CN110672857A - TNF-alpha high-sensitivity Elisa detection kit and use method and application thereof - Google Patents
TNF-alpha high-sensitivity Elisa detection kit and use method and application thereof Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G01N33/532—Production of labelled immunochemicals
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- 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
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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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- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention discloses a high-sensitivity Elisa detection kit for TNF-alpha, and a use method and application thereof, and belongs to the technical field of biomedical detection. The Elisa detection kit comprises: the kit comprises an ELISA plate coated with a TNF-alpha capture antibody, a TNF-alpha detection antibody, a TNF-alpha antibody standard substance, avidin-horseradish peroxidase, an ECL reagent, a PBS buffer solution, a cleaning solution, a confining liquid and a sample diluent; the kit, the use method and the application are based on the characteristic that ECL has high sensitivity and the like, and the ECL is used for ELISA fluorescence detection of the TNF-alpha antibody, so that the minimum detection limit of the TNF-alpha antibody in an Elisa detection kit is effectively improved, and the kit is suitable for detection of samples with low clinical sample amount to be detected or low TNF-alpha content; meanwhile, the detection kit can effectively improve the detection sensitivity and the detection limit.
Description
Technical Field
The invention relates to an Elisa detection kit for TNF-alpha and a use method and application thereof, in particular to a chemiluminescence detection kit for high-sensitivity Elisa with low content and less amount of TNF-alpha in a sample and a use method and application thereof, belonging to the technical field of biomedical detection.
Background
TNF-alpha (tumor necrosis factor alpha) is a cytokine which can directly kill tumor cells without obvious toxicity to normal cells, and is one of the bioactive factors which have the strongest effect of directly killing tumors and are discovered so far.
The Elisa detection principle comprises the steps of adding a capture antibody into a pore of a carrier enzyme label plate, coating the capture antibody on the carrier to form a solid phase antibody, and keeping the immunocompetence of the solid phase antibody; then adding a sample into the hole of the enzyme-labeled plate, specifically combining the protein to be detected in the sample with the solid-phase antibody to form a solid-phase antigen complex, and washing to remove impurities; then adding a detection antibody with biotin into the hole of the ELISA plate, and combining the detection antibody with the solid-phase antigen complex; finally, horseradish peroxidase with avidin is added into the wells of the enzyme label plate. And after the substrate is added, the substrate is catalyzed by enzyme to be changed into a colored product, and the amount of the colored product is directly related to the amount of the protein to be detected in the sample, so that quantitative analysis can be carried out according to the shade of the color reaction.
At present, the adopted TNF-alpha Elisa detection kit generally detects a sample to be detected according to a TMB substrate color development principle, but the sensitivity is not high, and certain limitations exist, and the detection kit specifically comprises:
the method has minimum requirements on the content of TNF-alpha in a sample to be detected, such as: the lowest detection limit of the kit with the commodity number of DTA00D of the American R & D company is 6.23 pg/mL, the lowest detection limit of the kit with the commodity number of 555212 of the American BD company is 4 pg/mL, the lowest detection limit of the kit with the commodity number of EHC103 of Shenzhen Xinbo of China is 7.8 pg/mL, and the lowest detection limit of the kit with the commodity number of EH009 of Shanghai Keke of China is 7 pg/mL;
secondly, the minimum requirement is provided for the required volume of the sample to be detected, namely: under the condition that the sample retest number is n =2, the final adding volume of a sample to be detected is required to be at least 200 mu L by an Elisa detection kit, so that the sample to be detected with high content and small quantity of TNF-alpha can meet the detection requirement after being diluted; however, for some samples to be detected with low content and small amount of TNF-alpha, it is difficult to accurately measure the content of TNF-alpha in the samples by using the existing Elisa kit, such as: in clinic, the effective detection is difficult to carry out by using the conventional ELISA detection kit because the content of TNF-alpha in the embryo culture solution is low and the amount of TNF-alpha in the embryo culture solution is small.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an Elisa detection kit for TNF-alpha and a use method thereof. In the technical scheme, the ECL has the characteristics of high sensitivity and the like, and is used for ELISA chemiluminescence detection of TNF-alpha, so that the sensitivity of the TNF-alpha in an Elisa detection kit is effectively improved, and the ECL is suitable for detecting samples with low clinical sample amount to be detected or low content of the TNF-alpha; meanwhile, the detection kit can effectively improve the detection sensitivity and the detection limit.
