CN117452002B - Human chorionic gonadotrophin colloidal gold detection test strip and kit for urine saliva simultaneous detection - Google Patents
Human chorionic gonadotrophin colloidal gold detection test strip and kit for urine saliva simultaneous detection Download PDFInfo
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- CN117452002B CN117452002B CN202311789757.9A CN202311789757A CN117452002B CN 117452002 B CN117452002 B CN 117452002B CN 202311789757 A CN202311789757 A CN 202311789757A CN 117452002 B CN117452002 B CN 117452002B
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- 238000001514 detection method Methods 0.000 title claims abstract description 94
- 229960004407 chorionic gonadotrophin Drugs 0.000 title claims abstract description 93
- 210000003296 saliva Anatomy 0.000 title claims abstract description 56
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- 238000012360 testing method Methods 0.000 title claims abstract description 33
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- 229910052737 gold Inorganic materials 0.000 claims abstract description 63
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
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- 239000002253 acid Substances 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
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- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
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- 239000012153 distilled water Substances 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
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- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- 239000003381 stabilizer Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 3
- 229940038773 trisodium citrate Drugs 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 2
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- 230000035935 pregnancy Effects 0.000 description 3
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
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- 210000003079 salivary gland Anatomy 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 1
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- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 238000003317 immunochromatography Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
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- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a human chorionic gonadotrophin colloidal gold detection test strip and a kit for urine saliva simultaneous detection, and relates to the technical field of in-vitro diagnosis, wherein the human chorionic gonadotrophin colloidal gold detection test strip for urine saliva simultaneous detection comprises a bottom plate, a sample pad, a biotinylation pad, a targeting polypeptide gold pad, a nitrocellulose membrane and absorbent paper are sequentially lapped on the bottom plate from left to right, and a solid phase of the targeting polypeptide gold pad is provided with a human chorionic gonadotrophin antibody 2 marked by targeting polypeptide gold particles, and the concentration is 15 mug/mL; the biotinylated human chorionic gonadotrophin antibody 1 is immobilized on the biotinylated pad and has the concentration of 15 mug/mL, and the invention can be compatible with two sample types, so that the consumable cost, the labor cost and the detection time are saved in the production process.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a human chorionic gonadotrophin colloidal gold detection test strip and a kit for urine and saliva simultaneous detection.
Background
The detection of Human Chorionic Gonadotrophin (HCG) in urine has been very widely used clinically for early pregnancy diagnosis, but the detection of saliva has been less studied in the detection of HCG.
At present, the traditional test strip for detecting Human Chorionic Gonadotrophin (HCG) in urine in the market mainly judges whether pregnancy exists or not through urine test, but the collection of urine is limited by occasions, and pollution is easily caused if the operation is improper; in addition, whether pregnancy exists or not can be judged through a blood test as early diagnosis or OTC diagnosis, but the blood test has the problems that blood collection is inconvenient and a professional is required to operate, and rapid collection at any time and any place cannot be achieved.
Saliva contains many proteins and genetic molecules, such as enzymes, hormones, antibodies, growth factors, and other antimicrobial components. Often, the alteration of the saliva composition is due to abnormal functioning of the salivary glands themselves, or the presence of diseases affecting the function of the salivary glands, so that the saliva composition may reflect certain sides of the body. The use of saliva as a diagnostic medium is a great advantage in that simple, painless, atraumatic sampling can improve patient compliance. The part for collecting saliva is selected as gingival tissue, most of the collected saliva is oral mucosa leakage liquid, and the saliva is collected by a wiping method, so that a patient or a physical examination person can collect the saliva at any time and any place. However, there is no kit for detecting Human Chorionic Gonadotrophin (HCG) in urine by saliva, and there is no human chorionic gonadotrophin colloidal gold detection test strip and kit for detecting Human Chorionic Gonadotrophin (HCG) in urine simultaneously by saliva.
