CN111337690A - Chemiluminescence kit for quantitatively detecting human inhibin b and preparation method thereof - Google Patents
Chemiluminescence kit for quantitatively detecting human inhibin b and preparation method thereof Download PDFInfo
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- CN111337690A CN111337690A CN202010161910.3A CN202010161910A CN111337690A CN 111337690 A CN111337690 A CN 111337690A CN 202010161910 A CN202010161910 A CN 202010161910A CN 111337690 A CN111337690 A CN 111337690A
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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
- G01N33/74—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
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- 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
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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
- G01N33/53—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
Abstract
The invention relates to the technical field of immunoassay, and provides a chemiluminescence kit for quantitatively detecting human inhibin b, which comprises: the kit comprises a chemiluminescent plate coated with a human inhibin b antibody, a sample reaction solution, a biotin-labeled human inhibin b antibody, a streptavidin-labeled enzyme substrate and an enzymatic luminescent substrate.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a chemiluminescence kit for quantitatively detecting human inhibin b, and a preparation method and a use method thereof.
Background
Inhibin b (inhibin B) is secreted by cells of the reproductive system, has close relation with reproductive capacity, and has endocrine, paracrine and autocrine regulation effects on reproductive function.
Inhibin b is a glycoprotein hormone derived from testis, and the level of serum inhibin b in adult males is significantly negatively correlated with FSH, and plays a negative feedback role on FSH. Shortly after birth, serotonin b levels gradually rise, reaching adult levels during puberty stage ii, and a negative relationship between serotonin b and FSH is maintained from puberty stage iii to adulthood. At the age of 20-30, the inhibin b level reaches another peak, after which the inhibin b level gradually decreases with age.
The male with low spermatogenic function and spermatogenic arrest has lower serum inhibin b level than the normal spermatogenic function male, only the male with supporting cell Syndrome (SCO) has extremely low serum inhibin b level, the SCO generation is obviously related to the serum inhibin b level, and the serum inhibin b level is also obviously related to the testicle volume and the total number of sperms. Inhibin b levels reflect the function of the entire testicular tissue and are a direct product of the seminiferous tract, and maintenance of detectable levels of inhibin b in adult male serum requires the presence of spermatogenic cells, and thus inhibin b is considered a serum marker of male spermatogenesis. However, the existing detection method of the kit still has the problems of relatively low detection sensitivity and accuracy, very high requirements on equipment and sampling, and incapability of realizing large-flux detection.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a chemiluminescent kit for quantitatively detecting human inhibin b, comprising: the kit comprises a chemiluminescent plate coated with a human inhibin b antibody, a sample reaction solution, a biotin-labeled human inhibin b antibody, a streptavidin-labeled enzyme substrate and an enzymatic luminescent substrate.
As a preferred technical scheme, the concentration of the human inhibin b antibody in the chemiluminescent plate is 1-5 ug/mL.
In a preferred embodiment, the sample reaction solution of the present invention includes at least one of dextran, phosphate buffer, and tris buffer.
In a preferred embodiment, the buffer of the biotin-labeled human inhibin b antibody in the present invention comprises at least one of Tris buffer, phosphate buffer, and protein protectant.
As a preferred technical scheme, the streptavidin labeled enzyme substrate comprises at least one of phosphate buffer and protein protective agent.
As a preferred technical solution, the protein protectant in the present invention includes at least one of casein, bovine serum albumin, gelatin, animal serum, and human serum.
As a preferred technical scheme, the enzyme substrate in the invention comprises at least one of horseradish catalase and alkaline phosphatase.
As a preferred technical scheme, the enzymatic luminescent substrate in the invention is a horseradish peroxidase enzymatic luminescent substrate and/or an enzymatic luminescent substrate of alkaline phosphatase.
