CN113447660A - Human brain glioma diagnosis ELISA kit and application of cerebrospinal fluid APP - Google Patents
Human brain glioma diagnosis ELISA kit and application of cerebrospinal fluid APP Download PDFInfo
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Abstract
The invention provides a human brain glioma diagnosis ELISA kit and application of human cerebrospinal fluid APP as a marker in human brain glioma diagnosis, and belongs to the technical field of biological medicines. The kit has the advantages of simple operation, stable reagent, good repeatability, strong specificity, high sensitivity and the like, is easy to popularize and apply in a large range, and has wide market prospect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of cerebrospinal fluid APP as a marker in early diagnosis and recurrence diagnosis of human glioma, and a corresponding double-antibody sandwich ELISA detection kit for diagnosis of human glioma.
Background
Brain glioma originates from glial cells, is the most common malignant tumor of the central nervous system (35.2% -61.0% of intracranial tumor and 80% of intracranial malignant tumor), and is classified into WHO I-IV grade according to the malignancy degree. Glioma grows rapidly and has high recurrence rate after operation, and if the glioma cannot be diagnosed as soon as possible and treated in time, the median survival time of a patient does not exceed 15 months. The imaging proves that the brain glioma usually progresses to the middle and late stages during intracranial space occupying lesion, and the pathological examination as the gold standard completely depends on pathological tissues taken out by operation, so that the two methods cannot be used for early diagnosis of the brain glioma and cannot realize early warning. Currently, there is no effective method/tool for early diagnosis and recurrence diagnosis of brain glioma.
Amyloid Precursor Protein (APP) is a highly conserved transmembrane protein with a receptor-like structure, expressed at very low levels in human normal cells, and highly expressed in a variety of human tumor cells. The inventor discovers through clinical research that: the APP is highly expressed in the cerebrospinal fluid of a patient with the brain glioma as well as in the tissue of the human brain glioma, the protein level of the APP is positively correlated with the malignancy degree of the brain glioma and the recurrence of the glioma, and the APP is negatively correlated with the clinical prognosis of the patient with the brain glioma. Cerebrospinal fluid is easy to obtain, can reflect intracranial lesion nature and disease degree, and is a relatively common clinical examination mode. Therefore, detection of APP protein in cerebrospinal fluid can be used for early diagnosis and relapse diagnosis of brain glioma.
Disclosure of Invention
Based on the problems in the prior art, the invention provides the application of cerebrospinal fluid APP as a marker for early diagnosis and relapse diagnosis of glioma, and provides an ELISA kit for early diagnosis and relapse diagnosis of glioma.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an ELISA kit for diagnosing human brain glioma comprises a human cerebrospinal fluid APP recombinant monoclonal antibody.
According to the scheme, the human brain glioma diagnosis ELISA kit comprises an ELISA plate with a solid-phase coated human cerebrospinal fluid APP recombinant monoclonal antibody, an enzyme-labeled antibody working solution, a conjugate diluent, a washing solution, a developing solution ABTS substrate, a stop solution, a positive control and a negative control.
According to the scheme, the enzyme-labeled antibody working solution is Horse Radish Peroxidase (HRP) -labeled human APP recombinant monoclonal antibody reaction solution; the conjugate diluent is 0.1% bovine serum albumin-phosphate buffered saline (BSA-PBS); the washing solution is 0.05% Tween 20-phosphate buffered saline (Tween 20-PBS); the color development liquid ABTS substrate is a mixed solution of ABTS, a citric acid buffer solution with pH of 5.0 and final concentration of 0.1M and hydrogen peroxide with final concentration of 3% (ABTS +0.1M, pH of 5.0 and citric acid buffer solution +3% H)2O2) (ii) a The stop solution is 1-2M sulfuric acid; the positive control is human cerebrospinal fluid APP standard protein liquid; the negative control is standard negative cerebrospinal fluid.
The invention also discloses application of the cerebrospinal fluid APP as a brain glioma diagnosis marker.
According to the scheme, the cerebrospinal fluid APP is used as a marker of a double-antibody sandwich method for early diagnosis and relapse diagnosis of the brain glioma.
The invention also discloses application of the cerebrospinal APP antibody in preparation of a kit for diagnosis of human brain glioma.
