CN113156137A - Chemiluminescence immunoassay kit for detecting CD47, and preparation method and application thereof - Google Patents

Chemiluminescence immunoassay kit for detecting CD47, and preparation method and application thereof Download PDF

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CN113156137A
CN113156137A CN202110188926.8A CN202110188926A CN113156137A CN 113156137 A CN113156137 A CN 113156137A CN 202110188926 A CN202110188926 A CN 202110188926A CN 113156137 A CN113156137 A CN 113156137A
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diluent
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李文靖
欧兰香
陈振
王岩
寇宗阳
张绍明
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Shandong Big Health Precision Medical Industry Technology Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Abstract

The invention relates to a chemiluminescence immunoassay kit for detecting CD47, and a preparation method and application thereof. The chemiluminescence immunoassay kit comprises: the kit comprises a microporous plate, a CD47 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled CD47 monoclonal antibody 2, an enzyme-labeled diluent, a 20X concentrated washing solution, a luminescent solution A and a luminescent solution B; according to the chemiluminescence immunoassay kit for detecting CD47, the intensifier HIOP is added into the luminescent liquid B, and is a phenolic compound 4-hydroxy-4' -iodobiphenyl with an active substituent at the para-position, compared with the traditional intensifier, the intensifying effect is better, the detection limit is reduced, the linear range is widened, and the sensitivity, the precision and the detection speed of the chemiluminescence immunoassay kit for detecting CD47 are obviously improved.

Description

Chemiluminescence immunoassay kit for detecting CD47, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of in-vitro diagnostic kits, and particularly relates to a chemiluminescence immunoassay kit for detecting CD47, and a preparation method and application thereof.
Background
CD47, also known as Integrin-associated Protein (IAP), is a highly glycosylated transmembrane Protein that is widely expressed on the surface of a variety of cells. The molecular structure of CD47 belongs to transmembrane glycoprotein of immunoglobulin superfamily, and comprises 1 extracellular Ig-type variable domain, 3-5 hydrophobic transmembrane segments, 1 carboxyl terminal cytoplasmic tail region and the like, and the function of the molecular structure is related to integrin. CD47 is widely expressed on the cell surface of different tissues of human body, such as erythrocyte, lymphocyte, liver cell, placenta and tumor cell surface. Signal regulatory protein alpha (SIRP alpha) is a specific ligand of the protein and is mainly expressed on the surfaces of phagocytic cells such as macrophages and dendritic cells. CD47 and its ligand SIRP α can form a signaling complex, regulate immune response and mediate bidirectional signal modulation, and participate in a variety of physiological functions, such as: macrophage phagocytosis, neutrophil chemotaxis, development of the nervous system, and the like.
CD47 binds to SIRP alpha receptor on macrophage surface, can activate tyrosine phosphorylase, and inhibit myosin from gathering under macrophage protrusion, thereby avoiding macrophage phagocytosis of target cell. Such as: under normal physiological state, aged or damaged red blood cells express CD47 and lose, thereby releasing the negative regulation of macrophage phagocytosis and being metabolized by the body. Studies have shown that many tumors, including lymphomas and leukemias, sarcomas, and almost all solid tumors, unlike their corresponding normal cells, express CD47 at high levels, which mediate the "Don't eat me" signal by binding to macrophage surface SIRP α ligands, resulting in innate immune escape from the tumor cells, and at high levels, both in promoting tumor cell growth and tumor cell metastasis. The research shows that the average expression level of CD47 in tumor cells is about 2-10 times higher than that in normal cells. Another study showed that the urine expression level of CD47 in bladder cancer patients was higher than that in the normal group; abnormal expression of CD47 in serum of patients with gastric cancer and breast cancer; the concentration of CD47 in the serum of patients with lung cancer and helicobacter pylori infection is obviously higher than that of healthy people, and the concentration of CD47 in the serum is obviously reduced after drug treatment, so that CD47 can be used as a more reliable index for diagnosing tumors and judging prognosis. Therefore, the development of a detection kit capable of detecting the content of human CD47 is beneficial to detection, screening and prognosis monitoring of tumor patients in clinic.
In the prior art, foreign related chemiluminescent immunoassay kits are applied correspondingly, but no registration letters of the CD47 detection kit are searched for domestic reagents and imported reagents when a website of the national drug administration is searched.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a chemiluminescence immunoassay kit for detecting CD47, and a preparation method and application thereof, so as to overcome the defects of instability and low sensitivity of the existing chemiluminescence immunoassay kit.
The technical scheme of the invention is as follows:
a chemiluminescent immunoassay kit for detecting CD47 comprising: the kit comprises a microporous plate, a CD47 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled CD47 monoclonal antibody 2, an enzyme-labeled diluent, a 20X concentrated washing solution, a luminescent solution A and a luminescent solution B.
Further preferably, the microplate is a 96-well or 48-well microplate, and each well of the microplate is coated with the CD47 monoclonal antibody 1(Mab 1).
Further preferably, the CD47 standard gradient solution comprises 6 CD47 standard solutions with concentration gradients of 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL, and the CD47 standard gradient solution is prepared by a standard buffer solution which is a buffer solution containing 5 wt% bovine serum albumin, 0.85 wt% sodium chloride and 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate with pH 7.0.
