CN116482388A - Reagent, kit and detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A - Google Patents
Reagent, kit and detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A Download PDFInfo
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- CN116482388A CN116482388A CN202210039409.9A CN202210039409A CN116482388A CN 116482388 A CN116482388 A CN 116482388A CN 202210039409 A CN202210039409 A CN 202210039409A CN 116482388 A CN116482388 A CN 116482388A
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Classifications
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/743—Steroid hormones
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- 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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/471—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
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- 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]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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- General Physics & Mathematics (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Gynecology & Obstetrics (AREA)
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Abstract
The embodiment of the application belongs to the technical field of beta-HCG/Prog/PAPP-A joint measurement, and relates to a reagent, a kit and a detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy-related plasma protein-A, wherein the reagent comprises a reagent component A, a reagent component B, a reagent component C, a reagent component D and a reagent component E; the reagent component A comprises fluorescent marked anti-beta-HCG murine monoclonal IgG, fluorescent marked anti-Prog murine monoclonal IgG, fluorescent marked anti-PAPP-A murine monoclonal IgG and fluorescent marked sheep anti-chicken IgY sheep antibody; the reagent component B is a beta-HCG monoclonal antibody, the reagent component C is a progesterone-BSA coupled antigen, the reagent component D is a PAPP-A monoclonal antibody, and the reagent component E is a chicken IgY antibody. The application realizes one-time sample addition and simultaneously and rapidly, and accurately and quantitatively detects three early pregnancy indexes beta-HCG/Prog/PAPP-A.
Description
Technical Field
The application relates to the technical field of beta-HCG/Prog/PAPP-A combined detection, in particular to a reagent, a kit and a detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A.
Background
Beta-human chorionic gonadotrophin, progesterone and pregnancy related plasma protein-A are widely used in clinic as specific indexes for diagnosing pregnancy at present, and patients with ectopic pregnancy have obvious differences from normal pregnant women in the indexes, so that the beta-human chorionic gonadotrophin, progesterone and pregnancy related plasma protein-A can be used for auxiliary diagnosis of ectopic pregnancy. Beta-human chorionic gonadotropin (beta-HCG) is a glycoprotein secreted by placental trophoblasts, and the beta-HCG enters the ovary from the placental trophoblasts through the blood circulation, stimulates the luteal secretion of progesterone to maintain the luteal function, and keeps the endometrium in a state suitable for embryo implantation development. PAPP-A can coordinate proliferation and differentiation of cytotrophoblast, and plays a key physiological regulation role in early gamete development, fertilized egg implantation, pregnancy state maintenance and fetal placenta growth and development. beta-HCG is a main index for clinical diagnosis of early pregnancy, and normal intrauterine gestators have an increasing trend along with the prolongation of pregnancy. The secretion of ectopic pregnancy beta-HCG is reduced, and the greater the difference between the level of ectopic pregnancy beta-HCG and that of normal pregnant women is over time, when the intrauterine embryo is dysplasia, the activity of the trophoblast cells is far weaker than that of normal intrauterine early pregnancy, and the secretion of PAPP-A by the trophoblast cells and decidua cells is correspondingly reduced. Trophoblasts are underdeveloped due to ectopic disease and Prog levels are significantly reduced. The dynamic combined detection of the level change of the beta-human chorionic gonadotrophin, the progesterone and the pregnancy related plasma protein-A in early pregnancy can predict early pregnancy ending of early threatened abortion patients, remarkably improves the diagnostic value of ectopic pregnancy, and has great significance for clinical guidance and treatment.
Methods and limitations for the detection of beta-human chorionic gonadotrophin, progesterone and pregnancy related plasma protein-a:
the instrument analysis method comprises the following steps: the chromatography or chromatography-mass spectrometry is an important technical platform for modern high-efficiency separation detection, can quantify various components in a complex matrix in a short time, has high sensitivity and specificity, and is a detection method with high clinical application rate. However, the instrumental analysis method needs expensive high-precision instruments, the detection place is inflexible, the professional requirements of operators are high, and the ever-increasing detection needs are gradually difficult to deal with.
Immunoassay method: the immunoassay method utilizes the principle of antigen-antibody specific recognition and combination to realize the specific detection of the object to be detected. The immunoassay technology can be combined with a plurality of technologies such as a radioactive labeling technology, an enzyme labeling technology, a fluorescent labeling technology, electrochemistry, chemiluminescence and the like to develop a detection technology with high sensitivity and high specificity. However, the performance of the immunoassay method depends on the preparation of high-quality antibodies, which are proteins and are easily inactivated by interference of external conditions.
Disclosure of Invention
The embodiment of the application aims to provide a reagent, a kit and a detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, which realize one-time sample addition and simultaneously and rapidly, and accurately and quantitatively detect three early pregnancy indexes, namely beta-human chorionic gonadotrophin, progesterone and pregnancy related plasma protein-A.
In order to solve the technical problems, the embodiment of the application provides a reagent for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, which adopts the following technical scheme:
a reagent for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, wherein the reagent comprises a reagent component A, a reagent component B, a reagent component C, a reagent component D and a reagent component E; the reagent component A comprises fluorescent marked anti-beta-HCG murine monoclonal IgG, fluorescent marked anti-Prog murine monoclonal IgG, fluorescent marked anti-PAPP-A murine monoclonal IgG and fluorescent marked sheep anti-chicken IgY sheep antibody; the reagent component B is a beta-HCG monoclonal antibody, the reagent component C is a progesterone-BSA coupled antigen, the reagent component D is a PAPP-A monoclonal antibody, and the reagent component E is a chicken IgY antibody.
