔21，导液腔21位于储液腔20下方；导液腔21上部设有与储液腔20连通的第一通孔211，下部设有与检测腔19连通的第二通孔212。导液腔21内设有沿导液腔滑动的活塞22，活塞22具有使导液腔21与储液腔20连通的起始状态，以及使导液腔21与检测腔19连通的检测状态；杯体9远离检测腔19一侧的杯壁上设有第三通孔213，导液腔21与第三通孔213连通。活塞22上设有储液槽221，如图5所示，在起始状态下，活塞的储液槽221与第一通孔211对齐，从而使导液腔21与储液腔20连通；如图6所示，在检测状态下，活塞的储液槽221与第二通孔212对齐，从而使导液腔21与检测腔19连通。
进一步的，杯体9内底部设有导液槽26，所述导液槽26与所述检测腔19连通，同时所述导液槽26位于第二通孔212的下方，从而使导液槽26与第二通孔212连通。
卡壳17及试纸条的结构和个数与实施例4相同，不同之处在于，插置有试纸条的卡壳17竖直放置于检测腔19内部。
该试剂杯通过设置卡壳17，可以将试纸条固定于试剂杯的检测腔19内，使用时，杯体保持直立状态，可使试纸条也保持直立状态，方便对样品进行吸取；试剂杯将试纸条设置为6个，可以根据不同试纸条的显色情况，对尿液中的hCG浓度进行半定量检测，可检测的浓度范围更宽，且不需要通过显色程度来进行比较判断，检测结果更准确。通过设置包含卡壳和试纸的检测腔19以及用于收集尿液的储液腔20，可以在起始状态下使检测腔19中的试纸与尿液样品隔开，然后通过设置活塞22及活塞上的储液槽221，可以在起始状态下，尿液通过第一通孔211由储液腔20进入储液槽中221；通过使活塞22在导液腔21中滑动至检测状态，尿液通过第二通孔212由储液槽221流入导液槽26，然后再流入检测腔19中，从而使检测腔19中卡壳17内的试纸条与尿液样品接触，完成检测；通过以上结构，可以更加方便的控制检测开始的时机，可以在样品收集完成后，再开始检测，防止边收集样品，边进行检测容易出现的检测结果不够准确的问题，使得结果更为准确，且操作简便；通过将导液腔21与第三通孔213连通，可以方便控制活塞22的滑动；通过设置储液槽221，可以保证导入检测腔19的尿液样品量是固定的，从而防止进入检测腔19的尿液样品过多而影响检测结果，保证检测结果的准确性。使用该试剂杯使用时，只需将尿液样品直接收集于杯体内，然后推动活塞22，便可进行检测和结果读取，使用方便卫生。
该试剂杯还包括杯盖10，杯盖10可拆卸地盖合在杯体9上。该试剂杯的杯盖10上还设有用于助推活塞22的助推件23，助推件23可拆卸地嵌合在杯盖10上，通过设置助推件23，可以更加方便地将活塞22由起始状态推至检测状态，且在不使用时，可以将助推件23嵌合在杯盖10上，方便收纳，防止其丢失。
使用时，打开杯盖10，将尿液样本收集于杯体9的储液腔20中，将杯盖10盖合在杯体9上，将杯体9直立放置，此时，如图5所示，试剂杯处于初始状态，储液腔20中的尿液样本通过第一通孔211进入活塞22的储液槽221中，取下嵌合在杯盖10上的助推件23，将其插入第三通孔213，通过助推件23将活塞22推至检测状态，如图6所示，储液槽221中的尿液样本通过第二通孔212流入底部的导液槽26中，然后再流入检测腔19中，处于检测腔19中的六个试纸条的上样垫2均浸入样品中，样品由于毛细作用，由上样垫2吸入试纸条内，依次通过胶体金吸附垫3、抗体承载膜4，到达吸水垫5，样品中的hCG与胶体金吸附垫3中吸附的胶体金－抗β-hCG单克隆抗体结合物或游离β抗体结合。与胶体金－抗β-hCG单克隆抗体结合物结合的hCG随着液体的流动，到达抗体承载膜4，分别与检测线中包被的抗α-hCG单克隆抗体和质控线包被的抗鼠IgG多克隆抗体结合，由于其带有胶体金标记，因此会在检测线6和质控线7处显色。而与游离β抗体结合的hCG，由于其不带有胶体金标记，因此不会在检测线6处显色，且由于该游离β抗体不是鼠源抗体，不会与质控线7处包被的抗鼠IgG多克隆抗体结合，由于六个试纸条的胶体金吸附垫中胶体金－抗β-hCG单克隆抗体结合物和游离β抗体的含量比例不同，因此，六个试纸条的检测线6会呈现不同的显色情况，可以根据不同试纸条的显色情况，对尿液中的hCG浓度进行半定量检测，与两个试纸相比，可检测的浓度范围更宽，且不需要通过显色程度来进行比较判断，检测结果更准确。该试剂杯使用时，只需将尿液样品直接收集于杯体内，然后推动活塞22，便可进行检测和结果读取，使用方便卫生。
