CN114184785A - Kit for identifying cell species based on colloidal gold method - Google Patents
Kit for identifying cell species based on colloidal gold method Download PDFInfo
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- CN114184785A CN114184785A CN202111402490.4A CN202111402490A CN114184785A CN 114184785 A CN114184785 A CN 114184785A CN 202111402490 A CN202111402490 A CN 202111402490A CN 114184785 A CN114184785 A CN 114184785A
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 241000894007 species Species 0.000 claims abstract description 36
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 239000012528 membrane Substances 0.000 claims abstract description 21
- 238000012360 testing method Methods 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000013592 cell lysate Substances 0.000 claims abstract description 14
- 241000283707 Capra Species 0.000 claims abstract description 11
- 238000010521 absorption reaction Methods 0.000 claims abstract description 8
- 239000010931 gold Substances 0.000 claims abstract description 8
- 229910052737 gold Inorganic materials 0.000 claims abstract description 8
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- 239000000243 solution Substances 0.000 claims description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 239000011248 coating agent Substances 0.000 claims description 13
- 238000000576 coating method Methods 0.000 claims description 13
- 238000007865 diluting Methods 0.000 claims description 12
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- 229910021641 deionized water Inorganic materials 0.000 claims description 11
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- 239000000020 Nitrocellulose Substances 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
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Images
Classifications
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
-
- 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
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention provides a kit for identifying cell species based on a colloidal gold method, which comprises a colloidal gold test strip, cell lysate and PBS; the colloidal gold test strip comprises a sample pad, a gold label pad, an NC membrane and a water absorption pad; the gold-labeled pad comprises a colloidal gold-labeled internal reference universal antibody A, and the NC membrane is coated with a secondary goat anti-mouse IgG and an internal reference specific monoclonal antibody B. The sample is absorbed by the sample pad, is combined with the contained gold-labeled species universal internal reference antibody A through the gold-labeled pad, can display a T line when passing through a detection line marked with a species-specific internal reference antibody B, can display a C line when passing through a quality control line marked with a secondary antibody, and judges the species of the sample by observing the color development of the species-specific internal reference antibody.
Description
Technical Field
The invention relates to the technical field of cell species identification, in particular to a kit for identifying cell species based on a colloidal gold method.
Background
At present, the cell species identification is mainly carried out by using a PCR method and utilizing species-specific primers, the PCR-based method is mainly divided into conventional PCR and fluorescent quantitative PCR, both the conventional PCR and the fluorescent quantitative PCR need to design a plurality of pairs of primers for carrying out PCR, and the design of the primers and the experimental operation are complex. The conventional method needs to use EB with carcinogenicity to dye products, and the fluorescent quantitative PCR needs to use fluorescent primers and a fluorescent quantitative PCR instrument, so the cost is higher. Therefore, a detection kit and a detection method which are simple and low in cost are needed.
Disclosure of Invention
In view of the above, the invention provides a kit for identifying cell species based on a colloidal gold method, which is simple to operate, extremely short in detection time, sensitive in reaction, free of special instruments and free of harmful substances.
The technical scheme of the invention is realized as follows: the invention provides a kit for identifying cell species based on a colloidal gold method, which comprises a colloidal gold test strip, cell lysate and PBS; the colloidal gold test strip comprises a sample pad, a gold label pad, an NC membrane and a water absorption pad; the gold-labeled pad comprises a colloidal gold-labeled internal reference universal antibody A, and the NC membrane is coated with a secondary goat anti-mouse IgG and an internal reference specific monoclonal antibody B.
On the basis of the technical scheme, preferably, the preparation method of the internal reference universal antibody A comprises the following steps: selecting universal fragments of different species of reference protein beta-Actin or GAPDH to synthesize corresponding polypeptides, coupling BSA or KLH, immunizing a mouse, preparing a mouse antibody to obtain a reference universal antibody A, and diluting with a diluent for later use.