Disclosure of Invention
The invention aims to provide a human chorionic gonadotrophin colloidal gold detection test strip and a kit for urine and saliva simultaneous detection, which can be compatible with two sample types, so that consumable cost, labor cost and detection time are saved in the production process.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the human chorionic gonadotrophin colloidal gold detection test strip for urine saliva simultaneous detection comprises a bottom plate, wherein a sample pad, a biotinylation pad, a targeting polypeptide gold pad, a nitrocellulose membrane and absorbent paper are sequentially lapped on the bottom plate from left to right, and a human chorionic gonadotrophin antibody 2 marked by targeting polypeptide gold particles is immobilized on the targeting polypeptide gold pad, and the concentration is 15 mug/mL; the biotinylated hCG antibody 1 is immobilized on the biotinylated pad at a concentration of 15 μg/mL.
As a further improvement of the above technical scheme:
the preparation method of the targeting polypeptide gold pad comprises the following steps: preparing Jin Chong suspension, wherein Jin Chong suspension is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 8.5, 0.5% BSA (buffer solution), 10% sucrose, 10% trehalose and 0.2% Proclin300, diluting the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2 with gold suspension to obtain target polypeptide gold-labeled antibody working solution, and spraying the target polypeptide gold-labeled antibody working solution on a binding pad to obtain the target polypeptide gold pad.
The preparation method of the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2 comprises the following steps:
(1) Preparing a targeting polypeptide gold solution, wherein the prepared targeting polypeptide Jin Lijing is 200nm, and comprises the following steps:
A. 1.5mL of 1.0X10 were taken -4 Diluting g/mL chloroauric acid aqueous solution to 30mL with distilled water, stirring and heating to 80 ℃, adding 0.15mL of trisodium citrate with the mass percent of 1% after 5min, continuously heating for 15min, and cooling to room temperature to obtain gold solution;
B. 30mL of 1.0X10 were taken -4 Mixing g/mL chloroauric acid aqueous solution and 1mL gold solution prepared in the step A, stirring and heating to boiling, adding 0.1g of polyvinylpyrrolidone as a stabilizer, adding 0.6mL of potassium borohydride as a strong reducing agent after 8min, reacting for 2min under boiling, and naturally cooling to room temperature to obtain a target polypeptide gold solution;
(2) Labeling human chorionic gonadotrophin antibody 2 with the targeting polypeptide gold solution prepared in step (1), comprising the steps of:
taking 100mL of the target polypeptide gold solution obtained in the step (1), regulating the pH value to 8.0, adding 1.5mg of human chorionic gonadotrophin antibody 2, adding 1mL of 20% bovine serum albumin solution, centrifuging, discarding supernatant to obtain precipitate, and adding 10mL of Jin Chong suspension to obtain the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2.
The preparation method of the biotinylation pad comprises the following steps: preparing Jin Chong suspension, wherein Jin Chong suspension is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 8.5, 0.5% BSA (BSA) by mass percent, 10% sucrose by mass percent, 10% trehalose by mass percent and 0.2% Proclin300 by mass percent, diluting biotinylated human chorionic gonadotrophin antibody 1 by using gold suspension to obtain a biotinylated antibody working solution, and coating the biotinylated antibody working solution on a binding pad to obtain a biotinylated pad.
The preparation method of the biotinylated human chorionic gonadotrophin antibody 1 comprises the following steps:
(1) Measuring ultrapure water by a measuring cylinder and pouring the ultrapure water into a beaker;
(2) Cutting a dialysis bag with proper length by scissors, putting the dialysis bag into a beaker containing 0.1M PB buffer solution, and heating the beaker on a resistance furnace until the dialysis bag is boiled;
(3) Washing the dialysis bag with ultrapure water, spin-drying, and recovering room temperature;
(4) Clamping one side of the human chorionic gonadotrophin antibody 1 by a clamp, diluting the human chorionic gonadotrophin antibody to 2mg/mL, adding the human chorionic gonadotrophin antibody into a dialysis bag, clamping the human chorionic gonadotrophin antibody by a dialysis clamp, putting the human chorionic gonadotrophin antibody into 2000mL of 0.1M PB buffer solution, dialyzing the human chorionic gonadotrophin antibody for 8 hours at a temperature of between 2 and 8 ℃ in a refrigerator, and replacing the human chorionic gonadotrophin antibody by 0.1M PB buffer solution every 2 hours, wherein the dosage is 2000mL each time and 4 times;
(5) And taking out the human chorionic gonadotrophin antibody 1 after the dialysis is finished, thus obtaining the biotinylated human chorionic gonadotrophin antibody 1.