The second aspect of the present invention provides a method for preparing the chemiluminescent kit, comprising at least the following steps:
(1) preparing a chemiluminescent plate coated with a human inhibin b antibody;
(2) preparing an antibody working solution of a biotin-labeled human inhibin b antibody: firstly, preparing a biotin-labeled human inhibin b antibody, and diluting with a diluent in a dilution ratio of 1:500-1: 5000;
(3) preparing a streptavidin labeled enzyme substrate working solution: diluting a streptavidin labeled enzyme substrate with a diluent according to the dilution ratio of 1:500-1: 5000;
(4) preparation of a human inhibin b standard: the dilution concentration gradient was: 0ng/ml, 0.0312ng/ml, 0.0625ng/ml, 0.125ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, are used for making and detecting the standard curve;
(5) preparation of working washing liquid: 0.01M PBST solution, 1L of washing solution was prepared: sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride and Tween 20; when in use, the product is diluted by 15-25 times of ultrapure water.
The third aspect of the present invention provides a method for using the chemiluminescence kit, which at least comprises the following steps:
s1: diluting the standard substance of the human inhibin b into standard substances with different concentration gradients, adding the standard substances into a chemiluminescence plate coated with the antibody of the human inhibin b, and marking at 100 ul/hole;
s2: taking a standard substance gradient concentration and a serum/plasma sample according to 100 ul/hole, adding the standard substance gradient concentration and the serum/plasma sample into a corresponding plate hole, and marking;
s3: adding sample reaction liquid into the standard sample hole and the sample hole according to 50 ul/hole;
s4: incubating at 35-45 deg.C for 0.5-2.5h, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s5: preparing an antibody working solution of a biotin-labeled human inhibin b antibody, and adding the antibody working solution into corresponding plate holes according to 100 ul/hole; incubating at 35-45 deg.C for 0.5-2.5 hr, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s6: preparing working solution of a streptavidin-labeled enzyme substrate, and adding the working solution of the streptavidin-labeled enzyme substrate into corresponding reaction holes according to 50 ul/hole; 50 ul/hole, standing at 35-45 ℃ for reaction for 20-40 min; taking out the luminous plate, washing the luminous plate for 2-6 times by 250ul of washing working solution in each hole, and patting dry;
s7: adding 50ul enzyme luminescent substrate into each hole, and reading a luminescent value;
s8: and (5) making a standard curve, and calculating the concentration value of the sample to be detected.
Compared with the prior art, the invention has the following excellent beneficial effects:
the invention provides a chemiluminescence kit for quantitatively detecting human inhibin b, which has the advantages that an enzymatic chemiluminescence method is adopted, a human inhibin b (inhibin B) monoclonal antibody or a human inhibin b (inhibin B) polyclonal antibody is coated in a chemiluminescence plate, an enzymatic catalytic luminescence substrate is utilized, and a biotin amplification system is utilized, so that the sensitivity and the accuracy of the kit are improved; experiments prove that the chemiluminescence method is an effective method for detecting human inhibin b (inhibin B) by matching with a biotin amplification system. The chemiluminescence method is matched with a biotin amplification system detection system to make good result judgment on the sample human inhibin b (inhibin B); compared with a human inhibin b (inhibin B) enzyme-linked immunosorbent assay (ELISA) detection kit on the market, the kit has higher detection sensitivity and stronger specificity; the kit does not need blood sample treatment, is simple and convenient to operate, has no high requirement on required equipment, can realize large-flux detection, and can realize semi-automatic or full-automatic batch operation in the immunodiagnosis part.
Drawings
Fig. 1 is a standard curve plotted in example 1, with the regression equation being that y is 610052x +4883.9 and R2 is 0.9993.
Detailed Description
The technical features of the technical solutions provided by the present invention will be further clearly and completely described below with reference to the specific embodiments, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The words "preferred", "preferably", "more preferred", and the like, in the present invention, refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
The invention provides a chemiluminescence kit for quantitatively detecting human inhibin b, which comprises: the kit comprises a chemiluminescent plate coated with a human inhibin b antibody, a sample reaction solution, a biotin-labeled human inhibin b antibody, a streptavidin-labeled enzyme substrate and an enzymatic luminescent substrate.
Preparation of chemiluminescence plate coated with human inhibin b antibody
In the present invention, the chemiluminescent plate coated with the human inhibin b antibody is a chemiluminescent plate coated with a suitable concentration of human inhibin b monoclonal antibody or human inhibin b polyclonal antibody.