On the basis of the technical scheme, preferably, the preparation method of the reference-specific monoclonal antibody B comprises the following steps: selecting specific fragments of different species of reference protein beta-Actin or GAPDH to synthesize corresponding polypeptides, coupling BSA or KLH, immunizing a mouse, preparing a mouse antibody to obtain a reference specific monoclonal antibody B, and diluting with a diluent for later use.
On the basis of the technical scheme, preferably, the diluent consists of the following components in percentage by mass: 0.1-1% of OVA, 0.8% of sodium chloride, 0.1-1% of glacial acetic acid, 0.01-0.1% of sodium azide, 0.02% of potassium chloride, 0.144% of disodium hydrogen phosphate and 0.024% of potassium dihydrogen phosphate, and the solvent is deionized water.
On the basis of the technical scheme, preferably, the reference-specific monoclonal antibody B coated in the nitrocellulose membrane is a detection line.
On the basis of the technical scheme, preferably, the goat anti-mouse IgG secondary antibody coated in the nitrocellulose membrane is a quality control line.
On the basis of the technical scheme, preferably, the sample is treated by cell lysis solution, and is dripped onto the sample pad after being diluted by PBS.
On the basis of the technical scheme, preferably, the cell lysate consists of NP40 with the mass fraction of 0.2-2%, Tris-HCL with the final concentration of 10-100mmol/L, sodium chloride with the mass fraction of 0.8%, PMSF with the final concentration of 0.05-0.5mmol/L, pepsin inhibitor with the final concentration of 0.2-2mmol/L, leupeptin with the final concentration of 0.2-2mmol/L and aprotinin with the final concentration of 0.2-2mmol/L, and the solvent is deionized water.
The invention also provides a preparation method of the kit for identifying the cell species based on the colloidal gold method, which comprises the following steps:
s1, diluting the colloidal gold-labeled internal reference general antibody A by 5 times with a gold-labeled antibody storage solution, adding a trehalose aqueous solution with the mass fraction of 5%, and coating the gold-labeled antibody on a gold-labeled pad according to the amount of 10 mu L/cm to prepare the gold-labeled pad containing the colloidal gold-labeled internal reference general antibody A;
s2, coating an internal reference specificity monoclonal antibody B and a secondary goat anti mouse IgG on the detection line and the quality control line of the NC membrane respectively to prepare a coated nitrocellulose membrane;
and S3, sequentially adhering the water absorption pad, the NC film, the gold label pad and the sample pad from top to bottom by taking the lining plate as a bottom plate to finish assembly.
On the basis of the above technical scheme, preferably, the gold-labeled antibody storage solution is composed of the following components in parts by mass: 1% trehalose, 0.5% Tween-20 and 0.02% NaN3The pH value is 8.2, and the solvent is deionized water.
Compared with the prior art, the kit for identifying the cell species based on the colloidal gold method has the following beneficial effects:
(1) the sample is absorbed by the sample pad, is combined with the contained gold-labeled species universal internal reference antibody A through the gold-labeled pad, can display a T line when passing through a detection line marked with a species-specific internal reference antibody B, can display a C line when passing through a quality control line marked with a secondary antibody, and judges the species of the sample by observing the color development of the species-specific internal reference antibody.
(2) The samples of the invention are not limited to human, rat, mouse, hamster, pig and chicken species, and may be increased or decreased as desired; also, the detection of tissue samples is not limited to cell samples.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the results of the detection according to the embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious 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 obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
A kit for identifying cell species based on a colloidal gold method comprises a colloidal gold test strip, cell lysate and PBS; the colloidal gold test strip comprises a sample pad, a gold label pad, an NC membrane and a water absorption pad; the gold-labeled pad comprises a colloidal gold-labeled internal reference universal antibody A, and the NC membrane is coated with a secondary goat anti-mouse IgG and an internal reference specific monoclonal antibody B.