The targeted polypeptide gold pad and the biotinylation pad are placed in a baking oven at 37+/-2 ℃ for drying for 12 hours.
The preparation method of the nitrocellulose membrane comprises the following steps: diluting SA coupling antigen to 1.2mg/mL with a diluent to serve as detection line liquid; and (3) diluting the goat anti-mouse monoclonal antibody to 1.5mg/mL with a diluent to serve as a quality control line liquid, respectively spraying the quality control line liquid on a nitrocellulose membrane by using a film drawing instrument to serve as a detection line and a quality control line, and drying the mixture in a baking oven at 37 ℃ for 4 hours.
The preparation method of the sample pad comprises the following steps: firstly, preparing a treatment fluid, wherein the treatment fluid is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 9.0, PVP10 with mass percent of 1.0%, S17 with mass percent of 1.0%, casein with mass percent of 0.5%, EDTA with mass percent of 1.0%, blocking agent with mass percent of 0.05% and sodium chloride with mass percent of 1.5%; next, the treatment liquid was coated on the glass fiber to obtain a sample pad.
The preparation method of the human chorionic gonadotrophin colloidal gold test strip for urine and saliva simultaneous detection comprises the following steps: and sequentially overlapping the sample pad, the biotinylation pad, the targeting polypeptide gold pad, the nitrocellulose membrane and the absorbent paper on the bottom plate from left to right to obtain the human chorionic gonadotrophin colloidal gold detection test strip for urine and saliva simultaneous detection.
The utility model provides a human chorionic gonadotrophin colloidal gold detection kit of urine saliva with detection, including detecting pen and urine cup, the detecting pen includes casing, pen cap and above-mentioned human chorionic gonadotrophin colloidal gold detection test strip of urine saliva with detection, the human chorionic gonadotrophin colloidal gold detection test strip of urine saliva with detection sets up in the casing, the casing upper surface is equipped with the observation window, the one end of casing is equipped with the pad that absorbs water, the one end and the sample pad that absorbs water of pad are connected, make the sample that awaits measuring can chromatography to the sample pad on, the pad that absorbs water sets up in the pen cap, pen cap and casing buckle are connected.
The invention adopts the technical proposal and has the following advantages:
1. the test strip in the detection pen applies the biotin amplification system to a colloidal gold particle immunochromatography system, adopts the targeted polypeptide gold labeled antibody, improves the sensitivity of a detection reagent, effectively reduces omission, simultaneously adds a blocking agent into a sample pad to improve the specificity, can detect urine and saliva samples at the same time, and saves the consumable cost and the labor cost for production; for detection, the compliance of patients is high, and the detection can be performed without time and place limitation;
2. the invention has simple operation, no need of professional personnel and no need of instruments, and can be widely applied to the detection of the base layer.
The invention is further described below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a schematic diagram of the structure of a test pen in a test kit for detecting human chorionic gonadotrophin colloidal gold in urine and saliva;
fig. 2 is a schematic structural diagram of a test strip in a human chorionic gonadotrophin colloidal gold detection kit for urine and saliva simultaneous detection.