In some embodiments, the concentration of human inhibin b antibody in the chemiluminescent plate is 1-5 ug/mL; preferably, the concentration is 1-4 ug/mL; more preferably, the concentration is 2 ug/mL.
In some embodiments, the method of preparing the human inhibin b antibody-coated chemiluminescent plate is as follows:
diluting the monoclonal antibody or polyclonal antibody of the human inhibin b to 1-5ug/ml by using phosphate buffer solution or carbonate buffer solution, adding the diluted monoclonal antibody or polyclonal antibody into a chemiluminescence plate according to 100 ul/hole, standing at 2-10 ℃ for 16-20 hours, taking out the chemiluminescence plate, spin-drying residual liquid, carrying out sealing treatment by using a protein protective agent, adding the sample according to 250 ul/hole, standing at 35-40 ℃ for 1-3 hours or standing at 2-10 ℃ for 16-20 hours, spin-drying the residual liquid, and drying and storing at 2-8 ℃ for later use.
In some more embodiments, the method for preparing the human inhibin b antibody-coated chemiluminescent plate is as follows:
diluting the monoclonal antibody or polyclonal antibody of the human inhibin b to 1-4ug/ml by using phosphate buffer solution or carbonate buffer solution, adding the diluted monoclonal antibody or polyclonal antibody into a chemiluminescence plate according to 100 ul/hole, standing at 4 ℃ for 16-20 hours, taking out the chemiluminescence plate, spin-drying residual liquid, carrying out sealing treatment by using a protein protective agent, adding the sample according to 250 ul/hole, standing at 37 ℃ for 2 hours or 4 ℃ for 16-20 hours, spin-drying the residual liquid, drying and storing at 4 ℃ for later use.
The concentration of the phosphate buffer solution is 0.01mol/L, and the phosphate buffer solution is PBS for short.
In some embodiments, the protein protectant includes at least one of casein, bovine serum albumin, gelatin, animal serum, and human serum; preferably, the protein protectant is bovine serum albumin. The bovine serum albumin is abbreviated as BSA.
In some embodiments, the sample reaction solution comprises at least one of dextran, phosphate buffer, tris buffer components; preferably, the sample reaction solution is a phosphate buffer solution.
Preparation of biotin-labeled human inhibin b antibody working solution
In some embodiments, the buffer of the biotin-labeled human inhibin b antibody comprises at least one of a Tris salt buffer, a phosphate buffer, a protein protectant; preferably, the buffer of the biotin-labeled human inhibin b antibody is phosphate buffer.
In some embodiments, the biotin-labeled human inhibin b antibody is a biotin-labeled human inhibin b monoclonal antibody or a human inhibin b polyclonal antibody.
In some embodiments, the biotin-labeled human inhibin b antibody working solution is prepared as follows:
(1) preparation of biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody
A10 mM NHS-DPEG4-Biotin working solution was prepared using 0.1M PBS buffer, pH 7.2. Adding human inhibin b monoclonal antibody or polyclonal antibody into appropriate amount of 10mM NHS-DPEG4-Biotin working solution, reacting at room temperature for 1 hr, separating and purifying with dextran gel, and removing free Biotin. Add 0.1. mu.g/. mu.LBSA and stand at 4 ℃ for further use.
(2) Preparation of biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody working solution
1) Preparation of a diluent: adding 1 g/L% casein or 1 g/L% BSA, 0.1V/Vproclin300 into 0.01M PB buffer solution;
2) preparing a working solution: diluting the biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody with a diluent to obtain a biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody working solution, wherein the dilution ratio is 1:500-1: 5000.
In some embodiments, the dilution ratio in step 2) is 1: 1000.
preparation of streptavidin labeled enzyme substrate working solution
In some embodiments, the streptavidin-labeled enzyme substrate comprises at least one of a phosphate buffer, a protein protectant.
In some preferred embodiments, the streptavidin-labeled enzyme substrate comprises a phosphate buffer, a protein protectant.
