CN112462063A - Neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent and preparation method thereof - Google Patents
Neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent and preparation method thereof Download PDFInfo
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- CN112462063A CN112462063A CN202010721475.5A CN202010721475A CN112462063A CN 112462063 A CN112462063 A CN 112462063A CN 202010721475 A CN202010721475 A CN 202010721475A CN 112462063 A CN112462063 A CN 112462063A
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Abstract
The invention discloses a neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent and a preparation method thereof, belonging to the technical field of in vitro diagnosis, wherein the reagent is formed by sequentially sticking a coating film, absorbent paper and a glass fiber film coated with a gold-labeled antibody; the coating film is prepared by the following method: diluting PGP9.5 antigen to a concentration of 5-20 mug/mL, spraying or coating on a coating film, sealing after air drying, and sealing for later use; the gold-labeled antibody is prepared by the following method: adding PGP9.5 antibody into colloidal gold according to the ratio of 9-11 mu g/mL, uniformly mixing, standing, centrifuging, removing supernatant, and washing and dissolving the precipitate for later use. The invention solves the technical problems of long time and labor consumption in the conventional method for measuring the PGP9.5 protein antibody measurement sample, is convenient and quick to operate, does not need large instruments and consumables, saves equipment and labor cost, realizes field detection, has strong anti-interference capability, and can judge and monitor the health condition of a patient.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a 9.5 antibody detection reagent for immunoassay of a neuron cytoplasmic protein gene product by means of colloidal gold labeling and color development and a preparation method thereof.
Background
Lung cancer is one of the most common malignancies in the world today. In the early stages of tumor development, the body's immune system recognizes proteins, i.e., Tumor Associated Antigens (TAAs), that are abnormally expressed in tumor cells. Changes in TAAs such as over-expression, mutation, misfolding, abnormal degeneration, etc., can lead to a cancer patient developing a self-reactive immune response and producing autoantibodies against these antigens, with a small amount of TAAs stimulation leading to a large amount of autoantibodies produced by B cells. Therefore, the autoantibodies for detecting TAAs can be used as molecular markers for early diagnosis of lung cancer.
At present, lung cancer screening mainly combines a plurality of detection means such as a low-dose computed tomography (LDCT) technology of a breast, detection of blood molecular markers, sputum cytology detection, a fiberbronchoscope and the like, but the diagnosis effect on early lung cancer is not good enough.
The expression of the neuron cytoplasmic protein gene product 9.5(PGP9.5) is independent of the differentiation of nerves, is closely related to the pathological stage of tumors, and has a large amount of expression in primary lung cancer. PGP9.5 is a neural specific peptide, ubiquitin hydrolase, widely expressed in various stages of neuronal differentiation, and ubiquitination of cellular proteins and targeted degradation thereof by ubiquitin-mediated proteolytic enzymes is an important mechanism for regulating cell cycle genes. In tumors, PGP9.5 increased the de-ubiquitination of cyclin possibly leading to uncontrolled growth of somatic cells.
The current method for measuring the PGP9.5 protein antibody is an enzyme-linked immunosorbent assay, the time for measuring a sample by the enzyme-linked immunosorbent assay is long, the operation is time-consuming and labor-consuming, although quantitative measurement can be realized, the field detection of a POCT reagent cannot be realized, and mutual interference cannot be realized. Therefore, a convenient and fast PGP9.5 protein antibody detection reagent is urgently needed, field detection is achieved, and judgment and monitoring on the health condition of patients are carried out.
Disclosure of Invention
Aiming at the technical problems of long time for measuring a sample of the PGP9.5 protein antibody and time and labor consumption in operation, the invention provides a reagent suitable for detecting the PGP9.5 antibody by a colloidal gold chromatography, which realizes on-site detection, does not need large instruments and consumables, has strong anti-interference capability and can judge and monitor the health condition of a patient.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention firstly provides a neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent, which is formed by sequentially sticking a coating film, absorbent paper and a glass fiber film coated with a gold-labeled antibody;
the coating film is prepared by the following method: diluting PGP9.5 antigen to a concentration of 5-20 mug/mL, spraying or coating on a coating film, sealing after air drying, and sealing for later use;
the gold-labeled antibody is prepared by the following method: adding PGP9.5 antibody into colloidal gold according to the ratio of 9-11 mu g/mL, uniformly mixing, standing, centrifuging, removing supernatant, and washing and dissolving the precipitate for later use.