In the drawing the view of the figure,
1-sample pad, 2-white patch, 3-detection line, 4-quality control line, 5-absorbent paper, 6-nitrocellulose membrane, 7-targeting polypeptide gold pad, 8-biotinylation pad, 9-bottom plate, 10-shell, 11-absorbent pad, 12-observation window and 13-pen cap.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not noted, according to conventional conditions or manufacturer's recommended conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The described embodiments are intended to be illustrative of only some, but not all embodiments of the invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the invention.
Example 1
As shown in figures 1-2, the human chorionic gonadotrophin colloidal gold test strip for urine saliva simultaneous detection comprises a bottom plate 9, wherein a sample pad 1, a biotinylation pad 8, a targeting polypeptide gold pad 7, a nitrocellulose membrane 6 and a water absorbing paper 5 are sequentially overlapped on the bottom plate 9 from left to right, the water absorbing paper 5 is adhered to one side of the nitrocellulose membrane 6, the targeting polypeptide gold pad 7, the biotinylation pad 8 and the sample pad 1 are sequentially adhered to the other side of the nitrocellulose membrane 6, one end of the sample pad 1 presses one end of the biotinylation pad 8 by 1.5mm, one end of the biotinylation pad 8 presses one end of the targeting polypeptide gold pad 7 by 1.5mm, the other end of the targeting polypeptide gold pad 7 presses one end of the nitrocellulose membrane 6 by 1mm, one end of the water absorbing paper 5 presses the other end of the nitrocellulose membrane 6 by 2.0mm, and white patches 2 are arranged on the outer sides of the targeting polypeptide gold pad 7 and the biotinylation pad 8.
The targeting polypeptide gold particle-marked human chorionic gonadotrophin antibody 2 is immobilized on the targeting polypeptide gold pad 7, and the concentration is 15 mug/mL; the biotinylated human chorionic gonadotrophin antibody 1 is immobilized on the biotinylated cushion 8 at a concentration of 15 μg/mL; SA coupling antigen is respectively coated on the nitrocellulose membrane 6 as a detection line 3, sheep anti-mouse is used as a quality control line 4, the concentration of the SA coupling antigen is 1.2mg/mL, and the concentration of the sheep anti-mouse is 1.5mg/mL.
The preparation method of the targeting polypeptide gold pad 7 comprises the following steps: preparing Jin Chong suspension, wherein Jin Chong suspension is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 8.5, 0.5% BSA (buffer solution), 10% sucrose, 10% trehalose and 0.2% Proclin300, diluting the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2 with gold suspension to obtain target polypeptide gold-labeled antibody working solution, and spraying the target polypeptide gold-labeled antibody working solution on a binding pad to obtain the target polypeptide gold pad 7.
The preparation method of the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2 comprises the following steps:
(1) Preparing a targeting polypeptide gold solution, wherein the prepared targeting polypeptide Jin Lijing is 200nm, and comprises the following steps:
A. 1.5mL of 1.0X10 were taken -4 Diluting g/mL chloroauric acid aqueous solution to 30mL with distilled water, stirring and heating to 80 ℃, adding 0.15mL of trisodium citrate with the mass percent of 1% after 5min, continuously heating for 15min, and cooling to room temperature to obtain gold solution;
B. 30mL of 1.0X10 were taken -4 Mixing g/mL chloroauric acid aqueous solution and 1mL gold solution prepared in step A, stirring and heating to boil, adding stabilizer polyvinyl pyridineRapidly adding 0.6mL of strong reducing agent potassium borohydride after 0.1g of pyrrolidone is 8min, reacting for 2min under boiling, and naturally cooling to room temperature to obtain a target polypeptide gold solution;
(2) Labeling human chorionic gonadotrophin antibody 2 with the targeting polypeptide gold solution prepared in step (1), comprising the steps of:
taking 100mL of the target polypeptide gold solution obtained in the step (1), regulating the pH value to 8.0, adding 1.5mg of human chorionic gonadotrophin antibody 2, adding 1mL of 20% bovine serum albumin solution, centrifuging, discarding supernatant to obtain precipitate, and adding 10mL of Jin Chong suspension to obtain the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2.