In some embodiments, the protein protectant includes at least one of casein, bovine serum albumin, gelatin, animal serum, and human serum; preferably, the protein protectant comprises at least one of casein and bovine serum albumin; more preferably, the protein protectant is bovine serum albumin.
In some embodiments, the enzyme substrate comprises at least one of horseradish catalase, alkaline phosphatase.
In some embodiments, the streptavidin-labeled enzyme substrate is prepared as follows:
(1) preparation of streptavidin-labeled horseradish peroxidase or alkaline phosphatase
Adopting a glutaric alcohol method, preparing horseradish peroxidase or alkaline phosphatase into 50IU/ml, taking a proper amount, adding into PBS (phosphate buffer solution) containing 1.25% glutaraldehyde and having pH of 6.8, uniformly mixing to room temperature, and reacting overnight. Collecting reaction solution, dialyzing with PBS (pH7.2) for 4 times, dissolving appropriate amount of SA in 1mol/L carbonate buffer solution (pH9.5), mixing with the dialyzed reaction solution, reacting at 4 deg.C, and adding appropriate amount of 0.2mol/L lysine solution. After mixing, the mixture was reacted at room temperature for 2 hours, and dialyzed 4 times against 0.05mol/L PBS (pH 7.2). Centrifuging to obtain supernatant, and cooling to 4 deg.C.
(2) Preparation of streptavidin-labeled horseradish peroxide or alkaline phosphatase working solution
1) Preparation of a diluent: adding 1 g/L% casein or 1 g/L% BSA, 0.1V/Vproclin300 into 0.01M PB buffer solution;
2) preparing a working solution:
diluting streptavidin-labeled horseradish peroxidase or alkaline phosphatase with a diluent to obtain streptavidin-labeled horseradish peroxidase working solution or streptavidin-labeled alkaline phosphatase working solution, wherein the dilution ratio is 1:500-1:5000, and preferably 1: 1000.
In some embodiments, the streptavidin-labeled horseradish peroxidase or alkaline phosphatase is commercially available as a streptavidin labeling kit.
In some embodiments, the enzymatic luminescent substrate is a horseradish peroxidase enzymatic luminescent substrate and/or an enzymatic luminescent substrate for alkaline phosphatase; preferably, the enzymatic luminescent substrate is a horseradish peroxidase enzymatic luminescent substrate.
Preparation of standard substance of human inhibin b
In some embodiments, the kit further comprises a human inhibin b standard.
In some embodiments, the human inhibin b standard is prepared as follows:
the human inhibin b standard antigen is a human inhibin b protein with the concentration of more than or equal to 95 percent through gene recombination or a human inhibin b (inhibin B) standard substance provided by international (WHO/NIBSC) or national institute, and the diluent of the human inhibin b (inhibin B) standard substance in the kit is HEPES containing 1g/L percent BSA, PB buffer containing 1g/L percent BSA, or Tris buffer containing 1g/L percent BSA, and contains 0.1V/V percent proclin 300. The dilution concentration gradient was: 0ng/ml, 0.0312ng/ml, 0.0625ng/ml, 0.125ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, and is used for preparing a standard curve for detection.
Preparation of working washing liquid
In some embodiments, the working wash is prepared as follows:
taking the washing solution as 0.01M PBST solution, preparing 1L of washing solution: sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride and Tween 20; when in use, the product is diluted by 15-25 times of ultrapure water.
In some preferred embodiments, the working washing solution is prepared as follows:
taking the washing solution as 0.01M PBST solution, preparing 1L of washing solution: 9.75g of sodium dihydrogen phosphate dihydrate, 51g of disodium hydrogen phosphate dodecahydrate, 155g of sodium chloride and Tween 2010 ml, and 0.1V/V% proclin300 was contained, and the solution was diluted 20 times with ultrapure water when used.