Further preferably, the formulation of the sample diluent is: based on 1000mL of sterilized pure water, the composition contains 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、BSA 10g、ProClinTM950 1mL。
Further preferably, the enzyme-labeled diluent is prepared from the following formula: NaCl 8.0g, KH2PO4 0.2g、Na2HPO4·12H2O2.9 g, KCl 0.2g, newborn bovine serum 200mL, Twen 200.5 mL, ProClinTM9501 mL, purified water to 1000mL, pH 7.4.
Further preferably, the formula of the 20 × concentrated washing solution is: based on 50mL of sterilized pure water, the sample contains 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL。
More preferably, the luminescent solution a is a disodium hydrogen phosphate-citric acid buffer solution in which carbamide peroxide is dissolved at pH 5.0, and is prepared into a 0.1mol/L carbamide peroxide stock solution, and then the carbamide peroxide stock solution is gradually diluted to a working concentration of 4.0 × 10 by using the disodium hydrogen phosphate-citric acid buffer solution-3mol/L。
More preferably, the luminescent liquid B is 3.0 × 10-3mol/L luminol solution and 1.6X 10-3mixing mol/L4-hydroxy-4' -iodine-biphenyl solution according to a ratio of 1: 1 (v/v).
Further preferably, the luminol solution is prepared into 0.1mol/L luminol stock solution by using 0.05mol/L, pH 10.3.3 carbonate buffer solution, the luminol stock solution is placed for three days in a dark place, and then 0.1mol/L, pH 10.3.3 NaHCO is used3Dilution of-NaOH buffer solution to 3.0X 10-3mol/L。
Further preferably, the 4-hydroxy-4' -iodo-biphenyl solution is prepared into 1 × 10 with redistilled dimethyl sulfoxide-2Diluting 4-hydroxy-4' -iodo-biphenyl solution at mol/L to 1.6 × 10-3mol/L。
Further preferably, the diluent formulation is: contains 12mL of Tween20 (1% v/v) based on 1000mL of sterilized purified water.
The chemiluminescence immunoassay kit for detecting CD47 also comprises a kit body, a non-setting adhesive, a sealing bag and an instruction book.
The preparation method of the chemiluminescence immunoassay kit for detecting CD47 comprises the following steps:
1) preparing a microporous plate:
1.1) preparing a coating liquid: weighing Na2CO3 1.59g，NaHCO32.94g, adding purified water to a constant volume of 1L, adjusting the pH value to 9.6, and sterilizing at high temperature for later use;
1.2) preparing a washing solution (PBST), wherein the formula of the washing solution is as follows:
Figure BDA0002944462340000021
Figure BDA0002944462340000031
adjusting pH to 7.4, filtering, sterilizing, and storing at 4 deg.C;
1.3) preparing a sealing liquid, wherein the formula of the sealing liquid is as follows:
Figure BDA0002944462340000032
adjusting pH to 7.4, filtering, sterilizing, and storing at 4 deg.C;
1.4) diluting the CD47 monoclonal antibody 1(Mab1) to a working concentration of 10 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking a microporous plate, using a 12-pore row gun to point the plate to ensure that the antibody solution of Mab1 reaches 100 mu L/pore, covering a cover plate film, placing the plate in a refrigerator at the temperature of 2-6 ℃, and coating the plate overnight for 20-24 hours;
1.6) taking out the overnight coated microporous plate, balancing at room temperature for 30min, throwing away coating liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid per hole, standing at room temperature for 1min, washing for 3 times, beating the absorbent paper to be dry;
1.7) adding 200 mu L of sealing liquid into each hole, and sealing for 2 hours at 37 ℃;
1.8) taking out the sealed microporous plate, balancing the microporous plate at room temperature for 30min, throwing off sealing liquid, and patting the microporous plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.9) vacuum packaging with an aluminum foil bag, marking, and storing at 2-8 ℃ for later use.
2) Preparation of CD47 monoclonal antibody 2 horseradish peroxidase
Labeling of CD47 monoclonal antibody 2 horseradish peroxidase (Mab2+ HRP) was performed according to the sodium periodate method, and the label was stored at 4 ℃ until use.