The preparation method of the biotinylation pad 8 comprises the following steps: preparing Jin Chong suspension, wherein Jin Chong suspension is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 8.5, 0.5% BSA (BSA) by mass percent, 10% sucrose by mass percent, 10% trehalose by mass percent and 0.2% Proclin300 by mass percent, diluting biotinylated human chorionic gonadotrophin antibody 1 by using gold suspension to obtain a biotinylated antibody working solution, and coating the biotinylated antibody working solution on a binding pad to obtain a biotinylated pad 8.
The preparation method of the biotinylated human chorionic gonadotrophin antibody 1 comprises the following steps:
(1) Measuring ultrapure water by a measuring cylinder and pouring the ultrapure water into a beaker;
(2) Cutting a dialysis bag with proper length by scissors, putting the dialysis bag into a beaker containing 0.1M PB buffer solution, and heating the beaker on a resistance furnace until the dialysis bag is boiled;
(3) Washing the dialysis bag with ultrapure water, spin-drying, and recovering room temperature;
(4) Clamping one side of the human chorionic gonadotrophin antibody 1 by a clamp, diluting the human chorionic gonadotrophin antibody to 2mg/mL, adding the human chorionic gonadotrophin antibody into a dialysis bag, clamping the human chorionic gonadotrophin antibody by a dialysis clamp, putting the human chorionic gonadotrophin antibody into 2000mL of 0.1M PB buffer solution, dialyzing the human chorionic gonadotrophin antibody for 8 hours at a temperature of between 2 and 8 ℃ in a refrigerator, and replacing the human chorionic gonadotrophin antibody by 0.1M PB buffer solution every 2 hours, wherein the dosage is 2000mL each time and 4 times;
(5) And taking out the human chorionic gonadotrophin antibody 1 after the dialysis is finished, thus obtaining the biotinylated human chorionic gonadotrophin antibody 1.
The targeted polypeptide gold pads 7 and biotinylated pads 8 were dried in an oven at 37.+ -. 2 ℃ for 12 hours.
The preparation method of the nitrocellulose membrane 6 comprises the following steps: diluting SA coupling antigen to 1.2mg/mL with a diluent to serve as detection line liquid; and (3) diluting the goat anti-mouse monoclonal antibody to 1.5mg/mL with a diluent to serve as a quality control line liquid, respectively spraying the quality control line liquid on a nitrocellulose membrane by using a film drawing instrument to serve as a detection line 3 and a quality control line 4, and drying the mixture in a drying oven at 37 ℃ for 4 hours.
The preparation method of the sample pad 1 comprises the following steps: firstly, preparing a treatment fluid, wherein the treatment fluid is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 9.0, PVP10 with mass percent of 1.0%, S17 with mass percent of 1.0%, casein with mass percent of 0.5%, EDTA with mass percent of 1.0%, blocking agent with mass percent of 0.05% and sodium chloride with mass percent of 1.5%; next, the treatment liquid was applied to glass fibers to obtain a sample pad 1.
The preparation method of the test strip comprises the following steps: and sequentially overlapping the sample pad 1, the biotinylation pad 8, the targeting polypeptide gold pad 7, the nitrocellulose membrane 6 and the absorbent paper 5 on the bottom plate from left to right to obtain the test strip.
The utility model provides a human chorionic gonadotrophin colloidal gold detection kit that urine saliva was examined simultaneously, including detecting pen and urine cup, detecting pen includes casing 10, pen cap 13 and above-mentioned human chorionic gonadotrophin colloidal gold detection test strip that urine saliva was examined simultaneously, the test strip setting is in casing 10, casing 10 upper surface is equipped with observation window 12, casing 10's one end is equipped with water absorption pad 11, water absorption pad 11's one end is connected with sample pad 1, make the sample that awaits measuring can chromatography to sample pad 1, water absorption pad 11 sets up in pen cap 13, pen cap 13 and casing 10 buckle are connected.