Preferably, the PGP9.5 antigen is a purified PGP9.5 genetically engineered recombinant antigen, or a synthetic polypeptide, or an affinity purified antigen.
Preferably, the purity of the PGP9.5 antigen is more than or equal to 95 percent.
Preferably, the PGP9.5 antigen is diluted to a concentration of 8. mu.g/mL and sprayed or coated on a coating film.
Preferably, the gold-labeled antibody is added to the PGP9.5 antibody at 10. mu.g/mL of colloidal gold.
Preferably, the glass fiber membrane coated with the gold-labeled antibody is obtained by the following method: and uniformly spreading the prepared gold-labeled antibody on a glass fiber membrane, spreading the prepared gold-labeled antibody on the glass fiber membrane in a concentration of 4-8 square centimeters per milliliter of solution, freeze-drying, sealing, and standing at 4 ℃ for later use.
The invention also provides a preparation method of the neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent, which comprises the following steps:
s1, preparation of coating film: diluting the coating antigen with a coating buffer solution to a concentration of 5-20 mu g/mL, spraying or coating the coating antigen on a coating film, wherein the liquid spraying amount is 20 microliters/35 centimeters, airing, soaking in a sealing solution at 37 ℃ for 60 minutes, taking out, drying at 37 ℃ for 2 hours, and sealing the bag for later use; the coating buffer solution is 0.05M sulfate buffer solution with pH9.6; the confining liquid is 0.01M phosphate buffer solution with pH7.0 and containing 2% BSA and 2% skimmed milk;
s2, preparing a glass fiber membrane coated with gold-labeled antibodies: adjusting the pH value of the colloidal gold to 7.5 by using 0.1M potassium carbonate, adding a PGP9.5 antibody into the colloidal gold according to 9-11 mug/mL, uniformly mixing, standing, centrifuging, removing supernatant, washing a precipitate by using a labeled washing solution, and dissolving the precipitate by using a gold-labeled antibody preservation solution with 10% of the initial colloidal gold volume to obtain a gold-labeled antibody; uniformly spreading the prepared gold-labeled antibody on a glass fiber membrane, spreading the gold-labeled antibody on a solution per milliliter by 4-8 square centimeters, freeze-drying, sealing a bag, and standing at 4 ℃ for later use; the labeling washing solution is 0.01M PBS solution with pH7.0 and 2% BSA; the gold-labeled antibody preservation solution is 0.01M PBS (phosphate buffer solution) pH7.0 containing 1% BSA, 0.5% skimmed milk, 5/ten thousand NaN3 and 0.1% Tween-20;
s3, cutting the coating film, the absorbent paper and the glass fiber film coated with the gold-labeled antibody, then pasting the cut glass fiber film on a plastic bottom plate to form a large plate, and cutting the large plate into single human reagents.
Preferably, the PGP9.5 antigen is a PGP9.5 genetically engineered recombinant antigen with a purity of 95% or more, or a synthetic polypeptide, or an affinity purified antigen.
Preferably, the firing method of the colloidal gold comprises the following steps: diluting 1% chloroauric acid to 0.01% with double distilled water, boiling in electric furnace, adding 4 ml 1% disodium citrate per 100 ml 0.01% chloroauric acid, boiling until the liquid is bright red, stopping heating, cooling to room temperature, and supplementing water.
Preferably, the condition of the centrifugation treatment in step S2 is 13000rpm for 30-35 minutes.
Compared with the prior art, the neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent provided by the invention has the beneficial effects that:
1. the diagnosis is rapid, the detection can be completed within 10 minutes, the sensitivity is high, and the anti-interference capability is strong;
2. no instrument and equipment are needed, so that the cost of the instrument and the equipment is saved;
3. the operation is simple and convenient, the operation by professional personnel is not needed, and the labor cost is saved.