The second aspect of the present invention provides a method for preparing the chemiluminescent kit, comprising at least the following steps:
(1) preparing a chemiluminescent plate coated with a human inhibin b antibody;
(2) preparing an antibody working solution of a biotin-labeled human inhibin b antibody: firstly, preparing a biotin-labeled human inhibin b antibody, and diluting with a diluent in a dilution ratio of 1:500-1: 5000;
(3) preparing a streptavidin labeled enzyme substrate working solution: diluting a streptavidin labeled enzyme substrate with a diluent according to the dilution ratio of 1:500-1: 5000;
(4) preparation of a human inhibin b standard: the dilution concentration gradient was: 0ng/ml, 0.0312ng/ml, 0.0625ng/ml, 0.125ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, are used for making and detecting the standard curve;
(5) preparation of working washing liquid: 0.01M PBST solution, 1L of washing solution was prepared: sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride, Tween20 and proclin300 with the content of 0.1 percent; when in use, the product is diluted by 15-25 times of ultrapure water.
The third aspect of the present invention provides a method for using the chemiluminescence kit, which at least comprises the following steps:
s1: diluting the standard substance of the human inhibin b into standard substances with different concentration gradients, adding the standard substances into a chemiluminescence plate coated with the antibody of the human inhibin b, and marking at 100 ul/hole;
s2: taking a standard substance gradient concentration and a serum/plasma sample according to 100 ul/hole, adding the standard substance gradient concentration and the serum/plasma sample into a corresponding plate hole, and marking;
s3: adding sample reaction liquid into the standard sample hole and the sample hole according to 50 ul/hole;
s4: incubating at 35-45 deg.C for 0.5-2.5h, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s5: preparing an antibody working solution of a biotin-labeled human inhibin b antibody, and adding the antibody working solution into corresponding plate holes according to 100 ul/hole; incubating at 35-45 deg.C for 0.5-2.5 hr, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s6: preparing working solution of a streptavidin-labeled enzyme substrate, and adding the working solution of the streptavidin-labeled enzyme substrate into corresponding reaction holes according to 50 ul/hole; 50 ul/hole, standing at 35-45 ℃ for reaction for 20-40 min; taking out the luminous plate, washing the luminous plate for 2-6 times by 250ul of washing working solution in each hole, and patting dry;
s7: adding 50ul enzyme luminescent substrate into each hole, and reading a luminescent value;
s8: and (5) making a standard curve, and calculating the concentration value of the sample to be detected.
In some preferred methods of using the chemiluminescent kit, the steps include at least:
s1: diluting the standard substance of the human inhibin b into standard substances with different concentration gradients, adding the standard substances into a chemiluminescence plate coated with the antibody of the human inhibin b, and marking at 100 ul/hole;
s2: taking a standard substance gradient concentration and a serum/plasma sample according to 100 ul/hole, adding the standard substance gradient concentration and the serum/plasma sample into a corresponding plate hole, and marking;
s3: adding sample reaction liquid into the standard sample hole and the sample hole according to 50 ul/hole;
s4: incubating at 35-45 deg.C for 0.5-2.5h, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s5: preparing an antibody working solution of a biotin-labeled human inhibin b antibody, and adding the antibody working solution into corresponding plate holes according to 100 ul/hole; incubating at 35-45 deg.C for 0.5-2.5 hr, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s6: preparing working solution of a streptavidin-labeled enzyme substrate, and adding the working solution of the streptavidin-labeled enzyme substrate into corresponding reaction holes according to 50 ul/hole; 50 ul/hole, standing at 35-45 ℃ for reaction for 20-40 min; taking out the luminous plate, washing the luminous plate for 2-6 times by 250ul of washing working solution in each hole, and patting dry;
s7: adding 50ul enzyme luminescent substrate into each hole, and reading a luminescent value;
s8: respectively taking the concentration value of the gradient of the standard substance as an x value and a luminous value as a y value, and making a standard curve to obtain a unitary linear equation; and substituting the luminous value of the sample to be detected into the standard curve, and calculating the concentration value of the sample to be detected.
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
A chemiluminescent kit for quantitatively detecting human inhibin b comprising: the kit comprises a chemiluminescent plate coated with a human inhibin b antibody, a sample reaction solution, a biotin-labeled human inhibin b antibody, a streptavidin-labeled enzyme substrate and an enzymatic luminescent substrate.