实施例6
如图7所示，本实施例提供了一种人绒毛膜促性腺激素半定量检测试剂杯，包括，杯体9、卡壳17和实施例1中的试纸。该试剂杯还包括杯盖10，杯盖10可拆卸地盖合在杯体9上。卡壳17及试纸条的结构和个数与实施例4相同，不同之处在于，卡壳17平行固定于杯盖10内部。
该试剂杯通过设置卡壳17，可以将试纸条平行固定于试剂杯的杯盖10内，使用时，杯体9呈侧倒状态，方便试纸条对样品进行吸取；通过将试纸条设置为6个，可以根据不同试纸条的显色情况，对尿液中的hCG浓度进行半定量检测，可检测的浓度范围更宽，且不需要通过显色程度来进行比较判断，检测结果更准确。通过将卡壳17固定在杯盖10上，可以在检测未开始时，将卡壳17中的试纸条与杯体中的尿液样品分开，开始检测时，只需拧紧杯盖10，将试剂杯侧倒放置，即可开始 检测，可方便控制检测开始的时机，防止边收集样品，边进行检测容易出现检测结果不够准确的问题，使得结果更为准确，且操作简便。
该试剂杯的杯盖10的外部边缘设有支撑体24，用于固定侧倒放置的试剂杯，杯体9上设有刻度线25。通过在杯盖10边缘设置支撑体24，可以在将试剂杯侧倒放置检测时，用于固定试剂杯，防止试剂杯的滚动；通过设置刻度线25，收集尿液时需保证尿液超过刻度线25，可以在试剂杯侧倒放置时，保证有足够量的尿液样品，使试纸条顺利进行吸附检测。
使用时，打开杯盖10，将尿液样本收集于杯体9中，使尿液样本量超过杯体9上的刻度线25，将杯盖10盖合在杯体9上并拧紧，将试剂杯侧倒放置，并通过支撑体24将试剂杯固定，六个试纸条的上样垫2均浸入样品中，样品由于毛细作用，由上样垫2吸入试纸条内，依次通过胶体金吸附垫3、抗体承载膜4，到达吸水垫5，样品中的hCG与胶体金吸附垫3中吸附的胶体金－抗β-hCG单克隆抗体结合物或游离β抗体结合。与胶体金－抗β-hCG单克隆抗体结合物结合的hCG随着液体的流动，到达抗体承载膜4，分别与检测线中包被的抗α-hCG单克隆抗体和质控线包被的抗鼠IgG多克隆抗体结合，由于其带有胶体金标记，因此会在检测线6和质控线7处显色。而与游离β抗体结合的hCG，由于其不带有胶体金标记，因此不会在检测线6处显色，且由于该游离β抗体不是鼠源抗体，不会与质控线7处包被的抗鼠IgG多克隆抗体结合，由于六个试纸条的胶体金吸附垫中胶体金－抗β-hCG单克隆抗体结合物和游离β抗体的含量比例不同，因此，六个试纸条的检测线6会呈现不同的显色情况，可以根据不同试纸条的显色情况，对尿液中的hCG浓度进行半定量检测，与两个试纸相比，可检测的浓度范围更宽，且不需要通过显色程度来进行比较判断，检测结果更准确。该试剂杯使用时，只需将尿液样品直接收集于杯体内，然后拧紧杯盖，侧倒放置，便可进行检测和结果读取，使用方便卫生。
实施例7
本实施例提供了一种实施例4中人绒毛膜促性腺激素半定量检测试剂杯的使用方法，包括，将样本、试剂杯均在室温下平衡；然后打开杯盖10，将尿液样本收集于杯体9中，将杯盖10盖合在杯体9上，将杯体9直立放置，六个试纸条的上样垫2均浸入样品中，5分钟内观察六个试纸条的检测线和质控线是否显色。检测结果包括如下几种情况：
表1试纸条的显示结果说明
Figure PCTCN2020071034-appb-000001
Figure PCTCN2020071034-appb-000002
检测开始时间为月经应该来但没有来的当天。如果提前检测，需在预计的月经之日再次检测。如果月经推迟，需在3天后再次检测。如果经期不规律，检测前应以最近月份中最长的经期计算。如果完全不知道月经预计何时开始，则至少在上次未采取保护措施行房19天后再进行检测。
实施例8
本实施例提供了实施例4中人绒毛膜促性腺激素半定量检测试剂杯在检测人怀孕周期、异位妊娠、流产或滋养层病变中的用途。