Further, the reagent component A is prepared by the following steps:
adding a marking buffer solution and fluorescent microspheres with solid content of 1% into four centrifuge tubes with the numbers of 1,2,3 and 4;
after vortex mixing, adding a marked activating solution into each centrifuge tube, and centrifuging after mixing uniformly;
removing the supernatant, retaining the sediment, and adding a marking buffer solution into each centrifuge tube;
after ultrasonic mixing, respectively adding anti-beta-HCG murine monoclonal IgG, anti-Prog murine monoclonal IgG, anti-PAPP-A murine monoclonal IgG and goat anti-chicken IgY goat antibody into four centrifuge tubes, after mixing, adding marked sealing liquid, ultrasonic and centrifuging;
removing supernatant, retaining sediment, adding a marked preservation solution into each centrifuge tube, and respectively transferring the solutions in the centrifuge tubes with the numbers of 1,2,3 and 4 into the centrifuge tubes with the numbers of 5,6,7 and 8 after ultrasonic treatment;
absorbing the marked preservation solution, cleaning centrifuge tubes with the numbers of 1,2,3 and 4, respectively transferring the solutions in the cleaned centrifuge tubes with the numbers of 1,2,3 and 4 into centrifuge tubes with the numbers of 5,6,7 and 8, and respectively obtaining fluorescent marked anti-beta-HCG murine monoclonal IgG solution, fluorescent marked anti-Prog murine monoclonal IgG solution, fluorescent marked anti-PAPP-A murine monoclonal IgG solution and fluorescent marked sheep anti-chicken IgY sheep antibody solution after uniformly mixing;
and mixing the fluorescent-labeled anti-beta-HCG murine monoclonal IgG solution, the fluorescent-labeled anti-Prog murine monoclonal IgG solution, the fluorescent-labeled anti-PAPP-A murine monoclonal IgG solution and the fluorescent-labeled goat anti-chicken IgY goat antibody solution in equal proportion to obtain the reagent component A.
Further, the labeling buffer comprises: 1.067% of 2-morpholinoethanesulfonic acid;
the marking activation solution includes: 2% of 1-ethyl- (3-dimethylaminopropyl) carbodiimide;
the marking sealing liquid comprises: 10% bovine serum albumin;
the label-holding liquid includes: 2% bovine serum albumin, 2% sucrose, 0.5% casein sodium salt, 5% trehalose, 0.05% bacteriostatic preservative, 0.02MpH value of 8.0Tris buffer.
Further, the concentration of the beta-HCG monoclonal antibody in the reagent component B is 0.5mg/mL, and the reagent component B is diluted by an antibody coating diluent.
Further, the concentration of the beta-HCG monoclonal antibody in the reagent component B is 0.5mg/mL, and the reagent component B is diluted by an antibody coating diluent.
Further, the PAPP-A monoclonal antibody concentration in the reagent component D is 0.5mg/mL, and the reagent component D is diluted by an antibody coating diluent.
Further, the content of the chicken IgY antibody in the reagent component E is 1.0mg/mL, and the chicken IgY antibody is diluted by an antibody coating diluent.
Further, the antibody coating diluent comprises 1% sucrose and 0.01M PBS buffer at pH7.4, wherein the 0.01M PBS buffer at pH7.4 comprises 0.8% sodium chloride, 0.02% potassium chloride, 0.114% disodium hydrogen phosphate and 0.027% potassium dihydrogen phosphate.
In order to solve the technical problems, the embodiment of the application also provides a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, which adopts the following technical scheme:
the kit comprises a detection card, wherein the detection card comprises a PVC bottom plate, and a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped on the PVC bottom plate, wherein the binding pad contains fluorescent marked anti-beta-HCG murine monoclonal IgG, fluorescent marked anti-Prog murine monoclonal IgG, fluorescent marked anti-PAPP-A murine monoclonal IgG and fluorescent marked sheep anti-chicken IgY sheep antibody, the nitrocellulose membrane is provided with a first detection line, a second detection line, a third detection line and a quality control line, the first detection line is coated with the beta-HCG monoclonal antibody, the second detection line is coated with the progesterone-BSA coupling antigen, the third detection line is coated with the PAPP-A monoclonal antibody, and the quality control line is coated with the chicken IgY antibody.
In order to solve the technical problems, the embodiment of the application also provides a detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, which adopts the following technical scheme:
inserting the same chip as the reagent lot number in the kit into an instrument;
adding a sample to be measured into the diluent, and uniformly mixing to obtain a target sample;
and (3) sucking a target sample, adding the target sample into a sample adding hole of the detection card, and placing the detection card into an instrument card slot for testing after room temperature reaction to obtain a detection result.