The preparation method of the chemiluminescent plate coated with the human inhibin b antibody is as follows:
diluting the monoclonal antibody or polyclonal antibody of the human inhibin b to 1-4ug/ml by using phosphate buffer solution or carbonate buffer solution, adding the diluted monoclonal antibody or polyclonal antibody into a chemiluminescence plate according to 100 ul/hole, standing at 4 ℃ for 16-20 hours, taking out the chemiluminescence plate, spin-drying residual liquid, carrying out sealing treatment by using a protein protective agent, adding the sample according to 250 ul/hole, standing at 37 ℃ for 2 hours, spin-drying the residual liquid, drying and storing at 4 ℃ for later use.
The sample reaction solution is phosphate buffer solution.
The preparation method of the biotin-labeled human inhibin b antibody working solution comprises the following steps:
(1) preparation of biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody
A10 mM NHS-DPEG4-Biotin working solution was prepared using 0.1M PBS buffer, pH 7.2. Adding human inhibin b monoclonal antibody or polyclonal antibody into appropriate amount of 10mM NHS-DPEG4-Biotin working solution, reacting at room temperature for 1 hr, separating and purifying with dextran gel, and removing free Biotin. Add 0.1. mu.g/. mu.LBSA and stand at 4 ℃ for further use.
(2) Preparation of biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody working solution
1) Preparation of a diluent: adding 1 g/L% casein or 1 g/L% BSA, 0.1V/Vproclin300 into 0.01M PB buffer solution;
2) preparing a working solution: and diluting the biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody by using a diluent to obtain a biotin-labeled human inhibin b monoclonal antibody or human inhibin b polyclonal antibody working solution, wherein the dilution ratio is 1: 1000.
The preparation method of the streptavidin labeled enzyme substrate is as follows:
(1) preparation of streptavidin-labeled horseradish peroxidase or alkaline phosphatase
Adopting a glutaric alcohol method, preparing horseradish peroxidase or alkaline phosphatase into 50IU/ml, taking a proper amount, adding into PBS (phosphate buffer solution) containing 1.25% glutaraldehyde and having pH of 6.8, uniformly mixing to room temperature, and reacting overnight. Collecting reaction solution, dialyzing with PBS (pH7.2) for 4 times, dissolving appropriate amount of SA in 1mol/L carbonate buffer solution (pH9.5), mixing with the dialyzed reaction solution, reacting at 4 deg.C, and adding appropriate amount of 0.2mol/L lysine solution. After mixing, the mixture was reacted at room temperature for 2 hours, and dialyzed 4 times against 0.05mol/L PBS (pH 7.2). Centrifuging to obtain supernatant, and cooling to 4 deg.C.
(2) Preparation of streptavidin-labeled horseradish peroxide or alkaline phosphatase working solution
1) Preparation of a diluent: adding 1 g/L% casein or 1 g/L% BSA, 0.1V/Vproclin300 into 0.01M PB buffer solution;
2) preparing a working solution:
diluting streptavidin-labeled horseradish peroxidase or alkaline phosphatase with a diluent to obtain streptavidin-labeled horseradish peroxidase working solution or streptavidin-labeled alkaline phosphatase working solution, wherein the dilution ratio is 1: 1000.
The enzymatic luminescent substrate is a horseradish peroxidase enzymatic luminescent substrate.
The preparation method of the human inhibin b standard substance is as follows:
the human inhibin b standard antigen is a human inhibin b protein with the concentration of more than or equal to 95 percent through gene recombination or a human inhibin b (inhibin B) standard substance provided by international (WHO/NIBSC) or national institute, and the diluent of the human inhibin b (inhibin B) standard substance in the kit is HEPES containing 1g/L percent BSA, PB buffer containing 1g/L percent BSA, or Tris buffer containing 1g/L percent BSA, and contains 0.1V/V percent proclin 300. The dilution concentration gradient was: 0ng/ml, 0.0312ng/ml, 0.0625ng/ml, 0.125ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, and is used for preparing a standard curve for detection.
The preparation method of the working washing solution is as follows:
taking the washing solution as 0.01M PBST solution, preparing 1L of washing solution: 9.75g of sodium dihydrogen phosphate dihydrate, 51g of disodium hydrogen phosphate dodecahydrate, 155g of sodium chloride and Tween 2010 ml, and 0.1V/V% proclin300 was contained, and the solution was diluted 20 times with ultrapure water when used.