采用实施例7中试剂杯的使用方法，对尿液样品进行检测，根据表1中的说明，半定量检测出尿液样品中人绒毛膜促性腺激素的含量范围。
根据《全国临床检验操作规程》中怀孕妇女的hCG参考值，来判断人的怀孕周期，具体为：妊娠0.2-1周，hCG参考值为5-50mIU/ml；妊娠1-2周，hCG参考值为50-500mIU/ml；妊娠2-3周，hCG参考值为100-5000mIU/ml；妊娠3-4周，hCG参考值为500-10000mIU/ml；妊娠4-5周，hCG参考值为1000-50000mIU/ml；妊娠5-6周，hCG参考值为10000-100000mIU/ml；妊娠6-8周，hCG参考值为15000-200000mIU/ml；妊娠8-12周，hCG参考值为10000-100000mIU/ml。
在临床诊断方面，通过半定量检测出的尿液样品中人绒毛膜促性腺激素的含量范围，进行异位妊娠、流产、滋养层病变的诊断判别和病程观察。具体为，在异位妊娠诊断方面，宫外孕时，hCG常为312-625mIU/ml，在子宫出血3天后，hCG仍可为阳性，因此hCG检查可作为它与其它急腹症的鉴别。在流产诊断方面，发生不完全流产如子宫内尚有胎盘组织残存，则hCG检查仍可呈阳性，而完全流产或死胎时，hCG则由阳性转为阴性，因此，其可作为保胎或吸宫治疗的参考依据。在先兆流产诊断方面，如尿中hCG一直维持较高水平则不会发生难免流产，而如果hCG在2500mIU/ml以下，并逐渐下降，则有流产或死胎的可能，当降至600mIU/ml则难免流产，因此，在保胎治疗中，如果hCG仍然继续下降，则说明保胎无效，而如果hCG不断上升，则说明保胎成功。此外，葡萄胎、恶性葡萄胎、绒毛膜上皮癌等患者尿中hCG会显著升高，可达10万到数百万mIU/ml。滋养层细胞肿瘤患者术后3周后尿hCG应小于50mIU/ml，8-12周呈阴性，如果hCG不下降或不转阴，则提示可能有残留病变。
对比例1
本对比例提供了一种与实施例1相似的人绒毛膜促性腺激素半定量检测试纸，不同之处在于，所用的游离β抗体为鼠源抗体，用国家级人绒毛膜促性腺激素标准品，配成浓度为10000mIU/ml的标准液，采用该试纸对其进行检测，得到的结果为六个试纸条中，第四、第五、第六试纸条的质控线均不显色，显示结果为无效。
对比例2
用国家级人绒毛膜促性腺激素标准品，配成浓度为5mIU/ml的标准液，采用中国专利文献CN108761099A中公开的hCG周期检测试纸及包括该试纸的hCG周期检测试剂盒对其进行检测，得到的结果为两个试纸条的质控线显色，两个试纸条的检测线均无显色。
实验例
1、所需试剂如下:
0mIU/ml的样品液:含蛋白的磷酸盐缓冲液(PBS)即为0mIU/ml的HCG样品液。
5mIU/ml，25mIU/ml，100mIU/ml,500mIU/ml,2500mIU/ml,10000mIU/ml的人绒毛膜促性腺激素(HCG)样品液:将人绒毛膜促性腺激素(HCG)标准品用含蛋白的磷酸盐缓冲液(0.01M PBS)配制成相应浓度。
500mIU/ml人促黄体生成素(hLH)：将hLH标准品用含蛋白的磷酸盐缓冲液(PBS)复溶后，用0mIU/ml的HCG样品液将其配制成浓度为500mIU/ml的hLH A液；用5mIU/ml的人绒毛膜促性腺激素样品液将其配制成浓度为500mIU/ml的hLH B液。
1000mIU/ml人卵泡刺激素(hFSH)：将hFSH标准品用含蛋白的磷酸盐缓冲液(PBS)复溶后，用0mIU/ml的HCG样品液将其配制成浓度为1000mIU/ml的hFSH A液；用5mIU/ml的人绒毛膜促性腺激素样品液将其配制成浓度为1000mIU/ml的hFSH B液。
1000μIU/ml人促甲状腺激素(hTSH)：将hTSH标准品用含蛋白的磷酸盐缓冲液(PBS)复溶后，用0mIU/ml的HCG样品液将其配制成浓度为1000μIU/ml的hTSH A液；用5mIU/ml的人绒毛膜促性腺激素样品液将其配制成浓度为1000μIU/ml的hTSH B液。
2、物理性状检验：