3) Preparation of a kit solution:
3.1) preparing an enzyme-labeled diluent, wherein the formula of the enzyme-labeled diluent is as follows:
Figure BDA0002944462340000033
Figure BDA0002944462340000041
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C;
3.2) preparation of 20 × concentrated washing solution, wherein the formula of the 20 × concentrated washing solution is as follows: sterilized 50mL of purified water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL；
3.3) gradient solutions of CD47 standard were prepared as follows:
preparing a buffer solution containing 5 wt% of bovine serum albumin, 0.85 wt% of sodium chloride and 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate, wherein the pH value of the buffer solution is 7.0; preparing 6 CD47 standard substance gradient solutions with concentration gradient of 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL by using the standard substance buffer solution;
3.4) preparing a sample diluent, wherein the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、BSA 10g、ProClinTM950 1mL；
3.5) the luminescent liquid A is carbamide peroxide solution, and the preparation method is as follows:
dissolving urea peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare 0.1mol/L urea peroxide stock solution, and gradually diluting with disodium hydrogen phosphate-citric acid buffer solution to working concentration of 4.0 × 10-3mol/L；
3.6) the luminescent solution B is a mixed solution obtained by mixing a luminol solution and a 4-hydroxy-4' -iodine-biphenyl solution according to a ratio of 1: 1(v/v), and the preparation method is as follows:
firstly, the luminol solution is prepared into 0.1mol/L luminol stock solution by using 0.05mol/L, pH 10.3.3 carbonate buffer solution, the luminol stock solution is placed for three days in a dark place, and then 0.1mol/L, pH 10.3.3 NaHCO is used3Dilution of-NaOH buffer solution to 3.0X 10- 3mol/L；
② 4-hydroxy-4' -iodine-biphenyl solution is prepared into 1x 10 by redistilled dimethyl sulfoxide-2Diluting 4-hydroxy-4' -iodo-biphenyl solution at mol/L to 1.6 × 10-3mol/L; the dilution was prepared in sterile 1000mL pure water and contained 12mL of Tween20 (1% v/v).
In the invention, antibody titer, enzyme-labeled antibody titer and antibody sensitivity are measured according to a chessboard method, so that each point of a standard curve is selected, wherein:
coating antibody: the working concentration of the CD47 monoclonal antibody 1(Mab1) is 10 mug/mL, and the diluent is coating liquid;
enzyme-labeled CD47 monoclonal antibody 2: the Mab2+ HRP working titer is 1: 50000, and the diluent is enzyme-labeled diluent;
the concentrations of the standard curves are 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL respectively, and the diluent is standard buffer solution.
In the invention, the precision and accuracy of the kit are verified as follows: the precision of the kit is verified by testing the standard CD47 in parallel; the accuracy of the kit is verified from the aspect of sample recovery rate of the kit;
in the invention, the sensitivity of the kit is measured as follows: the reagent reaction sensitivity was determined by detecting different amounts of CD47 with a CD47 concentration gradient.
The chemiluminescence immunoassay kit is applied to the detection of CD 47.
The method for detecting CD47 by using the chemiluminescence immunoassay kit comprises the following steps:
(1) balancing: balancing the sample to be tested and the reagent in the reagent box at room temperature for 30 +/-5 minutes;
(2) sample adding: arranging a standard sample hole and a sample hole in the microporous plate, adding 50 mu L of sample diluent into the standard sample hole according to the concentration gradient, and then adding 50 mu L of CD47 standard sample gradient solution; respectively arranging a blank hole and a sample hole to be detected in the sample hole, adding 100 mu L of sample diluent into the blank hole, adding 50 mu L of sample diluent into the sample hole to be detected, and then adding 50 mu L of sample to be detected; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent;
(3) and (3) incubation: sealing the microporous plate with a self-adhesive sealing sheet, and performing oscillation incubation at 37 ℃ for 20 minutes;
(4) preparing liquid: diluting 20 times of the concentrated washing solution with distilled water to obtain washing solution for later use;
(5) washing: discarding liquid, spin-drying, adding 300 μ L of washing solution into each hole, standing for 20-40s, discarding, repeating the above steps for 5 times, and patting to dry;
(6) adding an enzyme: diluting Mab2+ HRP according to the dilution ratio of 1: 50000, wherein the diluent is enzyme-labeled diluent, and adding 100 mu L of diluted Mab2+ HRP solution into each hole of a microporous plate;
(7) and (3) incubation: sealing the microporous plate with a self-adhesive sealing sheet, and performing shaking incubation at 37 ℃ for 10 minutes;
(8) washing: carefully uncovering the adhesive sticker, discarding the liquid, spin-drying, filling washing liquid into each hole, standing for 20-40s, discarding, repeating the steps for 5 times, and patting to dry;
(9) luminescence: adding 50 mu L of luminous liquid A and 50 mu L of luminous liquid B, shaking gently, mixing, and keeping out of the sun for 4 minutes at 18-25 ℃;
(10) and (3) determination: adjusting the blank hole to zero, and sequentially measuring the luminous intensity of each hole at the wavelength of 425nm, namely the RLU value;
(11) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
Has the advantages that:
1. according to the chemiluminescence immunoassay kit for detecting CD47, the intensifier HIOP is added into the luminescent liquid B, and is a phenolic compound 4-hydroxy-4' -iodobiphenyl with an active substituent at the para-position, compared with the traditional intensifier, the intensifying effect is better, the detection limit is reduced, the linear range is widened, and the sensitivity, the precision and the detection speed of the chemiluminescence immunoassay kit for detecting CD47 are obviously improved.
2. The chemiluminescence immunoassay kit for detecting CD47 is a detection kit obtained based on a chemiluminescence immunoadsorption method, and has the advantages of high stability, wide linear range, high sensitivity, good reproducibility, strong selectivity, high detection speed, low cost, simple and portable instrument, easy operation and the like, and experiments prove that the chemiluminescence immunoassay kit for detecting CD47 can effectively detect the content of CD47 in a human body and has potential clinical application value.