The using method of the chemiluminescence kit at least comprises the following steps:
s1: diluting the standard substance of the human inhibin b into standard substances with different concentration gradients, adding the standard substances into a chemiluminescence plate coated with the antibody of the human inhibin b, and marking at 100 ul/hole;
s2: taking a standard substance gradient concentration and a serum/plasma sample according to 100 ul/hole, adding the standard substance gradient concentration and the serum/plasma sample into a corresponding plate hole, and marking;
s3: adding sample reaction liquid into the standard sample hole and the sample hole according to 50 ul/hole;
s4: incubating at 35-45 deg.C for 0.5-2.5h, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s5: preparing an antibody working solution of a biotin-labeled human inhibin b antibody, and adding the antibody working solution into corresponding plate holes according to 100 ul/hole; incubating at 35-45 deg.C for 0.5-2.5 hr, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s6: preparing working solution of a streptavidin-labeled enzyme substrate, and adding the working solution of the streptavidin-labeled enzyme substrate into corresponding reaction holes according to 50 ul/hole; 50 ul/hole, standing at 35-45 ℃ for reaction for 20-40 min; taking out the luminous plate, washing the luminous plate for 2-6 times by 250ul of washing working solution in each hole, and patting dry;
s7: adding 50ul enzyme luminescent substrate into each hole, and reading a luminescent value;
s8: respectively taking the concentration value of the gradient of the standard substance as an x value and a luminous value as a y value, and making a standard curve to obtain a unitary linear equation; and substituting the luminous value of the sample to be detected into the standard curve, and calculating the concentration value of the sample to be detected.
The kit of this embodiment uses the concentration of the calibrator as the X value, and uses the luminescence value RLU corresponding to the concentration detection as the Y value, so as to make a one-dimensional equation. As shown in fig. 1, the regression equation of the standard curve is plotted, wherein y is 610052x +4883.9, and R2 is 0.9993.
Minimum detection limit: the kit detects the reference substance with the lowest detection limit (0.0312ng/ml) for 10 times while detecting the standard curve, and calculates the average concentration value (x) and the Standard Deviation (SD) of the reference substance with the lowest detection limit through the standard curve. According to the formula: the lowest limit of detection of the sample is calculated as X + -2 SD, the result is shown in Table 1, and the lowest limit of detection of human inhibin b (inhibin B) by the kit of the present invention is 0.0311-0.0315 ng/ml.
TABLE 1 minimum detection limits of the human inhibin b (inhibin B) chemiluminescence detection kit
| Sample(s) | Mean value (ng/ml) | Standard deviation (ng/ml) | Minimum detection limit (ng/ml) |
| Reference product with minimum detection limit | 0.0313 | 0.002 | 0.0311-0.0315 |
Precision: the kit of the invention uses the ratio of Standard Deviation (SD)/average concentration value (x) as the precision value, namely: the standard deviation of 0.002ng/ml divided by the average concentration value of 0.0313ng/ml, multiplied by 100%, is 6.389% < 15%
Compared with the ELISA detection kit owned by the current market, the kit for detecting the human inhibin b (inhibin B) has the advantages of simple and convenient operation, high operation sensitivity, good specificity, low detection cost, less sample usage amount, low requirement on equipment and the like.
Claims (10)
1. A chemiluminescence kit for quantitatively detecting human inhibin b, which is characterized by comprising: the kit comprises a chemiluminescent plate coated with a human inhibin b antibody, a sample reaction solution, a biotin-labeled human inhibin b antibody, a streptavidin-labeled enzyme substrate and an enzymatic luminescent substrate.
2. A chemiluminescent kit of claim 1 wherein the concentration of human inhibin b antibody in the chemiluminescent plate is 1-5 ug/mL.
3. A chemiluminescent kit according to claim 1 wherein the sample reaction solution comprises at least one of dextran, phosphate buffer, tris buffer components.