Example 2
A detection method of a human chorionic gonadotrophin colloidal gold detection kit for urine and saliva simultaneous detection comprises the following steps:
(1) Sample preparation:
urine sample: middle urine can be left in the urine cup, urine within one day can be detected, but HCG concentration in urine is usually highest in the morning.
(2) And (3) detection:
and (3) directly spraying urine on a sample adding section of the front section of the detection pen, and maintaining for 5-8 seconds. Or the water absorption pad of the detection pen is directly immersed into the collected urine, the liquid is taken out after climbing up to the observation area, and the detection pen is covered with the pen cap and then is horizontally placed. Reading the result for 5 minutes, and invalidating the result after 10 minutes;
judging results;
a. positive (+): and a red line appears on each of the quality control line 4 and the detection line 3.
b. Negative (-). Only one red line appears on the quality control line 4, and no red line appears on the position of the detection line 3.
c. Invalidation: the position of the quality control line 4 is free from a line, which indicates misoperation or reagent failure.
Example 3
A detection method of a human chorionic gonadotrophin colloidal gold detection kit for urine and saliva simultaneous detection comprises the following steps:
(1) Sample preparation:
saliva sample: the mouth is rinsed with water 30 minutes before sampling, when saliva is reserved, the tip of the tongue is abutted against the upper tooth root and the lower tooth root to enrich the saliva, and then the saliva is gently spitted into the urine cup until the saliva fills the bottom of the urine cup and no bubbles exist.
(2) And (3) detection:
the end with the water absorbing pad is put into the inlet, so that the water absorbing pad can fully absorb saliva. Or directly immersing the water absorption pad of the detection pen into the collected saliva, taking out after the liquid climbs to the observation area, covering the pen cap, and horizontally placing. Reading the result for 5 minutes, and invalidating the result after 10 minutes;
judging results;
a. positive (+): and a red line appears on each of the quality control line 4 and the detection line 3.
b. Negative (-). Only one red line appears on the quality control line 4, and no red line appears on the position of the detection line 3.
c. Invalidation: the position of the quality control line 4 is free from a line, which indicates misoperation or reagent failure.
Experiment
1. The HCG standards were tested at different concentrations of 0.1mIU/mL, 0.5mIU/mL, 2.5mIU/mL, 5mIU/mL, 12.5mIU/mL, 25mIU/mL, 100mIU/mL,500mIU/mL, 3 replicates for each concentration, and the results are shown in the following Table:
remarks: "+" represents positive, "±" represents weak positive, and "-" represents negative.
As shown in the table, the detection limit of the HCG standard substance detected by the human chorionic gonadotrophin colloidal gold detection kit for urine and saliva simultaneous detection can reach 0.5mIU/mL, and the sensitivity is high.