In order to achieve the technical purpose, the following technical scheme is proposed:
a TNF- α hypersensitive Elisa test kit comprising:
(1) an ELISA plate coated with a TNF-alpha capture antibody;
(2) a TNF-alpha detection antibody;
(3) a TNF-alpha standard;
(4) avidin-horseradish peroxidase;
(5) an ECL reagent;
(6) PBS buffer solution, cleaning solution, confining solution and sample diluent.
Furthermore, in the Elisa detection kit, the ELISA plate is a 96-hole white ELISA plate or a black ELISA plate.
Further, in the Elisa test kit:
TNF-alpha capture antibody, concentration 0.5 mg/mL;
capture antibody coating solution of Na2CO315 mmol/L and NaHCO334.9 mmol/L of mixed solution, and the pH value is 9.6;
TNF-alpha detection antibody with the concentration of 0.2 mg/mL;
TNF-alpha standard substance with the concentration of 0.1 mg/mL;
cleaning fluid which is PBS buffer solution and 0.5 per mill tween-20, and the pH value is 7.2-7.4;
blocking solution, PBS buffer +4% sucrose + 3% BSA (bovine serum albumin) + 2% FBS (fetal bovine serum);
the sample diluent was PBS buffer + 0.2% Tween-20 +1% BSA (bovine serum albumin).
Further, the PBS buffer solution comprises NaCl with the concentration of 137mmol/L, KCl with the concentration of 2.7mmol/L and Na with the concentration of 10mmol/L2HPO4And KH at a concentration of 2mmol/L2PO4The pH of the mixed solution of (1) was 7.4.
A method for using a high-sensitivity Elisa detection kit for TNF-alpha comprises the following steps:
A. pretreatment
Preparing an ELISA plate coated with TNF-alpha capture antibody: diluting the TNF-alpha capture antibody by 500 times with a capture antibody coating solution to obtain TNF-alpha capture antibody working solution with the concentration of 1 mug/mL; adding 200 mu L of the obtained TNF-alpha capture antibody working solution into each hole of an enzyme label plate, and incubating overnight at 4 ℃, or incubating for 1h at 37 ℃;
removing the incubation liquid, adding 300 mu L PBS buffer solution into each hole of the ELISA plate, washing for 1 time, and printing dry by filter paper;
adding 300 mu L of sealing liquid into each hole of the enzyme label plate, sealing for 2h at 37 ℃, removing the sealing liquid, printing the sealing liquid with filter paper, air-drying for 2h in a drying oven at 37 ℃, and storing at 4 ℃ for later use;
preparing TNF-alpha antibody standard solution: respectively diluting the TNF-alpha antibody standard product into TNF-alpha antibody standard solutions with the concentrations of 0pg/mL, 0.05 pg/mL, 0.5 pg/mL, 5pg/mL, 10 pg/mL, 20 pg/mL, 30 pg/mL and 40 pg/mL by adopting a sample diluent for later use;
preparing a sample solution to be detected: diluting the sample to be detected by 5 times, 10 times and 20 times by using sample diluent to obtain sample solution to be detected for later use;
preparing TNF-alpha detection antibody working solution: diluting the TNF-alpha detection antibody by 400 times by adopting a sample diluent to obtain TNF-alpha detection antibody working solution with the concentration of 0.5 mug/mL for later use;
preparing an avidin-horseradish peroxidase working solution: diluting the avidin-horseradish peroxidase by 100 times by adopting a sample diluent to obtain avidin-horseradish peroxidase working solution for later use;
B. adding standard solution or sample solution to be tested of TNF-alpha antibody
Adding the TNF-alpha antibody standard solution obtained in the step A or the sample solution to be detected obtained in the step A into each hole of the elisa plate prepared in the step A, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
C. TNF-alpha detection antibody working solution
Adding 100 mu L of TNF-alpha detection antibody working solution obtained in the step A into each hole of the ELISA plate in the step B, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
D. avidin-horseradish peroxidase added working solution
C, adding 100 mu L of avidin-horseradish peroxidase working solution into each hole of the ELISA plate in the step C, and incubating for 0.5h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
E. detecting fluorescence intensity
Mixing the solution A and the solution B in the ECL reagent in equal volume to obtain a mixed solution; and D, adding 100 mu L of mixed solution into each hole of the ELISA plate obtained in the step D, standing for 3 minutes at normal temperature in a dark place for reaction, and detecting the light absorption value of the reaction product at 425nm by using a full-wavelength ELISA reader (Thermo, Varioskan flash).