检验外观性状：取出试纸条，用目力观察，纸条应整洁完整、无毛刺、无破损、无污染；材料附着牢固。
试纸膜的宽度：6种灵敏度的试纸条，每种取2条，用游标卡尺测量纸条上膜的宽度，计算2条测定结果的平均值。
液体移行速度：6种灵敏度的试纸条，每种取2条，按实施例7中的使用方法进行操作，从试纸条浸入样品液开始用秒表(精度为0.01s)计时，直至液体达到抗体承载膜与吸水垫的交界线时停止计时，所用的时间记为(t)，用游标卡尺(精密度0.02mm)测量上样垫的上样端到抗体承载膜与吸水垫的交界线的长度，记为(L)，计算L/t即为移行速度，6种灵敏度的试纸条，每种重复测量2条试纸条，各自取平均值，结果符合液体移行速度不低于10mm/min，其层析状态为：规则，胶金不与水分层，5分钟内本底基本清晰。
3、检验最低检测限
用国家级人绒毛膜促性腺激素标准品，配成浓度分别为2.5mIU/ml，5mIU/ml，10mIU/ml的标准液，采用本申请实施例4的试剂杯对每种浓度的标准液检测3次，5分钟内观察结果，第一试纸条的最低检测限为5mIU/ml；用国家级人绒毛膜促性腺激素标准品，配成浓度分别为12.5mIU/ml，25mIU/ml，50mIU/ml的标准液，每种浓度的标准液检测3次，5分钟内观察结果，第二试纸条的最低检测限为25mIU/ml；用国家级人绒毛膜促性腺激素标准品，配成浓度分别为50mIU/ml，100mIU/ml，200mIU/ml的标准液，每种浓度的标准液检测3次，5分钟内观察结果，第三试纸条的最低检测限为100mIU/ml；用国家级人绒毛膜促性腺激素标准品，配成浓度分别为400mIU/ml，500mIU/ml，600mIU/ml的标准液，每种浓度的标准液检测3次，5分钟内观察结果，第四试纸条的最低检测限为500mIU/ml；用国家级人绒毛膜促性腺激素标准品，配成浓度分别为2400mIU/ml，2500mIU/ml，2600mIU/ml的标准液，每种浓度的标准液检测3次，5分钟内观察结果，第五试纸条的最低检测限为2500mIU/ml；用国家级人绒毛膜促性腺激素标准品，配成浓度分别为9900mIU/ml，10000mIU/ml，10100mIU/ml的标准液，每种浓度的标准液检测3次，5分钟内观察结果，第六试纸条的最低检测限为10000mIU/ml；
4、检验准确性和重复性：
用国家级人绒毛膜促性腺激素标准品，配成浓度分别为5mIU/ml，25mIU/ml，100mIU/ml,500mIU/ml,2500mIU/ml,10000mIU/ml的标准液，每种浓度的标准液检测20次，检测结果与相应标准液标示值相差同向不超过一个量级，未出现反向相差，阳性标准液未出现阴性结果。
5、特异性：
阴性特异性：将浓度为500mIU/ml的hLH A液、1000mIU/ml的hFSH A液、1000μIU/ml的hTSH A液分别进行检测，各重复3次，各溶液的3次结果检测线均为阴性。
阳性特异性：将浓度为500mIU/ml的hLH B液、1000mIU/ml的hFSH B液、1000μIU/ml的hTSH B液分别进行检测，各重复3次，各溶液的3次结果检测线结果应均为阳性，
6、检验稳定性：取试纸条和试剂杯，在有效期后一个月进行检测，分别进行物理性状检验、准确性、重复性和特异性检验，结果均符合要求。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本申请创造的保护范围之中。

Claims (19)

  1. 一种人绒毛膜促性腺激素半定量检测试纸，其特征在于，包括至少三个试纸条，每个试纸条均包括，基片(1)以及依次粘附于所述基片(1)上的上样垫(2)、胶体金吸附垫(3)、抗体承载膜(4)和吸水垫(5)，所述上样垫(2)、胶体金吸附垫(3)、抗体承载膜(4)和吸水垫(5)依次通过部分重叠搭接；
    所述抗体承载膜(4)上，靠近所述胶体金吸附垫(3)的一端设置有检测线(6)，靠近所述吸水垫(5)的一端设置有质控线(7)；所述检测线(6)包被有抗人绒毛膜促性腺激素α-hCG单克隆抗体，所述质控线(7)包被有抗鼠IgG多克隆抗体；