2. The invention relates to a human chorionic gonadotrophin colloidal gold detection kit for urine and saliva simultaneous detection, which is used for detecting the coincidence rate of urine and saliva samples and serum detected by an ELISA detection kit (a marketed kit):
the urine, saliva and serum samples of 5 clinical diagnosis pregnant persons and the urine, saliva and serum samples of 5 clinical diagnosis non-pregnant persons are collected from the clinic, and the human chorionic gonadotrophin colloidal gold detection kit and the ELISA detection kit for simultaneous detection of urine and saliva are used for simultaneous detection respectively, and the specificity and the detection rate are as follows:
as shown in the table, the total coincidence rate of the urine, saliva and ELISA detection kit detected by the human chorionic gonadotrophin colloidal gold detection kit for urine and saliva is up to 100%, which shows that the self-detection result of the invention has high coincidence rate and good specificity. Meanwhile, the method is simple to operate, does not need professional personnel or instruments, and can be widely applied to basic layer detection.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention, but is merely illustrative of the embodiments, and all the details not described herein are common knowledge of a person skilled in the art, so that it is possible for a person skilled in the art to modify the technical solutions described in the foregoing embodiments or to make equivalent substitutions for some technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. The utility model provides a human chorionic gonadotrophin colloidal gold test strip of urine saliva simultaneous detection which characterized in that: the kit comprises a bottom plate (9), wherein a sample pad (1), a biotinylation pad (8), a targeting polypeptide gold pad (7), a nitrocellulose membrane (6) and absorbent paper (5) are sequentially lapped on the bottom plate (9) from left to right, and a targeting polypeptide gold particle-marked human chorionic gonadotrophin antibody 2 is immobilized on the targeting polypeptide gold pad (7) with the concentration of 15 mug/mL; the solid phase of the biotinylation pad (8) is provided with the biotinylation human chorionic gonadotrophin antibody 1, and the concentration is 15 mug/mL;
the preparation method of the targeting polypeptide gold pad (7) comprises the following steps: preparing Jin Chong suspension, namely preparing Jin Chong suspension from 0.1mol/L Tris-HCL buffer solution with pH of 8.5, 0.5% of BSA (BSA) by mass percent, 10% of sucrose by mass percent, 10% of trehalose by mass percent and 0.2% of Proclin300 by mass percent, diluting human chorionic gonadotrophin antibody 2 marked by target polypeptide gold particles by using gold suspension to obtain target polypeptide gold marked antibody working solution, and spraying the working solution on a bonding pad to obtain a target polypeptide gold pad (7);
the preparation method of the target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2 comprises the following steps:
(1) Preparing a targeting polypeptide gold solution, wherein the prepared targeting polypeptide Jin Lijing is 200nm, and comprises the following steps:
A. 1.5mL of 1.0X10 were taken -4 Diluting g/mL chloroauric acid aqueous solution to 30mL with distilled water, stirring and heating to 80 ℃, adding 0.15mL of trisodium citrate with the mass percent of 1% after 5min, continuously heating for 15min, and cooling to room temperature to obtain gold solution;
B. 30mL of 1.0X10 were taken -4 g/mL aqueous chloroauric acid solution and 1mL stepMixing the gold solution prepared in the step A, stirring and heating to boiling, adding 0.1g of polyvinylpyrrolidone serving as a stabilizer, adding 0.6mL of potassium borohydride serving as a strong reducing agent rapidly after 8min, reacting for 2min under boiling, and naturally cooling to room temperature to obtain a target polypeptide gold solution;
(2) Labeling human chorionic gonadotrophin antibody 2 with the targeting polypeptide gold solution prepared in step (1), comprising the steps of:
taking 100mL of the target polypeptide gold solution obtained in the step (1), regulating the pH value to 8.0, adding 1.5mg of human chorionic gonadotrophin antibody 2, adding 1mL of 20% bovine serum albumin solution, centrifuging, discarding supernatant to obtain precipitate, and adding 10mL of Jin Chong suspension to obtain target polypeptide gold particle-labeled human chorionic gonadotrophin antibody 2;
the preparation method of the biotinylation pad (8) comprises the following steps: preparing Jin Chong suspension, namely preparing Jin Chong suspension from 0.1mol/L Tris-HCL buffer solution with pH of 8.5, 0.5% BSA (BSA) by mass percent, 10% sucrose by mass percent, 10% trehalose by mass percent and 0.2% Proclin300 by mass percent, diluting a biotinylated human chorionic gonadotrophin antibody 1 by using gold suspension to obtain a biotinylated antibody working solution, and coating the biotinylated antibody working solution on a binding pad to obtain a biotinylated pad (8);
the preparation method of the biotinylated human chorionic gonadotrophin antibody 1 comprises the following steps:
(1) Measuring ultrapure water by a measuring cylinder and pouring the ultrapure water into a beaker;
(2) Cutting a dialysis bag with proper length by scissors, putting the dialysis bag into a beaker containing 0.1M PB buffer solution, and heating the beaker on a resistance furnace until the dialysis bag is boiled;
(3) Washing the dialysis bag with ultrapure water, spin-drying, and recovering room temperature;
(4) Clamping one side of the human chorionic gonadotrophin antibody 1 by a clamp, diluting the human chorionic gonadotrophin antibody to 2mg/mL, adding the human chorionic gonadotrophin antibody into a dialysis bag, clamping the human chorionic gonadotrophin antibody by a dialysis clamp, putting the human chorionic gonadotrophin antibody into 2000mL of 0.1M PB buffer solution, dialyzing the human chorionic gonadotrophin antibody for 8 hours at a temperature of between 2 and 8 ℃ in a refrigerator, and replacing the human chorionic gonadotrophin antibody by 0.1M PB buffer solution every 2 hours, wherein the dosage is 2000mL each time and 4 times;
(5) And taking out the human chorionic gonadotrophin antibody 1 after the dialysis is finished, thus obtaining the biotinylated human chorionic gonadotrophin antibody 1.