Compared with the prior art, the embodiment of the application has the following main beneficial effects:
the application belongs to detection products of the point of care or point of care (POCT) type, and adopts a time-resolved fluoroimmunoassay method. The method is a non-isotope immunoassay technology, and the fluorescence is measured by a time resolution technology according to the luminous characteristics of lanthanide chelates through labeling antigens or antibodies by lanthanide, and simultaneously, two parameters of detection wavelength and time are used for signal resolution, so that the interference of background fluorescence signals in biological fluid and serum can be effectively reduced, and the analysis sensitivity is greatly improved. Meanwhile, the kit provided by the application can realize one-time sample addition and simultaneously and rapidly, accurately and quantitatively detect the beta-human chorionic gonadotrophin, the progesterone and the pregnancy related plasma protein-A which are three early pregnancy indexes, has the advantages of simplicity in operation, short detection time, low comprehensive cost, no interference of natural fluorescence of a sample, no radioactive pollution and the like, and provides a more accurate reference for dynamically detecting the early pregnancy hormone level change and predicting the pregnancy ending of early threatened abortion patients.
Drawings
For a clearer description of the solution in the present application, a brief description will be given below of the drawings that are needed in the description of the embodiments of the present application, it being obvious that the drawings in the following description are some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a schematic structural diagram of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A according to the present application;
FIG. 2 is another schematic structural diagram of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A according to the present application;
FIG. 3 is a graph of the analysis of the results of a kit for the detection of beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A according to the present application with a contrast agent;
FIG. 4 is a further analysis of the results of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A according to the present application with a contrast agent;
FIG. 5 is a further analysis of the results of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A according to the present application with a contrast agent;
FIG. 6 is a graph of another analysis of the results of serum and whole blood of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy associated plasma protein-A according to the present application;
FIG. 7 is a graph of another analysis of the results of serum and whole blood of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy associated plasma protein-A according to the present application;
fig. 8 is a graph of another analysis of the results of serum and whole blood of a kit for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to the present application.
Reference numerals: 1. a PVC bottom plate; 2. a sample pad; 3. a bonding pad; 4. a nitrocellulose membrane; 5. a first detection line; 6. a second detection line; 7. a third detection line; 8. a quality control line; 9. a water absorbing pad; 10. a detection card; 11. a sample adding hole; 12. a reading area; 13. a hand-held region.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description of the applications herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having" and any variations thereof in the description and claims of the present application and in the description of the figures above are intended to cover non-exclusive inclusions. The terms first, second and the like in the description and in the claims or in the above-described figures, are used for distinguishing between different objects and not necessarily for describing a sequential or chronological order.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
The following examples facilitate a better understanding of the present application, but are not intended to limit the present application. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
In order to better understand the technical solutions of the present application, the following description will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings.
1. A reagent for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, wherein the reagent comprises a reagent component A, a reagent component B, a reagent component C, a reagent component D and a reagent component E; the reagent component A comprises fluorescent marked anti-beta-HCG murine monoclonal IgG, fluorescent marked anti-Prog murine monoclonal IgG, fluorescent marked anti-PAPP-A murine monoclonal IgG and fluorescent marked sheep anti-chicken IgY sheep antibody; the reagent component B is a beta-HCG monoclonal antibody, the reagent component C is a progesterone-BSA coupled antigen, the reagent component D is a PAPP-A monoclonal antibody, and the reagent component E is a chicken IgY antibody.
Specifically, the reagent component A is a fluorescent microsphere marked antibody solution, and is prepared by the following steps:
(1) taking four clean 2mL centrifuge tubes with the numbers of 1,2,3 and 4, and respectively adding 1mL of marking buffer MES into each tube; mixing fluorescent microspheres uniformly by vortex, taking 100 mu L of fluorescent microspheres with the solid content of 1% in a marking buffer solution, and mixing uniformly by vortex again;
(2) adding 100 mu L of marked activating solution into the centrifuge tube, covering a cover, slowly and reversely and uniformly mixing for 10-20 times; after the uniform mixing is finished, placing the centrifuge tube on a rotary mixing instrument according to the proportion, and carrying out rotary reaction for 30min;
(3) after the reaction is completed, the mixture is centrifuged at 4 ℃ (the centrifugal speed is 10000rpm, the centrifugal time is 30 min);
(4) after centrifugation, the centrifuge tube is gently taken out, and a pipetting gun is used for slowly sucking the supernatant; taking out sediment, sucking 1000 mu L of marking buffer MES, and adding into a centrifuge tube; completely scattering the powder by using ultrasonic (a mode of using an ultrasonic cell grinder to work for 2s, intermittently 5s and repeating 3-5 times according to 90W ultrasonic);
(5) 50 mug of anti-beta-HCG murine monoclonal IgG, 50 mug of anti-Prog murine monoclonal IgG, 50 mug of fluorescent-labeled anti-PAPP-A murine monoclonal IgG and 80 mug of goat anti-chicken IgY goat antibody are respectively added into centrifugal tubes with the numbers of 1,2,3 and 4, and then are evenly mixed for 20 times in an upside-down way, placed on a rotary mixer and subjected to rotary reaction for 30 minutes;
(6) after the rotation reaction is completed, absorbing 100 mu L of the marked sealing liquid into the centrifuge tube, and completely scattering the marked sealing liquid by ultrasonic waves; placing the mixture on a rotary mixing instrument again, and carrying out rotary reaction for 2h;
(7) after the sealing is completed, centrifuging at 4 ℃ for 30min; after centrifugation, discarding the supernatant; taking a precipitate, sucking 1mL of marked preservation solution by using a pipetting gun, adding the marked preservation solution into a centrifuge tube, and completely scattering the marked preservation solution by using ultrasound;
(8) after the precipitation is uniformly dispersed, the markers with the numbers of 1,2,3 and 4 are respectively moved into 10mL centrifuge tubes with the numbers of 5,6,7 and 8, respectively sucking 2mL centrifuge tubes for cleaning and marking the marker preservation solution with the number of 4mL, respectively moving the markers into the 10mL centrifuge tubes after cleaning, and vortex mixing uniformly to respectively obtain fluorescent marked anti-beta-HCG murine monoclonal IgG solution, fluorescent marked anti-Prog murine monoclonal IgG solution, fluorescent marked anti-PAPP-A murine monoclonal IgG solution and fluorescent marked goat anti-chicken IgY sheep antibody solution.