    所述试纸条中的一个试纸条的胶体金吸附垫(3)上吸附有胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物；其余试纸条的胶体金吸附垫(3)上均吸附有胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物和游离β抗体，且所述其余试纸条的胶体金吸附垫(3)上所吸附的胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的质量比各不相同；其中，所述游离β抗体为非鼠源抗体。
  2. 根据权利要求1所述的人绒毛膜促性腺激素半定量检测试纸，其特征在于，所述游离β抗体为人源化、马源、兔源或羊源抗体。
  3. 根据权利要求1或2所述的人绒毛膜促性腺激素半定量检测试纸，其特征在于，包括六个试纸条，分别为第一试纸条(11)、第二试纸条(12)、第三试纸条(13)、第四试纸条(14)、第五试纸条(15)和第六试纸条(16)。
  4. 根据权利要求3所述的人绒毛膜促性腺激素半定量检测试纸，其特征在于，所述第二试纸条(12)、第三试纸条(13)、第四试纸条(14)、第五试纸条(15)、第六试纸条(16)的胶体金吸附垫上所吸附的胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的质量比依次为1:1、1:2、1:40、1:80、1:100。
  5. 一种权利要求1-4任一项所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，其特征在于，包括，
    S1、将抗体承载膜分别包被抗人绒毛膜促性腺激素α-hCG单克隆抗体和抗鼠IgG多克隆抗体，得到检测线和质控线，并在封闭处理液中进行封闭处理，干燥后备用；
    S2、取胶体金，调节pH值后，加入抗人绒毛膜促性腺激素β-hCG单克隆抗体，加入稳定剂搅拌，离心，收集沉淀，用胶体金复溶液将沉淀复溶，得到胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液；
    S3、将S2得到的所述胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液浇制在一个试纸条的胶体金吸附垫上，干燥，密封保存备用；
    S4、向S2得到的所述胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物复溶液中加入游离β抗体混合均匀，得到胶体金－抗人绒毛膜促性腺激素β-hCG单克隆抗体结合物与游离β抗体的质量比各不相同的混合物复溶液，分别浇制在其余试纸条的胶体金吸附垫上，干燥，密封保存备用；
    S5、分别取上样垫、胶体金吸附垫、抗体承载膜、吸水垫各一个沿基片的长度方向上依次以部分重叠的方式粘贴，得到一个所述的试纸条，共制备至少三个试纸条；
    其中，所述游离β抗体为非鼠源抗体。
  6. 根据权利要求5所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，其特征在于，所述试纸条的个数为六个，分别为第一试纸条、第二试纸条、第三试纸条、第四试纸条、第五试纸条和第六试纸条。
  7. 根据权利要求6所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，其特征在于，所述第一、第二、第三试纸条中，所述抗人绒毛膜促性腺激素β-hCG单克隆抗体按4μg/ml标记胶体金，所述第四、第五、第六试纸条中，所述抗人绒毛膜促性腺 激素β-hCG单克隆抗体按5μg/ml标记胶体金。
  8. 根据权利要求6或7所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，其特征在于，所述第二、第三、第四、第五、第六试纸条中，所述抗人绒毛膜促性腺激素β-hCG单克隆抗体与所述游离β抗体的质量比分别为1:1、1:2、1:40、1:80、1:100。