Further, in step E, the same volume of solution a and solution B is taken and placed in a centrifuge tube and mixed to obtain a mixed solution.
When the kit is adopted to detect the concentration of the human TNF-alpha antibody, the following effects are achieved: the minimum detection limit is 70 fg/mL, the sensitivity is 70 fg/mL, and the stability CV value is 0.19-14.17%; compared with the existing kit, the sensitivity is improved by about 100 times.
The application of a high-sensitivity Elisa detection kit for TNF-alpha is disclosed, which is applied to the preparation of a kit for detecting serum of children with in vitro nerve developmental diseases, wherein the kit comprises the steps of detecting the content of TNF-alpha in the serum of children with in vitro nerve developmental diseases, and providing reference for a doctor for the index of the content of TNF-alpha in the serum; children with a neurodegenerative disease include autistic children.
The application of a high-sensitivity Elisa detection kit for TNF-alpha in the preparation of a kit for detecting embryo culture solution of assisted reproductive failure patients is disclosed, wherein the kit comprises the detection of the content of TNF-alpha in the embryo culture solution of assisted reproductive failure patients and provides reference for doctors on the index of the content of TNF-alpha in the embryo culture solution.
By adopting the technical scheme, the beneficial technical effects brought are as follows:
1. in the invention, the ECL-based reagent has the characteristics of high sensitivity and the like, and is used for the ELISA chemiluminescence detection of TNF-alpha, so that the sensitivity of TNF-alpha in an Elisa detection kit is effectively improved, and the ECL-based reagent is suitable for the detection of samples with low clinical sample volume to be detected or low content of TNF-alpha. The method specifically comprises the following steps: the method can effectively reduce the minimum detection limit in human TNF-alpha Elisa detection, and realizes that the minimum detection limit is 70 fg/mL, the sensitivity is 70 fg/mL, and the stability CV value is 0.19-14.17%; compared with the existing kit, the sensitivity is improved by about 100 times.
2. Compared with a TMB substrate color development method, the ECL chemiluminescence method disclosed by the invention has the advantages that TMB substrate incubation time is saved, and direct detection can be realized after ECL is added, so that the time is effectively saved, and the detection time of TNF-alpha is reduced.
Drawings
FIG. 1 is a standard curve diagram of TMB color development detection in the prior art
FIG. 2 is a graph showing a standard curve of the detection by ECL chemiluminescence in the present invention.
Detailed Description
In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
The following examples relate to reagents, including in particular:
TNF- α capture antibody: 100 μ g of TNF-. alpha.capture antibody powder was formulated with PBS buffer to 0.5mg/mL stock solution, and TNF-. alpha.capture antibody powder was purchased directly from R & D under the trade name MAB 610-100;
capture antibody coating solution: is composed of Na with a concentration of 15 mmol/L2CO3And NaHCO with concentration of 34.9 mmol/L3The pH of the mixed solution of (1) is 9.6;
TNF- α detection antibody: 50 μ g of TNF-. alpha.detection antibody powder was prepared as a 0.2mg/mL stock solution with PBS buffer, and 50 μ g of TNF-. alpha.detection antibody powder was purchased directly from R & D under the trade name BAF 210;
TNF- α standard: mu.g of TNF-. alpha.powder, purchased directly from R & D under the trade name 210-TA-100, was prepared as a 0.1mg/mL stock solution using PBS buffer containing 0.1% BSA (bovine serum albumin);
avidin-horseradish peroxidase: purchased directly from R & D, Cat number DY 998;
the ECL reagent: including solution A and solution B, purchased directly from Thermo Scientific Inc., cat # 37075;
PBS buffer (1)Phosphate buffered saline solution): comprises NaCl with the concentration of 137mmol/L, KCl with the concentration of 2.7mmol/L and Na with the concentration of 10mmol/L2HPO4And KH at a concentration of 2mmol/L2PO4The pH of the mixed solution of (1) is 7.4;
cleaning solution: PBS buffer solution and 0.5 per mill tween-20, and the pH is 7.2-7.4;
sealing liquid: PBS buffer +4% sucrose + 3% bovine serum albumin + 2% fetal bovine serum;
sample diluent: PBS buffer + 0.2% Tween-20 +1% bovine serum albumin.