(9) Respectively obtaining a fluorescent-marked anti-beta-HCG murine monoclonal IgG solution, a fluorescent-marked anti-Prog murine monoclonal IgG solution, a fluorescent-marked anti-PAPP-A murine monoclonal IgG solution and a fluorescent-marked sheep anti-chicken IgY sheep antibody solution according to the volume ratio of 1:1:1:1 (V: V: V) are uniformly mixed, and the mixture is subjected to ultrasonic treatment, works for 2 seconds, is intermittently 5 seconds and is repeated for 3 times, so that the fluorescent microsphere marked antibody solution is obtained, and the fluorescent microsphere marked antibody solution is stored at the temperature of 2-8 ℃ and has the effective period of 15 days.
Wherein, the mass fraction of the material is calculated,
labeling buffer: 2-morpholinoethanesulfonic acid (MES) 1.067% and purified water.
Marking an activating solution: 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) 2% and purified water.
Marking a sealing liquid: bovine serum albumin 10% and purified water.
Marking preservation solution: bovine serum albumin 2%, sucrose 2%, casein sodium salt 0.5%, trehalose 5%, antibacterial preservative (Proclin 300) 0.05%,0.02M 8.0Tris buffer.
The concentration of the beta-HCG monoclonal antibody in the reagent component B is 0.5mg/mL, and the reagent component B is diluted by an antibody coating diluent. The content of the progesterone-BSA coupled antigen in the reagent component C is 0.5mg/mL, and the reagent component C is diluted by an antibody coating diluent. The PAPP-A monoclonal antibody concentration in the reagent component D is 0.5mg/mL, and the reagent component D is diluted by an antibody coating diluent. The content of the chicken IgY antibody in the reagent component E is 1.0mg/mL, and the chicken IgY antibody is diluted by an antibody coating diluent. The antibody coating dilutions included 1% sucrose and 0.01MpH of 7.4 in PBS buffer, wherein the 0.01MpH of 7.4 in PBS buffer included 0.8% sodium chloride, 0.02% potassium chloride, 0.114% disodium hydrogen phosphate and 0.027% potassium dihydrogen phosphate.
2. With continued reference to fig. 1, fig. 1 shows a schematic structural diagram of a kit for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to the present application. The kit comprises a detection card, a chip and diluent, wherein the detection card comprises a PVC bottom plate 1, a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a water absorption pad 9 which are sequentially overlapped on the PVC bottom plate 1, wherein the binding pad 3 contains fluorescent-marked anti-beta-HCG murine monoclonal IgG, fluorescent-marked anti-Prog murine monoclonal IgG, fluorescent-marked anti-PAPP-A murine monoclonal IgG and fluorescent-marked goat anti-chicken IgY sheep antibody, the nitrocellulose membrane 4 is provided with a first detection line 5, a second detection line 6, a third detection line 7 and a quality control line 8, the first detection line 5 is coated with the beta-HCG monoclonal antibody, the second detection line 6 is coated with progesterone-BSA coupling antigen, the third detection line 7 is coated with the PAPP-A monoclonal antibody, and the quality control line 8 is coated with the chicken IgY antibody. The chip stores the relation curve information of the detection value-detection object standard concentration; the diluent is phosphate buffer solution, and specifically, the diluent is prepared from 0.05% of antibacterial preservative (Proclin 300) and 0.01M PBS buffer solution with pH of 7.4, wherein the 0.01M PBS buffer solution with pH of 7.4: sodium chloride 0.8%, potassium chloride 0.02%, disodium hydrogen phosphate 0.114%, potassium dihydrogen phosphate 0.027%, and purified water.
Specifically, the bonding pad 3 and the water absorbing pad 9 are respectively lapped at two ends of the nitrocellulose membrane 4, one side of the water absorbing pad 3 is aligned with the PVC base plate 1, the other side of the water absorbing pad is covered on the nitrocellulose membrane 4 by 2-3 mm, and one side of the bonding pad 3 is covered on the nitrocellulose membrane 4 by 1-2 mm; one side of the sample pad 2 is aligned with the PVC base plate 1, and the other side is covered on the bonding pad 3 by 1-2 mm.
Fig. 2 is a finished product diagram of the test card 10 after the test strip and the card case are assembled, the test card 10 includes a sample adding hole 11, a reading area 12 and a holding area 13, wherein the sample adding hole 11 corresponds to a sample pad area of the test strip, the reading area 12 corresponds to a nitrocellulose membrane area of the test strip, and the holding area 13 is located at one end of a water absorbing pad of the test strip.