  9. 根据权利要求5-8任一项所述的人绒毛膜促性腺激素半定量检测试纸的制备方法，其特征在于，所述抗人绒毛膜促性腺激素α-hCG单克隆抗体的浓度为2-3mg/ml，所述抗鼠IgG多克隆抗体的浓度为1-2mg/ml。
  10. 一种人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，包括权利要求1-4任一项所述的人绒毛膜促性腺激素半定量检测试纸或权利要求5-9任一项所述的制备方法制备得到的人绒毛膜促性腺激素半定量检测试纸。
  11. 根据权利要求10所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，还包括杯体(9)和卡壳(17)，所述卡壳(17)放置于所述试剂杯的内部；所述卡壳(17)中设有平行设置的插槽(18)，所述插槽(18)的一端设有开口，供所述试纸条插置于其中，所述试纸条的上样垫(2)由所述开口处延伸至所述卡壳(17)外部。
  12. 根据权利要求11所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，还包括杯盖(10)，所述杯盖(10)可拆卸地盖合在所述杯体(9)上。
  13. 根据权利要求11或12所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，所述卡壳(17)竖直放置于所述杯体(9)内部，所述插槽(18)的开口端靠近所述杯体(9)底部。
  14. 根据权利要求13所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，所述杯体(9)内部设有检测腔(19)、储液腔(20)和导液腔(21)；所述导液腔(21)上部设有与所述储液腔连通的第一通孔(211)，下部设有与所述检测腔(19)连通的第二通孔(212)；所述导液腔(21)内设有沿所述导液腔滑动的活塞(22)，所述活塞(22)具有使所述导液腔(21)与所述储液腔(20)连通的起始状态，以及使所述导液腔(21)与所述检测腔(19)连通的检测状态；所述杯体(9)远离所述检测腔(19)一侧的杯壁上设有第三通孔(213)，所述导液腔(21)与所述第三通孔(213)连通；所述卡壳(17)竖直放置于所述检测腔(19)内部。
  15. 根据权利要求14所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，所述活塞(22)上设有储液槽(221)；在所述起始状态下，所述活塞的储液槽(221)与所述第一通孔(211)对齐，从而使所述导液腔(21)与所述储液腔(20)连通；在所述检测状态下，所述活塞的储液槽(221)与所述第二通孔(212)对齐，从而使所述导液腔(21)与所述检测腔(19)连通。
  16. 根据权利要求14或15所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，所述杯盖(10)上设有用于助推活塞的助推件(23)，所述助推件(23)可拆卸地嵌合在所述杯盖(10)上。
  17. 根据权利要求11或12所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，所述卡壳(17)平行固定于所述杯盖(10)内部。
  18. 根据权利要求17所述的人绒毛膜促性腺激素半定量检测试剂杯，其特征在于，所述杯盖(10)的外部边缘设有支撑体(24)，用于固定侧倒放置的试剂杯，所述杯体(9)上设有刻度线(25)。
  19. 一种权利要求1-4任一项所述的人绒毛膜促性腺激素半定量检测试纸或权利要求5-9任一项所述的制备方法制备的人绒毛膜促性腺激素半定量检测试纸或权利要求10-18任一项所述的人绒毛膜促性腺激素半定量检测试剂杯，在检测人怀孕周期、异位妊娠、流产和滋养层病变中的用途。
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