Drawings
FIG. 1 is a standard curve of the chemiluminescent immunoassay kit of example 4.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
In the following examples, the experimental methods used, which are not specifically described, are all conventional methods. The CD47 monoclonal antibody 1(Mab1) and CD47 monoclonal antibody 2(Mab2) used in the examples were commercially available from Shandongbo Biotech, Inc. The reagents used in the examples are all commercially available products.
Example 1
A chemiluminescent immunoassay kit for detecting CD47 comprising: the kit comprises a microporous plate, a CD47 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled CD47 monoclonal antibody 2(Mab2+ HRP), an enzyme-labeled diluent, a 20 Xconcentrated washing solution, a luminescent solution A, a luminescent solution B, a self-adhesive sealing sheet, a sealing bag and an instruction book;
wherein:
the microplate is a white opaque polystyrene 96 or 48-well microplate, and each well of the microplate is coated with a CD47 monoclonal antibody 1(Mab 1);
the CD47 standard substance gradient solution comprises 6 CD47 standard substance solutions with concentration gradients of 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL, and the CD47 standard substance gradient solution is prepared by a standard substance buffer solution which is a buffer solution containing 5 wt% of bovine serum albumin, 0.85 wt% of sodium chloride and 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate with the pH value of 7.0;
the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、BSA 10g、ProClinTM950 1mL；
The Mab2+ HRP is a product labeled by CD47 monoclonal antibody 2 horseradish peroxidase (Mab2+ HRP) by a sodium periodate method;
the formula of the enzyme-labeled diluent is as follows: NaCl 8.0g, KH2PO4 0.2g、Na2HPO4·12H2O2.9 g, KCl 0.2g, newborn bovine serum 200mL, Twen 200.5 mL, ProClinTM9501 mL, purified water to 1000mL, pH 7.4;
the formula of the 20 multiplied concentrated washing solution is as follows: sterilization50mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL；
The luminescent liquid A is 4.0 multiplied by 10-3Dissolving urea peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to obtain urea oxide stock solution of 0.1mol/L, and diluting with disodium hydrogen phosphate-citric acid buffer solution to working concentration of 4.0 × 10-3mol/L。
The luminescent liquid B is 3.0 multiplied by 10-3mol/L luminol solution and 1.6X 10-3mixing mol/L4-hydroxy-4' -iodine-biphenyl solution according to a ratio of 1: 1 (v/v);
the luminol solution is prepared into 0.1mol/L luminol stock solution by using 0.05mol/L, pH 10.3.3 carbonate buffer solution, is placed for three days in a dark place, and is then added with 0.1mol/L, pH 10.3.3 NaHCO3Dilution of-NaOH buffer solution to 3.0X 10- 3mol/L；
The 4-hydroxy-4' -iodo-biphenyl solution is prepared into 1 × 10 with redistilled dimethyl sulfoxide-2Diluting 4-hydroxy-4' -iodo-biphenyl solution at mol/L to 1.6 × 10-3mol/Lmol/L; the formula of the diluent is as follows: contains 12mL of Tween20 (1% v/v) based on 1000mL of sterilized purified water.
Each kit of the kit comprises 1 piece of CD47 monoclonal antibody 1 coated microporous plate, 6 bottles of CD47 standard substance gradient solution, sample diluent, Mab2+ HRP, enzyme-labeled diluent, 20 Xconcentrated washing solution, luminescent liquid A, luminescent liquid B, 1 bottle of each, one part of instruction and 3 adhesive seal sheets.
Example 2
The method for preparing the chemiluminescent immunoassay kit of example 1 comprises the following steps:
1) preparing a microporous plate:
1.1) preparing a coating liquid: weighing Na2CO3 1.59g，NaHCO32.94g, adding purified water to a constant volume of 1L, adjusting the pH value to 9.6, and sterilizing at high temperature for later use;
1.2) preparing a washing solution (PBST), wherein the formula of the washing solution is as follows:
Figure BDA0002944462340000071
adjusting pH to 7.4, filtering, sterilizing, and storing at 4 deg.C;
1.3) preparing a sealing liquid, wherein the formula of the sealing liquid is as follows:
Figure BDA0002944462340000072
adjusting pH to 7.4, filtering, sterilizing, and storing at 4 deg.C;
1.4) diluting the CD47 monoclonal antibody 1(Mab1) to a working concentration of 10 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking a microporous plate, using a 12-pore row gun to point the plate to ensure that the antibody solution of Mab1 reaches 100 mu L/pore, covering a cover plate film, placing the plate in a refrigerator at the temperature of 2-6 ℃, and coating the plate overnight for 20-24 hours;
1.6) taking out the overnight coated microporous plate, balancing at room temperature for 30min, throwing away coating liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid per hole, standing at room temperature for 1min, washing for 3 times, beating the absorbent paper to be dry;
1.7) adding 200 mu L of sealing liquid into each hole, and sealing for 2 hours at 37 ℃;
1.8) taking out the sealed microporous plate, balancing the microporous plate at room temperature for 30min, throwing off sealing liquid, and patting the microporous plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.9) vacuum packaging with an aluminum foil bag, marking, and storing at 2-8 ℃ for later use.