The invention also provides a double-antibody sandwich method detection method of cerebrospinal fluid APP, which comprises the following steps:
step S1 coating: diluting the human cerebrospinal fluid APP monoclonal antibody with a coating buffer solution, adding the diluted human cerebrospinal fluid APP monoclonal antibody into a pore plate, incubating for 24 hours at 4 ℃, and removing liquid in the pore plate;
step S2 seals: adding the conjugate diluent into a pore plate, standing for reaction, and removing liquid in the pore plate;
step S3 sample addition: respectively adding the human cerebrospinal fluid APP standard protein diluted by the conjugate diluent and the cerebrospinal fluid sample to be detected into a pore plate, standing for reaction, and removing liquid in the pore plate;
step S4 adding an enzyme-labeled antibody: diluting a proper amount of enzyme-labeled antibody working solution to 1000 times by using a conjugate diluent, adding the diluted enzyme-labeled antibody working solution into a pore plate, standing for reaction, and removing liquid in the pore plate;
step S5 color development: adding a developing solution ABTS substrate into the pore plate, incubating, and adding 100ul of stop solution;
step S6, standard curve drawing and statistics: and respectively measuring the OD value of the solution of each hole in the hole plate at the position of 405nm, drawing a standard curve according to the OD value of the standard, checking the content of the sample to be detected on the standard curve, and converting the content of human cerebrospinal fluid APP in the sample.
According to the above scheme, the standing reaction in the steps S2-S4 refers to standing at 37 ℃ for 2 hours; the step of removing the liquid in the pore plate in the steps S1-S4 refers to throwing off the liquid in each pore, washing the pore plate 3-5 times by using the washing liquid, and then drying the washing liquid.
According to the scheme, the human cerebrospinal fluid APP monoclonal antibody is diluted to 1 mu g/ml in the step S1; the pore plate is a 96 pore plate; the addition amount of the corresponding reagent added to the well plate in the steps S1-S5 is 100. mu.l/well.
According to the above scheme, the incubation in the step S5 is carried out for 15-20 minutes at room temperature in the absence of light.
The invention has the beneficial effects that:
1. the invention provides a novel early diagnosis and recurrence diagnosis marker of brain glioma, namely cerebrospinal Amyloid Precursor Protein (APP), the level of the cerebrospinal APP is a better detection index of the brain glioma, and the marker can be used as a clinical auxiliary diagnosis means of the brain glioma, and particularly has important clinical application value for early disease early warning, early diagnosis and recurrence diagnosis of the brain glioma.
2. The cerebral glioma diagnostic kit provided by the invention can be used for quickly and accurately detecting the APP protein in cerebrospinal fluid, can be used for quickly and auxiliarily diagnosing cerebral gliomas at each stage (especially at the early stage and after operation), and has important clinical application value. The kit has the advantages of simple operation, stable reagent, good repeatability, strong specificity, high sensitivity and the like, is easy to popularize and apply in a large range, and has wide market prospect.
Drawings
FIG. 1, example APP protein levels in cerebrospinal fluid under different conditions or before and after treatment of brain gliomas.
Detailed Description
The technical solution of the present invention will be described below with reference to specific examples.
The first embodiment is as follows: an ELISA kit for diagnosing human brain glioma.
A human brain glioma diagnostic ELISA kit, comprising:
1. a 96-hole enzyme label plate coated with a human cerebrospinal fluid APP recombinant monoclonal antibody in a solid phase manner;
2. enzyme-labeled antibody working solution: horse Radish Peroxidase (HRP) labeled human APP recombinant monoclonal antibody reaction solution;
3. conjugate dilution: 0.1% BSA-PBS (0.1% bovine serum albumin-phosphate buffered saline);
4. washing liquid: 0.05% Tween20-PBS (0.05% Tween 20-phosphate buffered saline);
5. developing solution ABTS substrate: ABTS +0.1M pH5.0 citric acid buffer +3% H2O2(ABTS, mixture of 0.1M citrate buffer at pH5.0 and 3% hydrogen peroxide at final concentration);
6. stopping liquid: 1-2M sulfuric acid;
7. positive control: human cerebrospinal fluid APP standard protein fluid;
8. negative control: standard negative cerebrospinal fluid.
Example two: a double-antibody sandwich method detection method of cerebrospinal fluid APP.