Detailed Description
The invention provides a neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent, which is formed by sequentially sticking a coating film, absorbent paper and a glass fiber film coated with a gold-labeled antibody. In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
In this example, purified PGP9.5 genetically engineered recombinant antigen was used as a solid phase, and the indirect method was used to detect whether the serum contained PGP9.5 antibody. The manufacturing method of this example is as follows:
a. antigen selection
The PGP9.5 recombinant antigen expressed by genetic engineering with purity > 95% was used.
b. Preparation of antigen membranes
1. Preparation of coating buffer: 0.05M of sulfate buffer solution with pH of 9.6 is used as a coating solution, and the solution is filtered by a 0.22 mu membrane and kept at 4 ℃ for later use for one week.
2. Preparing a sealing liquid: 0.01M Phosphate Buffer (PBS) with pH7.0 is prepared, 0.22 mu membrane filtration is carried out, and the mixture is kept at 4 ℃ for standby and has one week expiration date. Preparing a closed working solution: 2% BSA, 2% skim milk, 0.01M PBS (pH7.0), 0.22 μmembrane filtration, standing at 4 deg.C for three days.
3. Preparation of a coating film: adjusting a film spraying machine, adjusting the liquid spraying amount to be 20 microliters/35 centimeters, diluting the coating antigen with a coating buffer solution, adjusting the concentration to be 8 micrograms/mL, marking by a machine, separating two lines by 5mm, finely and uniformly drying at room temperature for 20 minutes. Soaking in sealing liquid at 37 deg.C for 60 min, taking out, oven drying at 37 deg.C for two hours, and sealing.
c. Preparation of colloidal gold and colloidal gold-labeled antibody
1. Preparing chloroauric acid: dissolving chloroauric acid with double distilled water to prepare 1% solution, standing at 4 deg.C for three days. 1000mL of 1% chloroauric acid solution formula: 10g of chloroauric acid; 1000mL of double distilled water.
2. Preparation of trisodium citrate: dissolving trisodium citrate with double distilled water to obtain 1% solution, and standing at 4 deg.C for three days. 1000mL of 1% trisodium citrate solution formulation: l0g trisodium citrate; 1000mL of double distilled water.
3. Preparation of 0.1M potassium carbonate: prepared by double distilled water, filtered by a 0.22 mu membrane, and placed at 4 ℃ for later use, and the effective period is one week. 1000mL of 0.1M potassium carbonate solution formula: 13.8g of potassium carbonate; 1000na1 double distilled water.
4.3% PEG-20000 preparation: prepared by double distilled water, filtered by a 0.22 mu membrane, and placed at 4 ℃ for later use, and the effective period is one week. 1000mL 3% PEG solution formulation: 30g PEG-20000; 1000mL of double distilled water.
5. Preparation of a marking washing solution: 2% BSA, 0.01M PBS pH7.0, 0.22 μmembrane filtration, standing at 4 ℃ for two weeks. 1000mL of the washing solution: 20g BSA; 1000mL of 0.01M PBS solution, pH7.0.
6. Preparing a gold-labeled antibody preservation solution: 1% BSA, 0.5% skim milk, 5/ten thousand NaN3, 0.1% Tween-20, 0.01M PBS solution (pH7.0), 0.22 μmembrane filtration, standing at 4 deg.C for fifteen days. 1000mL of gold-labeled antibody preservation solution formula: 10g BSA; 5g of skim milk; 0.5g NaN 3; lmL Tween-20; 1000mL of 0.01M PBS solution, pH7.0.
7. Firing of the colloidal gold: diluting 1% chloroauric acid to 0.01% with double distilled water, boiling in electric furnace, adding 1% trisodium citrate 4 ml per 100 ml chloroauric acid 0.01%, boiling until the liquid is bright red, stopping heating, cooling to room temperature, and supplementing water. The appearance should be pure, transparent and clear without sediment and floating objects.
8. Preparation of gold-labeled antibody: adjusting the pH value of the colloidal gold to 7.5 by 0.1M potassium carbonate, adding PGP9.5 antibody according to 10 micrograms of antibody/milliliter of the colloidal gold, uniformly mixing, standing for 30 minutes, centrifuging at 13000rpm for 34 minutes, removing the supernatant, washing the precipitate twice by using a labeling washing solution, finally removing the supernatant, dissolving the precipitate by using a gold-labeled antibody preservation solution with one tenth of the volume of the initial colloidal gold, standing for later use at 4 ℃, and prolonging the period for one week.
d. Freeze-drying of gold-labeled antibodies
Spreading the marked colloidal gold on glass fiber membrane uniformly, spreading 6 square cm per ml solution, freeze drying, sealing, and standing at 4 deg.C.
e. Production of test paper boards
1. Cutting a gold-labeled antibody glass fiber membrane: and cutting the gold-labeled antibody glass fiber film according to the width determined by the dripping experiment, and placing the gold-labeled antibody glass fiber film in a dry room for later use.