2) Preparation of CD47 monoclonal antibody 2 horseradish peroxidase
Labeling the CD47 monoclonal antibody 2 horseradish peroxidase (Mab2+ HRP) according to a sodium periodate method, and storing the label at 4 ℃ for later use;
3) preparation of a kit solution:
3.1) preparing an enzyme-labeled diluent, wherein the formula of the enzyme-labeled diluent is as follows:
Figure BDA0002944462340000081
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C;
3.2) preparation of 20 × concentrated washing solution, wherein the formula of the 20 × concentrated washing solution is as follows: sterilized 50mL of purified water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL；
3.3) gradient solutions of CD47 standard were prepared as follows:
preparing a buffer solution containing 5 wt% of bovine serum albumin, 0.85 wt% of sodium chloride and 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate, wherein the pH value of the buffer solution is 7.0; preparing 6 CD47 standard substance gradient solutions with concentration gradient of 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL by using the standard substance buffer solution;
3.4) preparing a sample diluent, wherein the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、BSA 10g、ProClinTM950 1mL；
3.5) the luminescent liquid A is carbamide peroxide solution, and the preparation method is as follows:
dissolving urea peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare 0.1mol/L urea peroxide stock solution, and gradually diluting with disodium hydrogen phosphate-citric acid buffer solution to working concentration of 4.0 × 10-3mol/L；
3.6) the luminescent solution B is a mixed solution obtained by mixing a luminol solution and a 4-hydroxy-4' -iodine-biphenyl solution according to a ratio of 1: 1(v/v), and the preparation method is as follows:
firstly, the luminol solution is prepared into 0.1mol/L luminol stock solution by using 0.05mol/L, pH 10.3.3 carbonate buffer solution, the luminol stock solution is placed for three days in a dark place, and then 0.1mol/L, pH 10.3.3 NaHCO is used3Dilution of-NaOH buffer solution to 3.0X 10- 3mol/L。
② 4-hydroxy-4' -iodine-biphenyl solution is prepared by redistilled dimethyl sulfoxide1×10-2Diluting 4-hydroxy-4' -iodo-biphenyl solution at mol/L to 1.6 × 10-3mol/L; the dilution was prepared in sterile 1000mL pure water and contained 12mL of Tween20 (1% v/v).
Antibody titers and antibody sensitivity were determined according to a checkerboard method, selecting the standard curve points, where:
coating antibody: the working concentration of the CD47 monoclonal antibody 1(Mab1) is 10 mug/mL, and the diluent is coating liquid;
the concentrations of the standard curves are 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL respectively, and the diluent is standard buffer solution.
Example 3
The method for detecting CD47 by using the chemiluminescence immunoassay kit of the embodiment 1 comprises the following steps:
(1) balancing: balancing the sample to be tested and the reagent in the reagent box at room temperature for 30 +/-5 minutes;
(2) sample adding: arranging a standard sample hole and a sample hole in the microporous plate, adding 50 mu L of sample diluent into the standard sample hole according to the concentration gradient, and then adding 50 mu L of CD47 standard sample gradient solution; respectively arranging a blank hole and a sample hole to be detected in the sample hole, adding 100 mu L of sample diluent into the blank hole, adding 50 mu L of sample diluent into the sample hole to be detected, and then adding 50 mu L of sample to be detected; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent;
(3) and (3) incubation: sealing the microporous plate with a self-adhesive sealing sheet, and performing oscillation incubation at 37 ℃ for 20 minutes;
(4) preparing liquid: diluting 20 times of the concentrated washing solution with distilled water to obtain washing solution for later use;
(5) washing: discarding liquid, spin-drying, adding 300 μ L of washing solution into each hole, standing for 20-40s, discarding, repeating the above steps for 5 times, and patting to dry;
(6) adding an enzyme: diluting Mab2+ HRP according to the dilution ratio of 1: 50000, wherein the diluent is enzyme-labeled diluent, and adding 100 mu L of diluted Mab2+ HRP solution into each hole of a microporous plate;
(7) and (3) incubation: sealing the microporous plate with a self-adhesive sealing sheet, and performing shaking incubation at 37 ℃ for 10 minutes;
(8) washing: carefully uncovering the adhesive sticker, discarding the liquid, spin-drying, filling washing liquid into each hole, standing for 20-40s, discarding, repeating the steps for 5 times, and patting to dry;
(9) luminescence: adding 50 mu L of luminous liquid A and 50 mu L of luminous liquid B, shaking gently, mixing, and keeping out of the sun for 4 minutes at 18-25 ℃;
(10) and (3) determination: adjusting the blank hole to zero, and sequentially measuring the luminous intensity of each hole at the wavelength of 425nm, namely the RLU value;
(11) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
The titer of the enzyme-labeled antibody and the sensitivity of the antibody were determined according to a checkerboard method, whereby each point of the standard curve was selected, wherein:
enzyme-labeled CD47 monoclonal antibody 2: the Mab2+ HRP working titer is 1: 50000, and the diluent is enzyme-labeled diluent.