The specific components of the kit are shown in Table 1:
TABLE 1
The kit provided by the application can realize the joint detection of three early pregnancy indexes (total beta-human chorionic gonadotrophin, progesterone and pregnancy-related plasma protein-A) in a human serum/plasma/whole blood sample, and has the advantages of simplicity and convenience in operation, short detection time, high sensitivity, no interference from natural fluorescence of the sample, no radioactive pollution and the like.
1. Preparation of sample pad:
the sample pad is made of glass fiber cotton, a power supply of a paper dipping pad pressing machine is started, the height of a wheel shaft is set, the height of a micrometer on the left side is 9mm, and the height of a micrometer on the right side is 9.5mm; secondly, pouring sample pad treatment liquid into a stainless steel liquid storage container, soaking an initial sample pad, and preventing overflow because the solution cannot be too full; and taking out the processed initial sample pad, and placing the initial sample pad on a clean, dry and flat airing frame. And (3) placing the airing rack in an environment with the temperature of 18-26 ℃ and the relative humidity of less than or equal to 30%, drying overnight (16-24) h, cutting, sealing, drying and preserving to obtain the sample pad.
Wherein, the sample pad treatment liquid is prepared from 1% of bovine serum albumin, 0.5% of Tween-20 and 0.2M borax borate buffer solution by mass fraction. 0.2M borax borate solution: 0.05M borax and 0.2M boric acid 3:7. 0.05M borax solution: sodium tetraborate 1.907% and purified water. 0.2M boric acid solution: boric acid 1.237% and purified water.
2. Preparation of bond pads
2.1 cutting of initial bond pads:
the glass fiber model 8964 was cut into (10.+ -. 0.5) mm× (300.+ -. 10) mm to obtain an initial bonding pad.
2.2 spray pad:
the reagent component A (namely fluorescent microsphere marked antibody solution) is sprayed from a metal spraying scribing machine onto the initial bonding pad obtained after cutting according to the spraying amount of 4.0 mu L/cm, and is dried overnight (16-24) in the environment with the temperature (37+/-2) ℃ and the humidity less than or equal to 30 percent. Taking out the dried initial bonding pad, obtaining the bonding pad, putting a proper amount of drying agent, and sealing in a self-sealing bag.
3. Preparation of the reaction plate:
in the process of preparing the reaction plate, except for coating the working solution, the solution used for coating is filtered by a microporous filter membrane with the pore diameter of 0.22 m. After dissolving the solid chemical reagent, the fine impurities are found, and if other liquid components are needed to be added, the solution is filtered by a microporous filter membrane with the pore diameter of 0.22m and then added.
And lightly uncovering a protective film at the NC film adhesion part on the PVC bottom plate, and uniformly pushing the initial nitrocellulose film from left to right to ensure that the initial nitrocellulose film is firm, thereby obtaining the reaction plate. Then streaking, namely diluting the beta-HCG monoclonal antibody, the progesterone-BSA coupled antigen, the PAPP-A monoclonal antibody and the chicken IgY antibody to 0.5mg/mL, 0.5mg/mL and 1.0mg/mL by using an antibody coating diluent to obtain a first detection line coating working solution (a reagent component B), a second detection line coating working solution (a reagent component C), a third detection line coating working solution (a reagent component D) and a quality control line coating working solution (a reagent component E); according to the film dividing amount of 1 mu L/cm and the film dividing length of 300mm, a detection line coating working solution, a second detection line coating working solution, a third detection line coating working solution and a quality control line coating working solution are respectively used for dividing a first detection line (T1) 5, a second detection line (T2) 6, a third detection line (T3) 7 and a quality control line (C line) 8 on an initial nitrocellulose film of a reaction plate, a reaction plate adhered with the nitrocellulose film is obtained, the distance between the quality control line and the third detection line is (4+/-0.5) mm, the distance between the third detection line and the second detection line is (4+/-0.5) mm, the distance between the second detection line and the first detection line is (4+/-0.5) mm, the distance between the quality control line and the upper edge (one end) of the nitrocellulose film is (9+/-0.5) mm, the distance between the first detection line and the lower edge (the other end) of the nitrocellulose film is (4+/-0.5) mm, and the temperature (37+/-2) and humidity in an electrothermal drying box is dried (24-16 h) in a temperature (16+/-30%). Taking out the dried reaction plate, sealing in a self-sealing bag, and putting a proper amount of drying agent.
And assembling the large plate under the environment of the temperature (18-26) ℃ and the humidity less than or equal to 30 percent. The reaction plate is spread on the working table, the protective film at the pasting position of the absorption pad on the reaction plate is uncovered, the absorption pad is adhered on the reaction plate, and the other side of the reaction plate is partially covered on the NC film (nitrocellulose film) for 2-3 mm. And then the protective film at the adhesion part of the bonding pad and the sample pad on the reaction plate is uncovered, the bonding pad is adhered to the upper end of the reaction plate, the bonding pad is ensured to face upwards, one end of the bonding pad is covered on the NC film by 1-2 mm, the sample pad is adhered to the reaction plate, the lower end of the sample pad is ensured to be aligned with the adhesion part of the sample pad of the PVC bottom plate, and the upper end of the sample pad is ensured to be covered on the bonding pad. And finally, placing the adhered large plate into an aluminum foil bag, adding a drying agent, and sealing by a sealing machine.