Example 1
A TNF- α hypersensitive Elisa test kit comprising:
(1) an ELISA plate coated with a TNF-alpha capture antibody;
(2) a TNF-alpha detection antibody;
(3) a TNF-alpha standard;
(4) avidin-horseradish peroxidase;
(5) an ECL reagent;
(6) PBS buffer solution, cleaning solution, confining solution and sample diluent.
When the kit is adopted to detect the concentration of the human TNF-alpha antibody, the following effects are achieved: the minimum detection limit is 70 fg/mL, the sensitivity is 70 fg/mL, and the stability CV value is 0.19-14.17%; compared with the existing kit, the sensitivity is improved by about 100 times.
Example 2
On the basis of example 1, further:
in the Elisa detection kit, the ELISA plate is a 96-hole white ELISA plate.
Example 3
On the basis of embodiment 2, the present embodiment is different in that:
in the Elisa detection kit, the ELISA plate is a 96-hole black ELISA plate.
Example 4
A method for using a high-sensitivity Elisa detection kit for TNF-alpha comprises the following steps:
A. pretreatment
Preparing an ELISA plate coated with TNF-alpha capture antibody: diluting the TNF-alpha capture antibody by 500 times with a capture antibody coating solution to obtain TNF-alpha capture antibody working solution with the concentration of 1 mug/mL; adding 200 mu L of the obtained TNF-alpha capture antibody working solution into each hole of a 96-hole black enzyme label plate, and incubating overnight at 4 ℃;
removing the incubation liquid, adding 300 mu L PBS buffer solution into each hole of the ELISA plate, washing for 1 time, and printing dry by filter paper;
adding 300 mu L of sealing liquid into each hole of the enzyme label plate, sealing for 2h at 37 ℃, removing the sealing liquid, printing the sealing liquid with filter paper, air-drying for 2h in a drying oven at 37 ℃, and storing at 4 ℃ for later use;
preparing TNF-alpha antibody standard solution: respectively diluting the TNF-alpha antibody standard product into TNF-alpha antibody standard solutions with the concentrations of 0pg/mL, 0.05 pg/mL, 0.5 pg/mL, 5pg/mL, 10 pg/mL, 20 pg/mL, 30 pg/mL and 40 pg/mL by adopting a sample diluent for later use;
preparing a sample solution to be detected: diluting the sample to be detected by 5 times, 10 times and 20 times by using sample diluent to obtain sample solution to be detected for later use;
preparing TNF-alpha detection antibody working solution: diluting the TNF-alpha detection antibody by 400 times by adopting a sample diluent to obtain TNF-alpha detection antibody working solution with the concentration of 0.5 mug/mL for later use;
preparing an avidin-horseradish peroxidase working solution: diluting the avidin-horseradish peroxidase by 100 times by adopting a sample diluent to obtain avidin-horseradish peroxidase working solution for later use;
B. adding standard solution or sample solution to be tested of TNF-alpha antibody
Adding the TNF-alpha antibody standard solution obtained in the step A or the sample solution to be detected obtained in the step A into each hole of the elisa plate prepared in the step A, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
C. TNF-alpha detection antibody working solution
Adding 100 mu L of TNF-alpha detection antibody working solution obtained in the step A into each hole of the ELISA plate in the step B, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
D. avidin-horseradish peroxidase added working solution
C, adding 100 mu L of avidin-horseradish peroxidase working solution into each hole of the ELISA plate in the step C, and incubating for 0.5h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
E. detecting fluorescence intensity
Mixing the solution A and the solution B in the ECL reagent in equal volume to obtain a mixed solution; and D, adding 100 mu L of mixed solution into each hole of the ELISA plate obtained in the step D, standing for 3 minutes at normal temperature in a dark place for reaction, and detecting the light absorption value of the reaction product at 425nm by using a full-wavelength ELISA reader (Thermo, Varioskan flash).