2. The test strip for detecting human chorionic gonadotrophin colloidal gold for urine and saliva co-detection according to claim 1, wherein: the targeting polypeptide gold pad (7) and the biotinylation pad (8) are placed in a baking oven at 37+/-2 ℃ to be dried for 12 hours.
3. The test strip for detecting human chorionic gonadotrophin colloidal gold for urine and saliva co-detection according to claim 2, wherein: the preparation method of the nitrocellulose membrane (6) comprises the following steps: diluting SA coupling antigen to 1.2mg/mL with a diluent to serve as detection line liquid; and (3) diluting the goat anti-mouse monoclonal antibody to 1.5mg/mL with a diluent to serve as a quality control line liquid, respectively spraying the quality control line liquid on a nitrocellulose membrane by using a film drawing instrument to serve as a detection line (3) and a quality control line (4), and drying the mixture in a drying oven at 37 ℃ for 4 hours.
4. A human chorionic gonadotrophin colloidal gold test strip for urine and saliva co-detection according to claim 3, wherein: the preparation method of the sample pad (1) comprises the following steps: firstly, preparing a treatment fluid, wherein the treatment fluid is prepared from 0.1mol/L Tris-HCL buffer solution with pH of 9.0, PVP10 with mass percent of 1.0%, S17 with mass percent of 1.0%, casein with mass percent of 0.5%, EDTA with mass percent of 1.0%, blocking agent with mass percent of 0.05% and sodium chloride with mass percent of 1.5%; next, the treatment liquid was applied to glass fibers to obtain a sample pad (1).
5. The test strip for detecting human chorionic gonadotrophin colloidal gold according to claim 4, wherein said test strip is characterized in that: the preparation method of the human chorionic gonadotrophin colloidal gold test strip for urine and saliva simultaneous detection comprises the following steps: and sequentially overlapping the sample pad (1), the biotinylation pad (8), the targeting polypeptide gold pad (7), the nitrocellulose membrane (6) and the absorbent paper (5) on the bottom plate from left to right to obtain the human chorionic gonadotrophin colloidal gold detection test strip for urine and saliva simultaneous detection.
6. A human chorionic gonadotrophin colloidal gold detection kit for urine and saliva simultaneous detection is characterized in that: the urine saliva detection test strip comprises a detection pen and a urine cup, wherein the detection pen comprises a shell (10), a pen cover (13) and the human chorionic gonadotrophin colloidal gold detection test strip for urine saliva detection according to one of claims 1-5, the human chorionic gonadotrophin colloidal gold detection test strip for urine saliva detection is arranged in the shell (10), an observation window (12) is arranged on the upper surface of the shell (10), a water absorption pad (11) is arranged at one end of the shell (10), one end of the water absorption pad (11) is connected with a sample pad (1), a sample to be detected can be chromatographed on the sample pad (1), the water absorption pad (11) is arranged in the pen cover (13), and the pen cover (13) is connected with the shell (10) in a buckling mode.
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