Cutting the prepared large plate into strips, loading each test strip into the lower cover of the fluorescent card shell, covering the upper cover after loading, placing the test strip on a card pressing machine, adjusting the height of the card pressing machine to be 3.5mm, and tightly combining the upper cover and the lower cover of the fluorescent card shell to obtain the detection card.
Wherein, the antibody coating diluent comprises the following components in percentage by mass: sucrose 1%,0.01M PBS buffer pH 7.4. 0.01M PBS buffer pH 7.4: sodium chloride 0.8%, potassium chloride 0.02%, disodium hydrogen phosphate 0.114%, potassium dihydrogen phosphate 0.027%, and purified water.
4. Preparation of time-resolved fluorescence immunochromatography chip for detecting beta-HCG/Prog/PAPP-A
4.1 preparation of chips
Preparing curve data: taking a beta-HCG enterprise calibrator, a Prog enterprise calibrator and PAPP-A enterprise calibrators (S1-S5), and operating according to a reagent instruction; and (3) carrying out test operation on a dry type fluorescence immunoassay management tool, repeatedly detecting the samples with each concentration for 3 times, and calculating the average value of the T/C value obtained by test. The formulation concentration and corresponding T/C mean values were taken as beta-HCG, prog, PAPP-A as linear data, respectively.
4.2 configuration of chip data
And selecting project parameters on a dry type fluorescence immunoassay experiment management tool, setting related parameters by using new construction or editing, clicking for storage after the editing is completed and confirmed without errors, and finishing the project parameter setting. Selecting a project beta-HCG/Prog/PAPP-A on a dry type fluorescence immunity experiment management tool, completing a new experiment, generating a selection curve, and selecting the newly-built experiment in the last step from a left experiment list; respectively filling the beta-HCG, prog, PAPP-A curve data prepared in the first step into a table, and sequentially clicking and automatically selecting, automatically sorting and storing after filling the data; after the preservation is completed, the reaction value is selected to be converted into logarithm and decimal number which are 4, the concentration is selected to be converted into logarithm and decimal number which are 2, and the fitting algorithm is selected to be MMF Model four-parameter growth curve fitting; the preparation of three curves of beta-HCG, prog, PAPP-A is completed. Selecting and generating an ID file, selecting a newly-built item, and automatically displaying all parameters set by the steps on an interface; setting production year, production month and batch in turn, automatically generating a bar code at the bar code after setting, and confirming whether the bar code is accurate; clicking to generate an ID file to complete the configuration of the chip data.
4.3 firing of chips
Returning to the desktop, selecting a dry type fluorescence immunoassay experiment management tool, and selecting an open file position. Double-clicking the Item folder. And finding the latest HEX file corresponding to the bar code data in the Item file. And burning 2-3 chips in the programmer from the chip data file, reading the chip data in the opened single-channel fluorescence analyzer, verifying whether the data is successfully written in, and determining whether the following information is wrong. After confirming the data and parameters, the chips can be burned in a large scale.
3. The operation method for detecting the beta-HCG/Prog/PAPP-A by time-resolved fluoroimmunoassay comprises the following steps:
when the product is used, the instruction manual and the instruction manual of the matched instrument are carefully read, and the test is carried out at room temperature (18-30 ℃).
1) Opening the instrument, and inserting a chip identical to the reagent batch number;
2) Sucking 100 mu L of sample by a pipette, adding the sample into the diluent, and mixing the sample upside down;
3) Opening an aluminum foil bag, taking out the detection card, and horizontally placing the detection card on a tabletop;
4) Sucking 100 mu L of the diluted and uniformly mixed sample by using a pipette, and adding the 100 mu L of the diluted and uniformly mixed sample into a sample adding hole of a detection card;
5) Selecting a sample type of plasma, serum or whole blood on a companion instrument;
6) And (3) testing in real time: after reacting for 15min at room temperature, placing the detection card into an instrument card slot, selecting an 'instant test' mode, and clicking a 'test'; standard test: placing the detection card into an instrument card slot, selecting a standard test mode, clicking a test mode, automatically timing the instrument, automatically testing and displaying a result after timing is finished;
7) Clicking "print" may print the test result report.
4. Time-resolved fluorescence immunochromatography kit product performance verification of beta-HCG/Prog/PAPP-A
1. Accuracy of
a) beta-HCG: the national standard 150535 for measuring beta-human chorionic gonadotrophin or the reference for measuring beta-human chorionic gonadotrophin with the concentration of about 25mIU/mL and 2500mIU/mL are detected according to the steps in the specification, and after 3 times of repeated measurement, the relative deviation is calculated according to the formula (1), wherein the relative deviation is not more than +/-15%.
b) Prog: adding a certain volume of a progesterone standard solution or a high-concentration sample with traceability (the volume ratio of the standard solution to the volume of a human sample is not more than 1:9) into low-concentration serum (or other body fluid components), preparing an accuracy reference with the concentration of about 10nmol/L and 60nmol/L, repeatedly detecting for 3 times, taking an average value, and calculating the recovery rate according to a formula (2), wherein the recovery rate is in a range of 85% -115%.
c) PAPP-A, a pregnancy related plasma protein-A enterprise reference of 40mIU/L and 4000mIU/L is measured according to the steps in the specification, and after 3 times of repeated measurement, the relative deviation is calculated according to the formula (1), wherein the relative deviation is not more than +/-10%.