Example 5
On the basis of embodiment 4, the present embodiment is different in that:
in step A, 200. mu.L of the TNF-alpha capture antibody working solution was added to each well of the microplate and incubated at 37 ℃ for 1 h.
Example 6
The application of a high-sensitivity Elisa detection kit for TNF-alpha in the preparation of a kit for detecting the serum of children with nerve developmental diseases comprises the detection of the content of the TNF-alpha in the serum of children with in vitro nerve developmental diseases and the reference of the index of the content of the TNF-alpha in the serum provided for doctors. Such as: self-closing children, according to the existing research and literature (such as "Pro-inflammatory cytokines in autoimmune childern in central Saudi Arabia."), report that the expression of TNF-alpha is obviously increased in the serum of the self-closing children, and the change of the expression level is obviously and positively correlated with the neurobehavioral indexes of the self-closing children. The kit has the advantages that the TNF-alpha content in the serum of the children is low, and the sensitivity of TNF-alpha detection can be effectively improved by adopting the detection kit.
Example 7
The application of a high-sensitivity Elisa detection kit for TNF-alpha in the preparation of a kit for detecting embryo culture solution of assisted reproductive failure patients is disclosed, wherein the kit comprises the detection of the content of TNF-alpha in the embryo culture solution of assisted reproductive failure patients and provides reference for doctors on the index of the content of TNF-alpha in the embryo culture solution. In the assisted reproduction process, the content of TNF-alpha is obviously increased in the embryo culture solution of a patient with planting failure, and compared with the existing Elisa detection kit, the detection sensitivity is improved by more than 100 times.
Comparative example
Firstly, taking embryo culture solution and blood serum of autistic children as examples, detecting the content of TNF-alpha by adopting an Elisa detection kit based on TMB color development in the prior art, and taking the Elisa detection kit as a control example of example 4 to explain the technical method of the invention. The method specifically comprises the following steps:
1. diluting the TNF-alpha capture antibody by 125 times with a capture antibody diluent to obtain TNF-alpha capture antibody working solution with the concentration of 4 mug/mL; adding 200 mu L of the obtained TNF-alpha capture antibody working solution into each hole of a 96-hole transparent enzyme label plate, and incubating overnight at 4 ℃;
removing the incubation liquid, adding 300 mu L PBS buffer solution into each hole of the ELISA plate, washing for 1 time, and printing dry by filter paper;
adding 300 mu L of sealing liquid into each hole of the enzyme label plate, sealing for 2h at 37 ℃, removing the sealing liquid, printing the sealing liquid with filter paper, air-drying for 2h in a drying oven at 37 ℃, and storing at 4 ℃ for later use;
2. diluting the TNF-alpha antibody standard product into TNF-alpha antibody standard solutions with the concentrations of 100 pg/mL, 33.3 pg/mL, 11.1pg/mL, 3.7pg/mL, 1.2 pg/mL and 0pg/mL respectively by using a sample diluent for later use;
3. diluting the sample to be detected by 3 times, 6 times and 9 times respectively by using the sample diluent to obtain a sample solution to be detected for later use;
4. diluting the TNF-alpha detection antibody by 300 times by adopting a sample diluent to obtain TNF-alpha detection antibody working solution with the concentration of 0.7 mug/mL for later use;
5. diluting the avidin-horseradish peroxidase by 50 times by using a sample diluent to obtain avidin-horseradish peroxidase working solution for later use;
6. adding 100 mu L of TNF-alpha antibody standard solution or sample solution to be detected into each hole of the ELISA plate in the step 1, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
7. adding 100 mu L of TNF-alpha detection antibody working solution into each hole of the ELISA plate in the step 6, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
8. adding 100 mu L of avidin-horseradish peroxidase working solution into each hole of the ELISA plate in the step 7, and incubating for 0.5h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
9. adding 100 μ L of TMB reagent (thermo Co., 34018 cat.) to each well of the microplate of step 8, and incubating at 37 ℃ for 0.5 h; then 100. mu.L of stop solution (2N H) was added to each well of the microplate2SO4) And finally detecting the absorbance of the reactant at 450 nm by a microplate reader.