Preparing an internal reference general antibody A/an internal reference specific monoclonal antibody B:
s1, respectively selecting universal fragments/specific fragments of different species (human, rat, mouse, hamster, pig and chicken) according to corresponding NCBI information, synthesizing corresponding polypeptides, and immunizing a mouse after coupling BSA;
s2, when the serum titer of the mouse reaches more than 1:20000, taking the spleen of the mouse and SP2/0 to fuse by a PEG method to obtain corresponding hybridoma cells;
s3, screening the hybridoma cell supernatant, and selecting positive hybridoma cells;
s4, injecting the positive hybridoma cells into the abdominal cavity of the mouse to form ascites, and centrifuging the ascites to obtain a supernatant;
s5, purifying the ascites supernatant by protein A/G to obtain the internal reference general antibody A/internal reference specific monoclonal antibody B.
The diluent consists of the following components in percentage by mass: 0.1-1% of OVA, 0.8% of sodium chloride, 0.1-1% of glacial acetic acid, 0.01-0.1% of sodium azide, 0.02% of potassium chloride, 0.144% of disodium hydrogen phosphate and 0.024% of potassium dihydrogen phosphate, and the solvent is deionized water.
The PBS consists of the following components in percentage by mass: 0.8 percent of sodium chloride, 0.02 percent of potassium chloride, 0.144 percent of disodium hydrogen phosphate and 0.024 percent of monopotassium phosphate, and the solvent is deionized water.
The cell lysate is composed of NP40 with the mass fraction of 0.2-2%, Tris-HCL with the final concentration of 10-100mmol/L, sodium chloride with the mass fraction of 0.8%, PMSF with the final concentration of 0.05-0.5mmol/L, pepsin inhibitor with the final concentration of 0.2-2mmol/L, leupeptin with the final concentration of 0.2-2mmol/L and aprotinin with the final concentration of 0.2-2mmol/L, and the solvent is deionized water.
The gold-labeled antibody storage solution comprises the following components in parts by mass: 1 percent of the trehalose, and the glucose is added,0.5% Tween-20 and 0.02% NaN3The pH value is 8.2, and the solvent is deionized water.
Antibody gold labeling:
s11, taking 1mL of 1% chloroauric acid solution, adding 99mL of ultrapure water to obtain a chloroauric acid solution with a final concentration of 0.01%, heating to boil, taking 1.6mL of 1% trisodium citrate, rapidly adding into the boiled chloroauric acid solution at one time, continuously heating until the solution is changed from light yellow to blue black, finally changing to bright red, continuing to heat for 5min after the color is stable, cooling at room temperature, and supplementing water to the original volume.
S12, 10mL of the above colloidal gold solution was put into a small clean beaker.
S13, adding 100 mu L of 0.1mol/LK under magnetic stirring2CO3The solution was stirred for 5 min.
S14, dropwise adding 100 mu L of the universal monoclonal antibody for the internal reference, and continuing stirring for 30min after the addition is finished; after 30min, 100 μ L of the solution is taken out and added with NaCl with the mass fraction of 10%, and whether the mark is saturated or not is detected.
S15, adding a BSA aqueous solution with the mass fraction of 15% dropwise to a final concentration of 1%, and continuing stirring for 5 min.
S16, purification: centrifuging at 12000rpm/min at 4 deg.C for 20min, removing supernatant, washing with colloidal gold stock solution for 2 times, and collecting 2 times of centrifugal precipitate.
S17, concentrating: the colloidal gold stock solution was added to a final volume of 1mL, and then stored at 4 ℃.
Coating:
s21, preparing a coating solution: the final concentration of the coating solution is 0.01mol/L PBS and 2.6mg/mL BSA, the pH value is 7.4, and the solvent is deionized water.
S22, coating concentration: the internal reference specificity monoclonal antibody B is diluted to the concentration of 1.5mg/mL by using a coating solution, and the secondary antibody goat anti-mouse IgG is diluted to the concentration of 1mg/mL by using the coating solution.
S23, coating: and coating a secondary goat anti-mouse IgG and an internal reference specific monoclonal antibody B on an NC membrane by using a membrane scribing instrument, wherein the concentrations are respectively 1 mu L/cm, the internal reference specific monoclonal antibody B is used as a T line, the secondary goat anti-mouse IgG is used as a C line, and then the membrane scribing instrument is placed at the temperature of 37 ℃ for 30-40 min.