A double-antibody sandwich method detection method for cerebrospinal fluid APP comprises the following steps:
step S1 coating: diluting the human cerebrospinal fluid APP monoclonal antibody to 1 mu g/ml by using a coating buffer solution, adding the diluted human cerebrospinal fluid APP monoclonal antibody into a 96-well plate, incubating for 24 hours at 4 ℃, throwing away liquid in each well, washing the 96-well plate for 5 times by using a washing solution, and then spin-drying the washing solution;
step S2 seals: adding 100 μ l/well of conjugate diluent into 96-well plate, standing at 37 deg.C for 2 hr, discarding the liquid in each well, washing the 96-well plate with washing solution for 5 times, and spin-drying the washing solution;
step S3 sample addition: respectively adding the human cerebrospinal fluid APP standard protein diluted by the conjugate diluent and a cerebrospinal fluid sample to be detected into a pore plate, standing for 2 hours at the temperature of 37 ℃, throwing away liquid in each pore, washing the 96 pore plate for 5 times by using washing liquid, and then spin-drying the washing liquid;
step S4 adding an enzyme-labeled antibody: diluting a proper amount of enzyme-labeled antibody working solution to 1000 times by using a conjugate diluent, adding the diluted solution into a 96-well plate, placing the plate at the temperature of 37 ℃ for 2 hours at the concentration of 100 mu l/well, throwing away liquid in each well, washing the 96-well plate for 5 times by using a washing solution, and then spin-drying the washing solution;
step S5 color development: adding a developing solution ABTS substrate into a 96-well plate, incubating for 20 minutes at room temperature in a dark place with 100 mu l/well, and adding 100ul of a stop solution;
step S6, standard curve drawing and statistics: and measuring the OD value of the solution of each hole in the hole plate at the position of 405nm by using a microplate reader, drawing a standard curve according to the OD value of the standard, checking the content of the sample to be detected on the standard curve, and converting the content of human cerebrospinal fluid APP in the sample.
Example three: testing the application of cerebrospinal fluid APP as a diagnostic marker for brain gliomas.
Selection of test samples:
1. the age is locked at 18-80 years;
2. normal 3 people who included no clinical symptoms and signs and were confirmed by imaging to be free of intracranial disease; patients with 3 clinically diagnosed migraine, 5 primary epilepsy, 4 autoimmune encephalitis, 4 bacterial meningitis, 4 viral meningitis patients; patients who are clinically diagnosed as brain tumors and are further diagnosed as brain gliomas in the pathology during operation comprise 6 persons, 8 persons, 10 persons and 9 persons at grade I, 3 persons and 2 persons at grade II, 7 days after primary brain gliomas operation and 3 persons, 6 persons, 8 persons and 6 persons at grade IV in recurrent brain gliomas.
Excluding gestation and lactation; eliminating the remarkable abnormality of the liver and kidney function and the blood coagulation function; eliminating organ diseases such as severe infection and severe cardiopulmonary distress; eliminating patients who can not or can not cooperate with lumbar puncture; excluding intracranial disease patients unrelated to the disease species of the study;
the test method comprises the following steps:
collecting cerebrospinal fluid of a test sample through lumbar puncture, detecting the content of APP protein in the cerebrospinal fluid by using the kit in the first embodiment and the detection method in the second embodiment, and comparing the levels of the APP protein in each group of cerebrospinal fluid through SPSS 12.0 software single-factor analysis of variance (One-Way).
And (3) analyzing a detection result:
table 1: APP protein level in cerebrospinal fluid (x + -s) for different diseases
And (4) conclusion: as shown in table 1 and fig. 1:
1. cerebrospinal fluid APP protein level can be used as early diagnosis marker of brain glioma: APP protein levels are lower in the cerebrospinal fluid of normal persons, migraine, epilepsy, autoimmune encephalitis, bacterial meningitis, viral meningitis patients, and are significantly elevated in the cerebrospinal fluid of brain glioma (level i-iv) patients;
2. cerebrospinal fluid APP protein levels are positively correlated with brain glioma malignancy: the higher the malignancy of brain glioma, the higher the cerebrospinal fluid APP protein level;
3. decreased cerebrospinal fluid APP protein levels reflect brain glioma resection: the level of APP protein in cerebrospinal fluid of a patient after brain glioma surgery is obviously reduced;
4. elevated cerebrospinal fluid APP protein levels are predictive of brain glioma recurrence: the APP protein level in cerebrospinal fluid of a patient with recurrent brain glioma is obviously increased.