For the Elisa test kit based on TMB visualization in the prior art (standard curve shown in fig. 1), we have found: the light absorption value of the embryo culture solution diluted by 4 times is 0.19, and the light absorption value of the undiluted blood serum of the autism children is 0.04, which are not in the effective detection range.
Secondly, taking embryo culture solution and autism children serum as examples, adopting the ECL chemiluminescence-based Elisa detection kit to detect the content of TNF-alpha, and the concrete steps and other conditions are the same as those in example 4, so as to explain the technical method in the invention.
For the Elisa test kit using ECL chemiluminescence (standard curve shown in fig. 2) of the present invention, the results are shown in table 1 below and are obtained: the absorbance of the 7-fold diluted embryo culture fluid was 1848, the absorbance of the 3-fold diluted serum from autistic children was 1931, and the concentrations fitted to the standard curve and the sample dilution were 5.77pg/mL and 1.25pg/mL, respectively.
Claims (10)
1. A high-sensitivity Elisa detection kit for TNF-alpha is characterized by comprising:
(1) an ELISA plate coated with a TNF-alpha capture antibody;
(2) a TNF-alpha detection antibody;
(3) a TNF-alpha standard;
(4) avidin-horseradish peroxidase;
(5) an ECL reagent;
(6) PBS buffer solution, cleaning solution, confining solution and sample diluent.
2. The highly sensitive Elisa detection kit for TNF-a of claim 1, wherein said TNF-a capture antibody concentration is 0.5 mg/mL;
the capture antibody coating solution for coating the TNF-alpha capture antibody is Na with the concentration of 15 mmol/L2CO3With NaHCO at a concentration of 34.9 mmol/L3The pH of the mixed solution of (1) is 9.6;
the concentration of the TNF-alpha detection antibody is 0.2 mg/mL;
the concentration of the TNF-alpha standard substance is 0.1 mg/mL;
the cleaning solution is PBS buffer solution containing 0.5 per mill of Tween-20, and the pH value is 7.2-7.4;
the blocking solution is PBS buffer solution containing 4% sucrose, 3% BSA and 2% FBS;
the sample diluent was PBS buffer containing 0.2% Tween-20 and 1% BSA.
3. The TNF-alpha high sensitivity Elisa kit according to claim 2, wherein the PBS buffer solution comprises NaCl with a concentration of 137mmol/L, KCl with a concentration of 2.7mmol/L and Na with a concentration of 10mmol/L2HPO4And KH at a concentration of 2mmol/L2PO4The pH of the mixed solution of (1) was 7.4.
4. The TNF-alpha high-sensitivity Elisa detection kit according to claim 1, characterized in that the ELISA plate is a 96-well white ELISA plate or a black ELISA plate.
5. The high-sensitivity Elisa kit for TNF-alpha according to claim 1, wherein the kit has a minimum detection limit of 70 fg/mL, a sensitivity of 70 fg/mL and a stability CV value of 0.19-14.17%.