2. Cutting the absorbent paper: the absorbent paper is cut into 35 cm long and 4.2 cm wide by a paper cutter and placed in a drying room for later use.
3. Cutting the glass fiber film: the glass fiber film was cut into strips of 35 cm in length and 2 cm in width, and placed in a dry room for future use.
4. And (3) pasting the large plate: the coating film, the absorbent paper, the freeze-dried gold-labeled antibody and the glass fiber film are adhered to a plastic bottom plate as required to form a large plate. The temperature of the assembly workshop is controlled at 25 ℃ and the humidity is controlled at 20-30%.
f. Slitting
The large plate is cut into single persons by a slitter, the width of each person is 2.5 mm, random sampling inspection is carried out, indoor quality control can be detected by sensitivity, the color development degree of a strip reaches a plus sign, no non-specific strip exists, and the strip can pass through Panel of Ministry of health.
g. Assembling and packaging
Packaging 50 cut test strips and a bag of drying agent in an aluminum foil plastic bag, putting the aluminum foil plastic bag into a test kit,
one instruction book per box. Storing at 4-25 deg.C in dark place without freezing.
The reagent of example 1 was used to test serum samples of 100 suspected lung cancer patients, and the enzyme-linked immunosorbent assay (ELISA) was used as a control to perform sensitivity detection (see Table 1) and anti-interference detection (see Table 2) on the present invention. The result shows that the sensitivity of the invention reaches 98.6 percent (75/76), and the anti-interference capability is strong.
TABLE 1100 serum sample PGP9.5 antibody test results of suspected lung cancer patients
TABLE 2 anti-interference results
Example 2
In this example, the PGP9.5 synthesized polypeptide was used as a solid phase, and the presence or absence of PGP9.5 antibody in serum was determined by the indirect method. The manufacturing method of this example is substantially the same as that of example 1, except that:
1. the PGP9.5 synthetic polypeptide was used as the coating antigen.
2. Preparation of coating film: adjusting a film spraying machine, adjusting the liquid spraying amount to be 20 microliters/35 centimeters, diluting the coating antigen with a coating buffer solution, adjusting the concentration to be 5 micrograms/mL, scribing by a machine, separating two lines by 5mm, thinning and uniformly, and airing for 20 minutes at room temperature. Soaking in sealing liquid at 37 deg.C for 60 min, taking out, oven drying at 37 deg.C for two hours, and sealing.
3. Preparation of gold-labeled antibody: adjusting the pH value of the colloidal gold to 7.5 by 0.1M potassium carbonate, adding PGP9.5 antibody according to 11 micrograms of antibody/milliliter of the colloidal gold, uniformly mixing, standing for 30 minutes, centrifuging at 13000rpm for 30 minutes, removing the supernatant, washing the precipitate twice by using a labeling washing solution, finally removing the supernatant, dissolving the precipitate by using a gold-labeled antibody preservation solution with one tenth of the volume of the initial colloidal gold, standing at 4 ℃ for later use, and prolonging the period for one week.
4. Freeze-drying of gold-labeled antibody: the marked colloidal gold is evenly spread on a glass fiber membrane, each milliliter of solution is spread with 4 square centimeters, and the solution is frozen, dried, sealed and placed at 4 ℃ for standby.
The reagent prepared in example 2 was used to test serum samples of 100 suspected lung cancer patients, and the enzyme-linked immunosorbent assay (ELISA) was used as a control to perform sensitivity detection (see Table 3) and anti-interference detection (see Table 4) on the present invention. The result shows that the sensitivity of the invention reaches 100.0 percent (76/76), and the anti-interference capability is strong.