The above embodiments are only used for illustrating but not limiting the technical solutions of the present invention, and although the above embodiments describe the present invention in detail, those skilled in the art should understand that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and any modifications and equivalents may fall within the scope of the claims.
Claims (10)
1. A human brain glioma diagnosis ELISA kit is characterized by comprising a human cerebrospinal fluid APP recombinant monoclonal antibody.
2. The human brain glioma diagnostic ELISA kit of claim 1, which comprises an ELISA plate with a solid phase coated with a human cerebrospinal fluid APP recombinant monoclonal antibody, an enzyme-labeled antibody working solution, a conjugate diluent, a washing solution, a developing solution ABTS substrate, a stop solution, a positive control and a negative control.
3. The human brain glioma diagnostic ELISA kit of claim 2 wherein: the enzyme-labeled antibody working solution is a horse radish peroxidase-labeled human APP recombinant monoclonal antibody reaction solution; the conjugate diluent is 0.1% bovine serum albumin-phosphate buffered saline; the washing solution is 0.05% of Tween 20-phosphate buffer salt solution; the color development liquid ABTS substrate is a mixed solution of ABTS, a citric acid buffer solution with the pH value of 5.0 and the final concentration of 0.1M and hydrogen peroxide with the final concentration of 3 percent; the stop solution is 1-2M sulfuric acid; the positive control is human cerebrospinal fluid APP standard protein liquid; the negative control is standard negative cerebrospinal fluid.
4. Application of cerebrospinal fluid APP as a brain glioma diagnosis marker.
5. The use according to claim 4, characterized by the use of cerebrospinal APP as marker for the double antibody sandwich method for early diagnosis and relapse diagnosis of brain gliomas.
6. The application of cerebrospinal APP antibody in the preparation of a kit for diagnosing human brain glioma.
7. A double-antibody sandwich method detection method for cerebrospinal fluid APP is characterized by comprising the following steps:
step S1 coating: diluting the human cerebrospinal fluid APP monoclonal antibody with a coating buffer solution, adding the diluted human cerebrospinal fluid APP monoclonal antibody into a pore plate, incubating for 24 hours at 4 ℃, and removing liquid in the pore plate;
step S2 seals: adding the conjugate diluent into a pore plate, standing for reaction, and removing liquid in the pore plate;
step S3 sample addition: respectively adding the human cerebrospinal fluid APP standard protein diluted by the conjugate diluent and the cerebrospinal fluid sample to be detected into a pore plate, standing for reaction, and removing liquid in the pore plate;
step S4 adding an enzyme-labeled antibody: diluting a proper amount of enzyme-labeled antibody working solution to 1000 times by using a conjugate diluent, adding the diluted enzyme-labeled antibody working solution into a pore plate, standing for reaction, and removing liquid in the pore plate;
step S5 color development: adding a developing solution ABTS substrate into the pore plate, incubating, and adding 100ul of stop solution;
step S6, standard curve drawing and statistics: and respectively measuring the OD value of the solution of each hole in the hole plate at the position of 405nm, drawing a standard curve according to the OD value of the standard, checking the content of the sample to be detected on the standard curve, and converting the content of human cerebrospinal fluid APP in the sample.
8. The method for detecting cerebrospinal APP by the double antibody sandwich method according to claim 7, wherein the standing reaction in steps S2-S4 is at 37 ℃ for 2 hours; the step of removing the liquid in the pore plate in the steps S1-S4 refers to throwing off the liquid in each pore, washing the pore plate 3-5 times by using the washing liquid, and then drying the washing liquid.
9. The method for detecting cerebrospinal fluid APP by the double antibody sandwich method according to claim 7, wherein the human cerebrospinal fluid APP monoclonal antibody is diluted to 1 μ g/ml in step S1; the pore plate is a 96 pore plate; the addition amount of the corresponding reagent added to the well plate in the steps S1-S5 is 100. mu.l/well.
10. The method for detecting cerebrospinal APP by the double antibody sandwich method according to claim 7, wherein the incubation in step S5 is performed at room temperature in the absence of light for 15-20 minutes.
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