6. A method of using the TNF- α hypersensitive Elisa test kit according to any of claims 1-5 comprising the steps of:
A. pretreatment
Preparing an ELISA plate coated with TNF-alpha capture antibody: diluting the TNF-alpha capture antibody with a capture antibody coating solution to obtain TNF-alpha capture antibody working solution with the concentration of 1 mug/mL; adding 200 mu L of the obtained TNF-alpha capture antibody working solution into each hole of an enzyme label plate, and incubating overnight at 4 ℃, or incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L PBS buffer solution into each hole of the ELISA plate, washing for 1 time, and printing dry by filter paper; adding 300 mu L of sealing liquid into each hole of the enzyme label plate, sealing for 2h at 37 ℃, removing the sealing liquid, printing the sealing liquid with filter paper, air-drying for 2h in a drying oven at 37 ℃, and storing at 4 ℃ for later use;
preparing TNF-alpha antibody standard solution: sampling a sample diluent, namely respectively diluting the TNF-alpha standard substance into TNF-alpha antibody standard liquids with the concentrations of 0pg/mL, 0.05 pg/mL, 0.5 pg/mL, 5pg/mL, 10 pg/mL, 20 pg/mL, 30 pg/mL and 40 pg/mL for later use;
preparing a sample solution to be detected: taking a sample diluent, and diluting a sample to be detected to 5 times, 10 times and 20 times respectively to obtain a sample solution to be detected for later use;
preparing TNF-alpha detection antibody working solution: taking a sample diluent, and diluting the TNF-alpha detection antibody to obtain TNF-alpha detection antibody working solution with the concentration of 0.5 mug/mL for later use;
preparing an avidin-horseradish peroxidase working solution: taking a sample diluent, and diluting the avidin-horseradish peroxidase by 100 times to obtain avidin-horseradish peroxidase working solution for later use;
B. adding standard solution or sample solution to be tested of TNF-alpha antibody
Adding the TNF-alpha antibody standard solution obtained in the step A or the sample solution to be detected obtained in the step A into each hole of the elisa plate prepared in the step A, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
C. TNF-alpha detection antibody working solution
Adding 100 mu L of TNF-alpha detection antibody working solution obtained in the step A into each hole of the ELISA plate in the step B, and incubating for 1h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
D. avidin-horseradish peroxidase added working solution
C, adding 100 mu L of avidin-horseradish peroxidase working solution into each hole of the ELISA plate in the step C, and incubating for 0.5h at 37 ℃; removing the incubation liquid, adding 300 mu L of cleaning liquid into each hole of the enzyme label plate, cleaning for 3 times, and drying the filter paper;
E. detecting fluorescence intensity
Mixing the solution A and the solution B in the ECL reagent in equal volume to obtain a mixed solution; and D, adding 100 mu L of mixed solution into each hole of the ELISA plate obtained in the step D, reacting for 3 minutes under the condition of normal temperature and light shielding, and detecting the light absorption value of a reaction product by using a full-wavelength ELISA reader.
7. The use method of the TNF-alpha high-sensitivity Elisa detection kit according to claim 6, characterized in that the detection wavelength of the full-wavelength microplate reader is 425 nm.
8. Use of a high sensitivity Elisa assay kit for TNF- α according to any of claims 1 to 5, characterized in that: the kit is applied to the preparation of the kit for detecting the serum of children with in vitro neural developmental diseases.
9. The use of the highly sensitive Elisa kit for TNF- α according to claim 8, characterized in that: the children with the neural developmental disease comprise autistic children.
10. Use of a high sensitivity Elisa assay kit for TNF- α according to any of claims 1 to 5, characterized in that: the kit is applied to the preparation of the kit for detecting the embryo culture solution of the assisted reproductive failure patient.