Assembling the test strip:
and S31, diluting the gold-labeled pad by 0.8cm in width, diluting the gold-labeled antibody by 5 times by using a gold-labeled antibody storage solution, adding a trehalose aqueous solution with the final concentration of 5%, coating the gold-labeled antibody on the gold-labeled pad according to the amount of 10 mu L/cm, placing the gold-labeled pad in a refrigerator at the temperature of minus 80 ℃ for one night, and freeze-drying the gold-labeled pad by using a freeze-dryer for later use.
And S32, sequentially adhering the water absorption pad, the NC film, the gold label pad and the sample pad from top to bottom by taking the lining plate as a bottom plate, completing assembly, and cutting into test strips with the width of 3mm by a slitter for detection. The test strip is divided into four parts: the upper section is a handheld part (a water absorption pad which absorbs redundant liquid in a detection sample), the middle section is an experimental reaction area (an NC membrane which comprises a detection zone T for judging results and a quality control zone C for indicating the quality of the test paper strip), the lower section is a sample area (a glass cellulose membrane which contacts the sample to be detected), and a gold-labeled pad soaked with a colloidal gold-labeled compound is arranged between the middle section and the lower section.
A detection step:
s41, selecting 2X 106Respectively adding 500uL of cell lysate to the cell precipitates of individuals, rats, mice, hamsters, pigs and chickens to lyse the cells, and diluting the cell lysates with PBS if the concentration is too high to perform experiments;
s42, taking out the test strips, and dripping 100uL of cell lysate on each test strip sample pad;
and S43, standing at room temperature for reaction for 3-5min, observing the species of the test strip with specific T-line color development, and observing within 10min, wherein the color development is ineffective after 10 min.
Human, rat, mouse, hamster, pig and chicken cell lysates are diluted to different concentrations and smeared to glass fiber to make sample pads, and the sample pads are assembled with other components to form reagent strips for detection, wherein the kit without cell lysates is used as a comparative example 1.
Examples 1-3, comparative example 1 provide several colloidal gold test strips prepared according to the above procedure, in which the respective components and concentrations of the cell lysate and diluent used for each are shown in tables 1-2, and the results are shown in tables 3-5 and fig. 1.
TABLE 1 cell lysate Components
| Reagent | Example 1 | Example 2 | Example 3 | Comparative example 1 |
| NP40(％) | 0.2 | 1 | 2 | 0 |
| Tris-HCl(mmol/L) | 10 | 50 | 100 | 0 |
| Sodium chloride (%) | 0.8 | 0.8 | 0.8 | 0 |
| PMSF(mmol/L) | 0.05 | 0.3 | 0.5 | 0 |
| Pepsin inhibitionReagent mmol/L | 0.2 | 1 | 2 | 0 |
| Leupeptin (mmol/L) | 0.2 | 2 | 0.5 | 0 |
| Aprotinin (mmol/L) | 0.2 | 1.5 | 2 | 0 |
TABLE 2 Diluent composition
| Reagent | Example 1 | Example 2 | Example 3 | Comparative example 1 |
| OVA(％) | 0.1 | 1 | 0.5 | 0.1 |
| Sodium chloride (%) | 0.8 | 0.8 | 0.8 | 0.8 |
| Glacial acetic acid (%) | 0.1 | 0.6 | 1 | 0.1 |
| Sodium azide (%) | 0.01 | 0.1 | 0.05 | 0.01 |
| Potassium chloride (%) | 0.02 | 0.02 | 0.02 | 0.02 |
| Disodium hydrogen phosphate (%) | 0.144 | 0.144 | 0.144 | 0.144 |
| Potassium dihydrogen phosphate (%) | 0.024 | 0.024 | 0.024 | 0.024 |
TABLE 3 comparison of detection sensitivity
| Group of | Diluting by 5 times | Diluting by 10 times | Diluted by 15 times | Diluting by 20 times |
| Example 1 | ++++ | ++++ | ++++ | +++ |
| Example 2 | ++++ | ++++ | +++ | +++ |
| Example 3 | ++++ | ++++ | ++++ | +++ |
| Comparative example 1 | ++ | + | +- | - |
TABLE 4 comparison of assay stability
| Group of | 1 day | 1 month | 6 months old | 12 months old | 24 months |
| Example 1 | ++++ | ++++ | ++++ | +++ | +++ |
| Example 2 | ++++ | ++++ | +++ | +++ | +++ |
| Example 3 | ++++ | ++++ | ++++ | +++ | +++ |
| Comparative example 1 | ++ | + | - | - | - |
TABLE 5 comparison of detection times
| Group of | 3min | 5min | 10min | 20min | 30min |
| Example 1 | +++ | +++ | +++ | +++ | +++ |
| Example 2 | +++ | +++ | +++ | +++ | +++ |
| Example 3 | +++ | +++ | +++ | +++ | +++ |
| Comparative example 1 | - | - | - | + | ++ |
Remarking: in tables 3 to 5, "-" indicates no color development and a negative result; "+" indicates the degree of color development, and the more "+" the darker the degree of color development and the higher the degree of positivity.