PGP9.5 antibody detection results of serum samples of suspicious lung cancer patients in Table 3100 cases
TABLE 4 anti-interference results
Example 3
In this example, PGP9.5 affinity purified antigen was used as a solid phase, and the indirect method was used to determine whether the serum contained PGP9.5 antibody. The manufacturing method of this example is basically the same as that of example 1, except for the following points:
1. PGP9.5 affinity purified antigen was used as the coating antigen.
2. Preparation of coating film: adjusting a film spraying machine, adjusting the liquid spraying amount to be 20 microliter/35 cm, diluting the coating antigen with a coating buffer solution, adjusting the concentration to be 20 microgram/mL, scribing by a machine, separating two lines by 5mm, finely and uniformly drying at room temperature for 20 minutes. Soaking in sealing liquid at 37 deg.C for 60 min, taking out, oven drying at 37 deg.C for two hours, and sealing.
3. Preparation of gold-labeled antibody: adjusting the pH value of the colloidal gold to 7.5 by 0.1M potassium carbonate, adding PGP9.5 antibody according to 9 mu g antibody/ml colloidal gold, uniformly mixing, standing for 30 minutes, centrifuging at 13000rpm for 30 minutes, discarding the supernatant, washing the precipitate twice by using a labeling washing solution, discarding the supernatant for the last time, dissolving the precipitate by using a gold-labeled antibody preservation solution with one tenth of the volume of the initial colloidal gold, standing at 4 ℃ for later use, and prolonging the period for one week.
4. Freeze-drying of gold-labeled antibody: the marked colloidal gold is evenly spread on a glass fiber membrane, 8 square centimeters of solution are spread in each milliliter, and the solution is frozen, dried, sealed and placed at 4 ℃ for standby.
The reagent prepared in example 3 was used to test serum samples of 100 suspected lung cancer patients, and the enzyme-linked immunosorbent assay (ELISA) was used as a control to perform sensitivity detection (see Table 5) and anti-interference detection (see Table 6) on the present invention. The result shows that the sensitivity of the invention reaches 98.6 percent (75/76), and the anti-interference capability is strong.
TABLE 5100 serum sample PGP9.5 antibody detection results of suspected lung cancer patients
TABLE 6 anti-interference results
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, but these modifications or substitutions do not substantially depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent is characterized by being formed by sequentially sticking a coating film, absorbent paper and a glass fiber film coated with a gold-labeled antibody;
the coating film is prepared by the following method: diluting PGP9.5 antigen to a concentration of 5-20 mug/mL, spraying or coating on a coating film, sealing after air drying, and sealing for later use;
the gold-labeled antibody is prepared by the following method: adding PGP9.5 antibody into colloidal gold according to the ratio of 9-11 mu g/mL, uniformly mixing, standing, centrifuging, removing supernatant, and washing and dissolving the precipitate for later use.
2. The reagent for detecting 9.5 antibody colloidal gold of gene product of neuronal cytosolic protein according to claim 1, wherein said PGP9.5 antigen is purified PGP9.5 genetically engineered recombinant antigen, or synthetic polypeptide, or affinity purified antigen.
3. The reagent for detecting 9.5 antibody colloidal gold of neuronal cytoplasmic protein gene product according to claim 1 or 2, wherein the purity of the PGP9.5 antigen is not less than 95%.
4. The reagent for detecting 9.5 antibody colloidal gold of a gene product of neuronal cytosolic protein according to claim 1, wherein said PGP9.5 antigen is diluted to a concentration of 8 μ g/mL and sprayed or coated on the coating film.
5. The reagent for detecting 9.5 antibody colloidal gold of neuronal cytosolic protein gene product according to claim 1, wherein said gold-labeled antibody is added to PGP9.5 antibody at 10 μ g/mL colloidal gold.
6. The reagent for detecting the antibody colloidal gold of the gene product of neuronal cytosolic protein 9.5 according to claim 1 or 5, wherein said glass fiber membrane coated with the gold-labeled antibody is obtained by: and uniformly spreading the prepared gold-labeled antibody on a glass fiber membrane, spreading the prepared gold-labeled antibody on the glass fiber membrane in a concentration of 4-8 square centimeters per milliliter of solution, freeze-drying, sealing, and standing at 4 ℃ for later use.