Formula (1): bi= (Xi-T)/t×100%, where Bi represents the relative deviation, xi represents the concentration measured each time; t represents the indicated concentration.
Formula (2):wherein R represents recovery rate; v represents the volume of the standard solution added; v (V) 0 Representing the volume of a human sample; c represents the average value of the detection concentration of the human source sample after the standard solution is added; c (C) 0 An average value representing the detected concentration of the human sample; cs represents the concentration of the standard solution.
TABLE 2 detection of Caliper-HCG, PAPP-A accuracy
TABLE 3 detection card Prog accuracy
2. Detection limit
5 low-value samples with approximate detection limits (beta-HCG: 2.5mIU/mL, prog:0.1nmol/L, PAPP-A:4 mIU/L) were tested, and each sample was tested 5 times. The following requirements are met, namely, the setting of the blank Limit (LOB) and the detection Limit (LOD) can be considered to be basically reasonable.
(1) The number of detection results of which the 25 detection values are smaller than a blank limit (beta-HCG: 1mIU/mL, prog:0.02nmol/L, PAPP-A:2 mIU/L) is smaller than or equal to 3;
(2) all 25 detection values were not higher than the detection limit (. Beta. -HCG:2.5mIU/mL, prog:0.1nmol/L, PAPP-A:4 mIU/L).
Table 4 detection limits for each item of the detection card
3. Linear range
High-value samples near the upper limit of a linear interval are diluted according to a certain proportion (beta-HCG: 10000mIU/mL, 2500mIU/mL, 250mIU/mL, 25mIU/mL and 2.5mIU/mL, prog:100nmol/L, 60nmol/L, 10nmol/L, 1nmol/L and 0.1nmol/L, PAPP-A:10000mIU/L, 4000mIU/L, 400mIU/L, 40mIU/L and 4 mIU/L), wherein samples with low-value concentration need to be near the lower limit of the linear interval, the operation is performed on a fluorescence immunoassay analyzer according to a reagent specification, the samples with each concentration are repeatedly detected for 3 times, the average value of the samples is calculated, the linear fitting is performed by a least square method, the linear correlation coefficient r is calculated according to a formula (3), and the linear correlation coefficient (r) is not lower than 0.9900.
Equation (3):wherein n represents the number of measurement samples; xi represents the concentration of the measurement sample; yi represents the mean of measured values corresponding to the measured sample concentrations for 3 replicates; r represents a linear correlation coefficient. />
Table 5 test card each item linearity
4. Repeatability of
The same batch of reagent is randomly extracted, enterprise references (beta-HCG: 25mIU/mL, 2500mIU/mL; prog:10nmol/L, 60nmol/L; PAPP-A:40mIU/L, 4000 mIU/L) are detected, 10 times of repetition are respectively carried out, the average value M and the standard deviation SD of the 10 times of measurement results are respectively calculated, the variation Coefficient (CV) is obtained according to the formula (3), and the results are all in accordance with the requirements.
a) beta-HCG: the Coefficient of Variation (CV) in the batch is less than or equal to 15 percent.
b) Prog: the Coefficient of Variation (CV) in the batch should be less than or equal to 10%.
c) PAPP-A: the Coefficient of Variation (CV) in the batch should be less than or equal to 10%.
Equation (3): cv=sd/mx100%, where CV represents a coefficient of variation; SD represents the standard deviation of the 10 measurements; m represents the average of 10 measurements.
Table 6 test card item reproducibility
5. Comparison of the results of the kit and the contrast agent
The kit and the contrast reagent are used for simultaneously testing 96 serum samples, the beta-HCG detection result is compared with the Rogowski test value, the Prog detection result is compared with the Rogowski test value, the PAPP-A detection result is compared with the Rogowski test value, and the correlation is shown in figures 3 to 5. Specifically, FIG. 3 is a graph showing the clinical relevance of the kit of the present application to Roche beta-HCG. FIG. 4 is a scatter plot of clinical results of the subject kit and rogowski Prog. FIG. 5 is a scatter plot of clinical results of the subject kit and Roche PAPP-A.
6. The reagent serum and whole blood sample consistency analysis
The reagent simultaneously tests 48 cases of homologous serum and whole blood samples, and the correlation is shown in fig. 6 to 8. Specifically, fig. 6 is a scatter diagram of the detection results of the beta-HCG in whole blood and serum samples of the kit. FIG. 7 is a scatter plot of the Prog detection results of whole blood and serum samples of the subject kit. FIG. 8 is a scatter plot of PAPP-A assay results for whole blood and serum samples from the subject kit.
It is apparent that the embodiments described above are only some embodiments of the present application, but not all embodiments, the preferred embodiments of the present application are given in the drawings, but not limiting the patent scope of the present application. This application may be embodied in many different forms, but rather, embodiments are provided in order to provide a more thorough understanding of the present disclosure. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing, or equivalents may be substituted for elements thereof. All equivalent structures made by the specification and the drawings of the application are directly or indirectly applied to other related technical fields, and are also within the protection scope of the application.