Example 4
Detection of linearity, accuracy, sensitivity, precision of the chemiluminescent immunoassay kit of example 1
1. Linearity of the kit
6 concentration gradients of CD47 standard were used, respectively: CD47 standard gradient concentrations 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL, 0ng/mL, RLU value readings were taken for the standard solutions using the detection method of example 3 (see Table 1) and linearity calculated. The linear analysis curve of the kit is shown in FIG. 1.
Table 1: linear assay reading of the kit
Standard substance (ng/mL) 560 280 140 70 35 0
RLU value 80782 41828 20953 11043 5769 875
As can be seen from fig. 1, the curve equation is: y 143.41x +941.34, R2When the concentration is 0.9994, the concentration is in the range of 0-560ng/mL, and the kit has good linearity.
2. The accuracy of the kit is verified by sample recovery rate
The CD47 standard was added to a 140ng/mL CD47 standard solution to give final concentrations of 300ng/mL, 200ng/mL, and 160ng/mL CD47 standard, respectively, and each sample was tested in 3 replicates and the calculated recovery statistics are shown in Table 2.
Table 2: recovery of CD47 standard
Figure BDA0002944462340000101
The result shows that the recovery rate of the CD47 standard sample with high, medium and low concentration is 88.1-93.6%, the average recovery rate is 90.0-92.2%, and the overall average recovery rate is 91.0%.
3. Precision of the kit
The CD47 standard solution 140ng/mL was tested 10 times in parallel, and the results are shown in Table 3.
Table 3: results of precision test
Plate hole number 1 2 3 4 5 6 7 8 9 10
RLU value 23035 22826 20048 21538 23621 22445 21438 20833 21423 21650
Precision: CV% ═ 4.96%.
4. Sensitivity of the kit:
the sample diluent (CD47 concentration is 0ng/mL) is used for repeated measurement for 20 times, the light emitting value corresponding to the addition of 2 times of standard deviation SD to the average value M is obtained, the light emitting value is substituted into a curve (y is 139.83x +875) fitted with the first two points (0 and 35ng/mL) of the standard curve in the detection process, the detection sensitivity of the method is calculated, the calculated concentration is the lowest detection limit of the kit, the lowest detection limit is 0.422ng/mL, and the test is shown in Table 4.
Table 4: results of sensitivity test
Item Numerical value
RLU value: m +2SD (n 20) 934
Corresponding concentration value (ng/mL) 0.422
The test result shows that the sensitivity is 0.422 ng/mL.
Example 5 CD47 detection assay
Subject:
experimental group of non-small cell lung cancer patients 15, wherein 10 men, 5 women, mean age 58 ± 10; the average age of the healthy control group was 59 + -6, and the healthy control group had no history of diabetes, hypertension, and coronary heart disease among the 15 men, 8 men and 7 women.
And (3) determination: collecting peripheral blood of an experimental group (15 persons) of a patient with non-small cell lung cancer and fasting peripheral blood of a healthy control group (15 persons), standing for 2 hours at normal temperature, centrifuging at 3000r/min and 4 ℃ for 8 minutes, collecting serum, packaging the serum into an EP (EP) tube, placing all blood samples in a refrigerator at the temperature of-80 ℃ for storage and detection in order to reduce errors between groups and measurement errors, balancing the blood samples at room temperature for 30 minutes before measurement, and measuring by adopting the method of example 3, wherein the measurement results are shown in Table 5.
Table 5: CD47 detection analysis of control group and experimental group
Group of Concentration (ng/mL) Group of Concentration (ng/mL)
Control group 1 15.66 Experimental group 1 31.99
Control group 2 12.59 Experimental group 2 48.25
Control group 3 10.92 Experimental group 3 43.48
Control group 4 15.91 Experimental group 4 59.16
Control group 5 11.50 Experimental group 5 47.59
Control group 6 12.95 Experimental group 6 52.20
Control group 7 15.82 Experimental group 7 51.03
Control group 8 13.57 Experimental group 8 35.27
Control group 9 12.20 Experimental group 9 52.02
Control group 10 15.20 Experimental group 10 39.93
Control group 11 12.27 Experimental group 11 39.28
Control group 12 13.00 Experimental group 12 35.41
Control group 13 14.77 Experimental group 13 46.31
Control group 14 12.20 Experimental group 14 48.97
Control group 15 14.88 Experimental group 15 33.95
As can be seen from Table 5, the concentration of CD47 in the healthy control group is 10-16ng/mL, and the concentration of CD47 in the experimental group of the patient with non-small cell lung cancer is 30-60ng/mL, which is consistent with that the expression level of CD47 in tumor cells is averagely 2-10 times higher than that of normal cells reported in the literature, and the detection kit has potential application value in clinical detection.

Claims (10)

1. A chemiluminescent immunoassay kit for detecting CD47, wherein the chemiluminescent immunoassay kit for detecting CD47 comprises: the kit comprises a microporous plate, a CD47 standard substance gradient solution, a sample diluent, a horseradish peroxidase-labeled CD47 monoclonal antibody 2, an enzyme-labeled diluent, a 20X concentrated washing solution, a luminescent solution A and a luminescent solution B.
2. The chemiluminescent immunoassay kit for detecting CD47 of claim 1 wherein the microplate is a 96 or 48 well microplate and each well is coated with CD47 monoclonal antibody 1(Mab 1).