7. The method for preparing the reagent for detecting the colloidal gold of the 9.5 antibody of the gene product of neuronal cytoplasmic protein according to any of claims 1 to 6, comprising the steps of:
s1, preparation of coating film: diluting the coating antigen with a coating buffer solution to a concentration of 5-20 mu g/mL, spraying or coating the coating antigen on a coating film, wherein the liquid spraying amount is 20 microliters/35 centimeters, airing, soaking in a sealing solution at 37 ℃ for 60 minutes, taking out, drying at 37 ℃ for 2 hours, and sealing the bag for later use; the coating buffer solution is 0.05M sulfate buffer solution with pH9.6; the confining liquid is 0.01M phosphate buffer solution with pH7.0 and containing 2% BSA and 2% skimmed milk;
s2, preparing a glass fiber membrane coated with gold-labeled antibodies: adjusting the pH value of the colloidal gold to 7.5 by using 0.1M potassium carbonate, adding a PGP9.5 antibody into the colloidal gold according to 9-11 mug/mL, uniformly mixing, standing, centrifuging, removing supernatant, washing precipitate by using a labeling washing solution, and dissolving the precipitate by using a gold-labeled antibody preservation solution with 10% of the initial colloidal gold volume to obtain a gold-labeled antibody; uniformly spreading the prepared gold-labeled antibody on a glass fiber membrane, spreading the gold-labeled antibody on a solution per milliliter by 4-8 square centimeters, freeze-drying, sealing, and standing at 4 ℃ for later use; the labeling washing solution is 0.01M PBS solution with pH7.0 and 2% BSA; the gold-labeled antibody preservation solution is 0.01M PBS (phosphate buffer solution) with pH7.0, and contains 1% BSA, 0.5% skimmed milk, 5/ten thousand NaN3 and 0.1% Tween-20;
s3, cutting the coating film, the absorbent paper and the glass fiber film coated with the gold-labeled antibody, then pasting the cut glass fiber film on a plastic bottom plate to form a large plate, and cutting the large plate into individual human reagents.
8. The method for preparing the reagent for detecting 9.5 antibody colloidal gold of neuronal cytoplasmic protein gene product according to claim 7, wherein the PGP9.5 antigen is a PGP9.5 genetically engineered recombinant antigen with a purity of 95% or more, or a synthetic polypeptide, or an affinity purified antigen.
9. The method for preparing the colloidal gold reagent for detecting 9.5 antibody of neuronal cytoplasmic protein gene product according to claim 7, wherein the firing method of the colloidal gold is: diluting 1% chloroauric acid to 0.01% with double distilled water, boiling in electric furnace, adding 1% trisodium citrate 4 ml per 100 ml chloroauric acid 0.01%, boiling until the liquid is bright red, stopping heating, cooling to room temperature, and supplementing water.
10. The method for preparing the reagent for detecting colloidal gold as claimed in claim 7, wherein the centrifugation in step S2 is carried out at 13000rpm for 30-35 minutes.
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Cited By (2)
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| CN113759112B (en) * | 2021-09-27 | 2022-05-24 | 北京美联泰科生物技术有限公司 | Freeze-drying method of brain specific protein product 9.5 |
| CN114563570A (en) * | 2022-01-29 | 2022-05-31 | 北京美联泰科生物技术有限公司 | Application of signal amplification technology in PGP9.5 detection kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453589A (en) * | 2003-06-18 | 2003-11-05 | 广州万孚生物技术有限公司 | Reagent for colloidal gold chromatographic analysis of SARS coronavirus antibody |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| CN104987366A (en) * | 2014-08-21 | 2015-10-21 | 上海市东方医院 | Rheumatoid arthritis autoantibody conjugated antigen and applications thereof |
| CN107561288A (en) * | 2017-08-30 | 2018-01-09 | 上海市肺科医院 | A kind of pulmonary cancer diagnosis kit for detecting blood autoantibody and its application |
| CN109001472A (en) * | 2018-08-03 | 2018-12-14 | 迪瑞医疗科技股份有限公司 | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method |
-
2020
- 2020-07-24 CN CN202010721475.5A patent/CN112462063A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453589A (en) * | 2003-06-18 | 2003-11-05 | 广州万孚生物技术有限公司 | Reagent for colloidal gold chromatographic analysis of SARS coronavirus antibody |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| CN104987366A (en) * | 2014-08-21 | 2015-10-21 | 上海市东方医院 | Rheumatoid arthritis autoantibody conjugated antigen and applications thereof |
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