Claims (10)
1. A reagent for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A, which is characterized by comprising a reagent component A, a reagent component B, a reagent component C, a reagent component D and a reagent component E;
the reagent component A comprises fluorescent marked anti-beta-HCG murine monoclonal IgG, fluorescent marked anti-Prog murine monoclonal IgG, fluorescent marked anti-PAPP-A murine monoclonal IgG and fluorescent marked sheep anti-chicken IgY sheep antibody; the reagent component B is a beta-HCG monoclonal antibody, the reagent component C is a progesterone-BSA coupled antigen, the reagent component D is a PAPP-A monoclonal antibody, and the reagent component E is a chicken IgY antibody.
2. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to claim 1, wherein said reagent component a is prepared by the steps of:
adding a marking buffer solution and fluorescent microspheres with solid content of 1% into four centrifuge tubes with the numbers of 1,2,3 and 4;
after vortex mixing, adding a marked activating solution into each centrifuge tube, and centrifuging after mixing uniformly;
removing the supernatant, retaining the sediment, and adding a marking buffer solution into each centrifuge tube;
after ultrasonic mixing, respectively adding anti-beta-HCG murine monoclonal IgG, anti-Prog murine monoclonal IgG, anti-PAPP-A murine monoclonal IgG and goat anti-chicken IgY goat antibody into four centrifuge tubes, after mixing, adding marked sealing liquid, ultrasonic and centrifuging;
removing supernatant, retaining sediment, adding a marked preservation solution into each centrifuge tube, and respectively transferring the solutions in the centrifuge tubes with the numbers of 1,2,3 and 4 into the centrifuge tubes with the numbers of 5,6,7 and 8 after ultrasonic treatment;
absorbing the marked preservation solution, cleaning centrifuge tubes with the numbers of 1,2,3 and 4, respectively transferring the solutions in the cleaned centrifuge tubes with the numbers of 1,2,3 and 4 into centrifuge tubes with the numbers of 5,6,7 and 8, and respectively obtaining fluorescent marked anti-beta-HCG murine monoclonal IgG solution, fluorescent marked anti-Prog murine monoclonal IgG solution, fluorescent marked anti-PAPP-A murine monoclonal IgG solution and fluorescent marked sheep anti-chicken IgY sheep antibody solution after uniformly mixing;
and mixing the fluorescent-labeled anti-beta-HCG murine monoclonal IgG solution, the fluorescent-labeled anti-Prog murine monoclonal IgG solution, the fluorescent-labeled anti-PAPP-A murine monoclonal IgG solution and the fluorescent-labeled goat anti-chicken IgY goat antibody solution in equal proportion to obtain the reagent component A.
3. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to claim 2, wherein said labeling buffer comprises: 1.067% of 2-morpholinoethanesulfonic acid;
the marking activation solution includes: 2% of 1-ethyl- (3-dimethylaminopropyl) carbodiimide;
the marking sealing liquid comprises: 10% bovine serum albumin;
the label-holding liquid includes: 2% of bovine serum albumin, 2% of sucrose, 0.5% of casein sodium salt, 5% of trehalose, 0.05% of antibacterial preservative and 0.02M of Tris buffer with pH value of 8.0.
4. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to claim 1, wherein the concentration of said β -HCG monoclonal antibody in said reagent component B is 0.5mg/mL, diluted by antibody coating dilution.
5. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to claim 1, wherein the amount of said progesterone-BSA conjugate antigen in said reagent component C is 0.5mg/mL, diluted by an antibody coating diluent.
6. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to claim 1, wherein said PAPP-a monoclonal antibody concentration in said reagent component D is 0.5mg/mL, diluted by an antibody coating diluent.
7. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to claim 1, wherein the content of said chicken IgY antibody in said reagent component E is 1.0mg/mL, diluted by antibody coating dilution.
8. The reagent for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a according to any one of claims 4 to 7, wherein said antibody coating dilution comprises 1% sucrose and 0.01M PBS buffer at pH7.4, wherein said 0.01M PBS buffer at pH7.4 comprises 0.8% sodium chloride, 0.02% potassium chloride, 0.114% disodium hydrogen phosphate and 0.027% monopotassium phosphate.
9. A kit for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a comprising the reagent of claim 1, wherein the kit comprises a detection card comprising a PVC substrate and a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorbing pad sequentially lapped on the PVC substrate, wherein the conjugate pad comprises a fluorescent-labeled anti- β -HCG murine monoclonal IgG, a fluorescent-labeled anti-Prog murine monoclonal IgG, a fluorescent-labeled anti-PAPP-a murine IgG and a fluorescent-labeled goat anti-chicken IgY sheep antibody, the nitrocellulose membrane has a first detection line coated with β -HCG monoclonal antibody, a second detection line coated with a progesterone-BSA conjugated chicken antigen, a third detection line coated with a PAPP-a monoclonal antibody, and a quality control line coated with IgY antibody.
10. A method for detecting β -human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-a using the kit of claim 9, comprising the steps of:
inserting the same chip as the reagent lot number in the kit into an instrument;
adding a sample to be measured into the diluent, and uniformly mixing to obtain a target sample;
and (3) sucking a target sample, adding the target sample into a sample adding hole of the detection card, and placing the detection card into an instrument card slot for testing after room temperature reaction to obtain a detection result.
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| CN202210039409.9A CN116482388A (en) | 2022-01-13 | 2022-01-13 | Reagent, kit and detection method for detecting beta-human chorionic gonadotrophin/progesterone/pregnancy related plasma protein-A |
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