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Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050272110A1 (en) * | 2004-03-01 | 2005-12-08 | Dr. Andrzej Drukier | Supersensitive immunoassays |
| WO2009063307A2 (en) * | 2007-11-14 | 2009-05-22 | Bio-Technology General (Israel) Ltd. | Inflammatory activity detection method using il-6 |
| CN102175868A (en) * | 2011-01-07 | 2011-09-07 | 中国医学科学院肿瘤研究所 | Application of combined quantitative detection of IL-1[beta], IL-6, IL-10, IL-17, IL-23 and TNF-[alpha] in serum in early-stage prediction of liver cancer |
| CN102495215A (en) * | 2011-12-06 | 2012-06-13 | 任庆远 | Kit for quantitatively detecting tumor necrosis factor alpha |
| CN102539778A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting recombinant human tumor necrosis factor-alpha |
| CN103604933A (en) * | 2013-11-27 | 2014-02-26 | 华中科技大学同济医学院附属同济医院 | Kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and application thereof |
| CN106018825A (en) * | 2016-05-23 | 2016-10-12 | 黄官友 | Method for improving test-tube baby clinical outcome by selecting embryo with good development potential |
| CN106432491A (en) * | 2016-06-03 | 2017-02-22 | 东南大学 | TNF-alpha protein nanobody and coding sequences and application thereof |
| CN106596978A (en) * | 2017-02-20 | 2017-04-26 | 成都朴华科技有限公司 | ECM1 protein antibody and applications thereof, and kit |
| CN106932593A (en) * | 2017-03-03 | 2017-07-07 | 四川大学华西第二医院 | The double-antibody sandwich enzyme-linked immunologic adsorption detection kit as Testing index and its application with NCAM 1 |
| CN109164090A (en) * | 2018-08-08 | 2019-01-08 | 福建医科大学 | The electrochemiluminescdetection detection method and its kit of tumor necrosis factor α |
| CN109789208A (en) * | 2016-07-26 | 2019-05-21 | 新加坡科技研究局 | Anti- Annexin A2 monoclonal antibody |
-
2019
- 2019-10-10 CN CN201910957223.XA patent/CN110672857A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050272110A1 (en) * | 2004-03-01 | 2005-12-08 | Dr. Andrzej Drukier | Supersensitive immunoassays |
| WO2009063307A2 (en) * | 2007-11-14 | 2009-05-22 | Bio-Technology General (Israel) Ltd. | Inflammatory activity detection method using il-6 |
| CN102539778A (en) * | 2010-12-24 | 2012-07-04 | 北京义翘神州生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting recombinant human tumor necrosis factor-alpha |
| CN102175868A (en) * | 2011-01-07 | 2011-09-07 | 中国医学科学院肿瘤研究所 | Application of combined quantitative detection of IL-1[beta], IL-6, IL-10, IL-17, IL-23 and TNF-[alpha] in serum in early-stage prediction of liver cancer |
| CN102495215A (en) * | 2011-12-06 | 2012-06-13 | 任庆远 | Kit for quantitatively detecting tumor necrosis factor alpha |
| CN103604933A (en) * | 2013-11-27 | 2014-02-26 | 华中科技大学同济医学院附属同济医院 | Kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and application thereof |
| CN106018825A (en) * | 2016-05-23 | 2016-10-12 | 黄官友 | Method for improving test-tube baby clinical outcome by selecting embryo with good development potential |
| CN106432491A (en) * | 2016-06-03 | 2017-02-22 | 东南大学 | TNF-alpha protein nanobody and coding sequences and application thereof |
| CN109789208A (en) * | 2016-07-26 | 2019-05-21 | 新加坡科技研究局 | Anti- Annexin A2 monoclonal antibody |
| CN106596978A (en) * | 2017-02-20 | 2017-04-26 | 成都朴华科技有限公司 | ECM1 protein antibody and applications thereof, and kit |
| CN106932593A (en) * | 2017-03-03 | 2017-07-07 | 四川大学华西第二医院 | The double-antibody sandwich enzyme-linked immunologic adsorption detection kit as Testing index and its application with NCAM 1 |
| CN109164090A (en) * | 2018-08-08 | 2019-01-08 | 福建医科大学 | The electrochemiluminescdetection detection method and its kit of tumor necrosis factor α |
Non-Patent Citations (5)
| Title |
|---|
| ELIF BURCU AYDIN 等: "A highly sensitive immunosensor based on ITO thin films covered by a new semi-conductive conjugated polymer for the determination of TNF alpha in human saliva and serum samples", 《BIOSENSORS AND BIOELECTRONICS》 * |
| LAILA Y. AL-AYADHI 等，: "Pro-inflammatory cytokines in autistic children in central Saudi Arabia", 《NEUROSCIENCES》 * |
| LINAN SONG 等: "Single molecule measurements of tumor necrosis factor α and interleukin-6 in the plasma of patients with Crohn"s disease", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| 李中燕等: "麻黄碱对TNF-α诱导人支气管上皮细胞eotaxin表达的影响", 《重庆医学》 * |
| 罗丽荣: ""化学发光成像分析的研究"", 《中国优秀博士学位论文全文数据库 工程科技I辑》 * |
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Application publication date: 20200110 |