3. The chemiluminescent immunoassay kit for detecting CD47 of claim 1, wherein the CD47 standard gradient solution comprises 6 CD47 standard solutions with concentration gradients of 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL, the CD47 standard gradient solution is prepared by using a standard buffer solution, and the standard buffer solution is a buffer solution containing 5 wt% bovine serum albumin, 0.85 wt% sodium chloride and pH 7.0 of 1.0mmol/L disodium hydrogen phosphate-sodium dihydrogen phosphate.
4. The chemiluminescent immunoassay kit for detecting CD47 of claim 1 wherein the sample diluent is formulated as: based on 1000mL of sterilized pure water, the composition contains 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、BSA 10g、ProClinTM950 1mL。
5. The chemiluminescent immunoassay kit for detecting CD47 of claim 1 wherein the enzyme-labeled diluent is formulated as follows: NaCl 8.0g, KH2PO4 0.2g、Na2HPO4·12H2O2.9 g, KCl 0.2g, newborn bovine serum 200mL, Twen 200.5 mL, ProClinTM9501 mL, purified water to 1000mL, pH 7.4;
preferably, the formula of the 20 × concentrated washing solution is as follows: based on 50mL of sterilized pure water, the sample contains 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL。
6. The chemiluminescence immunoassay kit for detecting CD47, according to claim 1, wherein the luminescent solution A is urea peroxide dissolved in disodium hydrogen phosphate-citric acid buffer solution with pH 5.0, prepared into 0.1mol/L urea peroxide stock solution, and then diluted stepwise to working concentration of 4.0 x 10 by disodium hydrogen phosphate-citric acid buffer solution-3mol/L。
7. The chemiluminescent immunoassay kit for detecting CD47 of claim 1 wherein the luminescent solution B is 3.0 x 10-3mol/L luminol solution and 1.6X 10-3mixing mol/L4-hydroxy-4' -iodine-biphenyl solution according to a ratio of 1: 1 (v/v);
further preferably, the luminol solution is prepared into 0.1mol/L luminol stock solution by using 0.05mol/L, pH 10.3.3 carbonate buffer solution, the luminol stock solution is placed for three days in a dark place, and then 0.1mol/L, pH 10.3.3 NaHCO is used3Dilution of-NaOH buffer solution to 3.0X 10-3mol/L；
Further preferably, the 4-hydroxy-4' -iodo-biphenyl solution is prepared into 1 × 10 with redistilled dimethyl sulfoxide- 2Diluting 4-hydroxy-4' -iodo-biphenyl solution at mol/L to 1.6 × 10-3mol/L; the formula of the diluent is as follows: contains 12mL of Tween20 (1% v/v) based on 1000mL of sterilized purified water.
8. A method for preparing a chemiluminescent immunoassay kit for detecting CD47 of claim 1, comprising the steps of:
1) preparing a microporous plate:
1.1) preparing a coating liquid: weighing Na2CO3 1.59g，NaHCO32.94g, adding purified water to a constant volume of 1L, and adjustingAdjusting pH to 9.6, and sterilizing at high temperature;
1.2) preparing a washing solution (PBST), wherein the formula of the washing solution is as follows:
Figure FDA0002944462330000021
adjusting pH to 7.4, filtering, sterilizing, and storing at 4 deg.C;
1.3) preparing a sealing liquid, wherein the formula of the sealing liquid is as follows:
Figure FDA0002944462330000022
adjusting pH to 7.4, filtering, sterilizing, and storing at 4 deg.C;
1.4) diluting the CD47 monoclonal antibody 1(Mab1) to a working concentration of 10 mug/mL by using a coating solution, uniformly mixing, and standing for 15 minutes;
1.5) taking a microporous plate, using a 12-pore row gun to point the plate to ensure that the antibody solution of Mab1 reaches 100 mu L/pore, covering a cover plate film, placing the plate in a refrigerator at the temperature of 2-6 ℃, and coating the plate overnight for 20-24 hours;
1.6) taking out the overnight coated microporous plate, balancing at room temperature for 30min, throwing away coating liquid, beating the absorbent paper to be dry, adding 300 mu L of washing liquid per hole, standing at room temperature for 1min, washing for 3 times, beating the absorbent paper to be dry;
1.7) adding 200 mu L of sealing liquid into each hole, and sealing for 2 hours at 37 ℃;
1.8) taking out the sealed microporous plate, balancing the microporous plate at room temperature for 30min, throwing off sealing liquid, and patting the microporous plate dry by absorbent paper; drying in a silica gel dryer at room temperature under humidity below 30% for 5 hr;
1.9) vacuum packaging with an aluminum foil bag, marking, and storing at 2-8 ℃ for later use;
2) preparation of CD47 monoclonal antibody 2 horseradish peroxidase
Labeling the CD47 monoclonal antibody 2 horseradish peroxidase (Mab2+ HRP) according to a sodium periodate method, and storing the label at 4 ℃ for later use;
3) preparation of a kit solution:
3.1) preparing an enzyme-labeled diluent, wherein the formula of the enzyme-labeled diluent is as follows:
Figure FDA0002944462330000023
Figure FDA0002944462330000031
adjusting pH to 7.4, filtering for sterilization, and storing at 4 deg.C;
3.2) preparation of 20 × concentrated washing solution, wherein the formula of the 20 × concentrated washing solution is as follows: sterilized 50mL of purified water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、Tween20 0.5mL；