4. The chemiluminescent kit of claim 1 wherein the buffer of the biotin-labeled human inhibin b antibody comprises at least one of a Tris buffer, a phosphate buffer, and a protein protectant.
5. The chemiluminescent kit of claim 1 wherein the streptavidin labeled enzyme substrate comprises at least one of a phosphate buffer, a protein protectant.
6. A chemiluminescent kit according to claim 4 or claim 5 wherein the protein protectant comprises at least one of casein, bovine serum albumin, gelatin, animal serum, human serum.
7. The chemiluminescent kit of claim 1 wherein the enzyme substrate comprises at least one of horseradish catalase, alkaline phosphatase.
8. A chemiluminescent kit according to claim 1 wherein the enzymatic luminogenic substrate is a horseradish peroxidase enzymatic luminogenic substrate and/or an enzymatic luminogenic substrate for alkaline phosphatase.
9. A method of preparing a chemiluminescent kit of any one of claims 1 to 9 comprising the steps of:
(1) preparing a chemiluminescent plate coated with a human inhibin b antibody;
(2) preparing an antibody working solution of a biotin-labeled human inhibin b antibody: firstly, preparing a biotin-labeled human inhibin b antibody, and diluting with a diluent in a dilution ratio of 1:500-1: 5000;
(3) preparing a streptavidin labeled enzyme substrate working solution: diluting a streptavidin labeled enzyme substrate with a diluent according to the dilution ratio of 1:500-1: 5000;
(4) preparation of a human inhibin b standard: the dilution concentration gradient was: 0ng/ml, 0.0312ng/ml, 0.0625ng/ml, 0.125ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, are used for making and detecting the standard curve;
(5) preparation of working washing liquid: 0.01M PBST solution, 1L of washing solution was prepared: sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dodecahydrate, sodium chloride and Tween 20; when in use, the product is diluted by 15-25 times of ultrapure water.
10. A method of using a chemiluminescent kit according to any one of claims 1 to 9 comprising at least the steps of:
s1: diluting the standard substance of the human inhibin b into standard substances with different concentration gradients, adding the standard substances into a chemiluminescence plate coated with the antibody of the human inhibin b, and marking at 100 ul/hole;
s2: taking a standard substance gradient concentration and a serum/plasma sample according to 100 ul/hole, adding the standard substance gradient concentration and the serum/plasma sample into a corresponding plate hole, and marking;
s3: adding sample reaction liquid into the standard sample hole and the sample hole according to 50 ul/hole;
s4: incubating at 35-45 deg.C for 0.5-2.5h, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s5: preparing an antibody working solution of a biotin-labeled human inhibin b antibody, and adding the antibody working solution into corresponding plate holes according to 100 ul/hole; incubating at 35-45 deg.C for 0.5-2.5 hr, taking out, washing with 250ul of working solution per well for 2-6 times, and drying;
s6: preparing working solution of a streptavidin-labeled enzyme substrate, and adding the working solution of the streptavidin-labeled enzyme substrate into corresponding reaction holes according to 50 ul/hole; 50 ul/hole, standing at 35-45 ℃ for reaction for 20-40 min; taking out the luminous plate, washing the luminous plate for 2-6 times by 250ul of washing working solution in each hole, and patting dry;
s7: adding 50ul enzyme luminescent substrate into each hole, and reading a luminescent value;
s8: and (5) making a standard curve, and calculating the concentration value of the sample to be detected.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111965361A (en) * | 2020-08-05 | 2020-11-20 | 上海长征医院 | Chemiluminescence kit for quantitatively detecting human chitinase 3-like protein 1 |
| CN112816694A (en) * | 2020-12-31 | 2021-05-18 | 泰州泽成生物技术有限公司 | Abnormal prothrombin detection kit and preparation method thereof |
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2020
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111965361A (en) * | 2020-08-05 | 2020-11-20 | 上海长征医院 | Chemiluminescence kit for quantitatively detecting human chitinase 3-like protein 1 |
| CN112816694A (en) * | 2020-12-31 | 2021-05-18 | 泰州泽成生物技术有限公司 | Abnormal prothrombin detection kit and preparation method thereof |
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