3.3) gradient solutions of CD47 standard were prepared as follows:
preparing a buffer solution containing 5 wt% of bovine serum albumin, 0.85 wt% of sodium chloride and 1.0mmol/L of disodium hydrogen phosphate-sodium dihydrogen phosphate, wherein the pH value of the buffer solution is 7.0; preparing 6 CD47 standard substance gradient solutions with concentration gradient of 560ng/mL, 280ng/mL, 140ng/mL, 70ng/mL, 35ng/mL and 0ng/mL by using the standard substance buffer solution;
3.4) preparing a sample diluent, wherein the formula of the sample diluent is as follows: sterilized 1000mL of pure water containing 8g of NaCl and NaH2PO4·2H2O 0.2g、Na2HPO4·12H2O 2.9g、KCl 0.2g、BSA 10g、ProClinTM950 1mL；
3.5) the luminescent liquid A is carbamide peroxide solution, and the preparation method is as follows:
dissolving urea peroxide in disodium hydrogen phosphate-citric acid buffer solution with pH of 5.0 to prepare 0.1mol/L urea peroxide stock solution, and gradually diluting with disodium hydrogen phosphate-citric acid buffer solution to working concentration of 4.0 × 10-3mol/L；
3.6) the luminescent solution B is a mixed solution obtained by mixing a luminol solution and a 4-hydroxy-4' -iodine-biphenyl solution according to a ratio of 1: 1(v/v), and the preparation method is as follows:
firstly, the luminol solution is prepared into 0.1mol/L luminol stock solution by using 0.05mol/L, pH 10.3.3 carbonate buffer solution, the luminol stock solution is placed for three days in a dark place, and then 0.1mol/L, pH 10.3.3 NaHCO is used3Dilution of-NaOH buffer solution to 3.0X 10-3mol/L；
② 4-hydroxy-4' -iodine-biphenyl solution is prepared into 1x 10 by redistilled dimethyl sulfoxide-2Diluting 4-hydroxy-4' -iodo-biphenyl solution at mol/L to 1.6 × 10-3mol/L; the dilution was prepared in sterile 1000mL pure water and contained 12mL of Tween20 (1% v/v).
9. Use of the chemiluminescent immunoassay kit of claim 1 for the detection of CD 47.
10. The method for detecting CD47 using the chemiluminescent immunoassay kit of claim 1 comprising the steps of:
(1) balancing: balancing the sample to be tested and the reagent in the reagent box at room temperature for 30 +/-5 minutes;
(2) sample adding: arranging a standard sample hole and a sample hole in the microporous plate, adding 50 mu L of sample diluent into the standard sample hole according to the concentration gradient, and then adding 50 mu L of CD47 standard sample gradient solution; respectively arranging a blank hole and a sample hole to be detected in the sample hole, adding 100 mu L of sample diluent into the blank hole, adding 50 mu L of sample diluent into the sample hole to be detected, and then adding 50 mu L of sample to be detected; adding a sample to the bottom of the hole of the plate during sample adding, slightly shaking and uniformly mixing the sample and the hole wall to the greatest extent;
(3) and (3) incubation: sealing the microporous plate with a self-adhesive sealing sheet, and performing oscillation incubation at 37 ℃ for 20 minutes;
(4) preparing liquid: diluting 20 times of the concentrated washing solution with distilled water to obtain washing solution for later use;
(5) washing: discarding liquid, spin-drying, adding 300 μ L of washing solution into each hole, standing for 20-40s, discarding, repeating the above steps for 5 times, and patting to dry;
(6) adding an enzyme: diluting Mab2+ HRP according to the dilution ratio of 1: 50000, wherein the diluent is enzyme-labeled diluent, and adding 100 mu L of diluted Mab2+ HRP solution into each hole of a microporous plate;
(7) and (3) incubation: sealing the microporous plate with a self-adhesive sealing sheet, and performing shaking incubation at 37 ℃ for 10 minutes;
(8) washing: carefully uncovering the adhesive sticker, discarding the liquid, spin-drying, filling washing liquid into each hole, standing for 20-40s, discarding, repeating the steps for 5 times, and patting to dry;
(9) luminescence: adding 50 mu L of luminous liquid A and 50 mu L of luminous liquid B, shaking gently, mixing, and keeping out of the sun for 4 minutes at 18-25 ℃;
(10) and (3) determination: adjusting the blank hole to zero, and sequentially measuring the luminous intensity of each hole at the wavelength of 425nm, namely the RLU value;
(11) drawing a standard curve on coordinate paper by taking each concentration of the standard substance as an abscissa and taking the RLU value as an ordinate, finding out the corresponding concentration of the sample to be detected from the standard curve according to the measured RLU value, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample; or calculating a linear regression equation of a standard curve by using the concentration of the standard substance and the RLU value, substituting the RLU value of the sample to be detected into the equation, calculating the concentration of the sample, and multiplying by the dilution factor to obtain the actual concentration of the sample.
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