FIG. 1 is a graph of the test result of example 1, and it can be seen from FIG. 1 that the sample tested in the example is human, the test result is visible with naked eyes, the test strip has darker color, which indicates that the degree of positive is high; the test paper strips of rats, mice, hamsters, pigs and chickens do not develop color, which shows that the detection by the kit of the invention has no pollution and other interference and high detection accuracy. The data in tables 3-5 prove that the kit has the advantages of extremely short detection time, sensitive reaction and stable reaction, the result can be judged and read after the reaction time is 3 minutes, an auxiliary instrument is not needed, the result can be directly observed by naked eyes, and the application range is wide.
The samples of the invention are not limited to human, rat, mouse, hamster, pig and chicken species, and may be increased or decreased as desired; also, the detection of tissue samples is not limited to cell samples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A kit for identifying cell species based on a colloidal gold method is characterized in that: comprises a colloidal gold test strip, cell lysate and PBS; the colloidal gold test strip comprises a sample pad, a gold label pad, an NC membrane and a water absorption pad; the gold-labeled pad comprises a colloidal gold-labeled internal reference universal antibody A, and the NC membrane is coated with a secondary goat anti-mouse IgG and an internal reference specific monoclonal antibody B.
2. The kit for identifying cell species according to claim 1, which is characterized in that: the preparation method of the internal reference universal antibody A comprises the following steps: selecting universal fragments of different species of reference protein beta-Actin or GAPDH to synthesize corresponding polypeptides, coupling BSA or KLH, immunizing a mouse, preparing a mouse antibody to obtain a reference universal antibody A, and diluting with a diluent for later use.
3. The kit for identifying cell species according to claim 1, which is characterized in that: the preparation method of the reference specificity monoclonal antibody B comprises the following steps: selecting specific fragments of different species of reference protein beta-Actin or GAPDH to synthesize corresponding polypeptides, coupling BSA or KLH, immunizing a mouse, preparing a mouse antibody to obtain a reference specific monoclonal antibody B, and diluting with a diluent for later use.
4. A kit for identifying cell species based on the colloidal gold method according to claim 2 or 3, wherein: the diluent consists of the following components in percentage by mass: 0.1-1% of OVA, 0.8% of sodium chloride, 0.1-1% of glacial acetic acid, 0.01-0.1% of sodium azide, 0.02% of potassium chloride, 0.144% of disodium hydrogen phosphate and 0.024% of potassium dihydrogen phosphate, and the solvent is deionized water.
5. The kit for identifying cell species according to claim 1, which is characterized in that: the reference specificity monoclonal antibody B coated in the nitrocellulose membrane is a detection line.
6. The kit for identifying cell species according to claim 1, which is characterized in that: the goat anti-mouse IgG secondary antibody coated in the nitrocellulose membrane is a quality control line.
7. The kit for identifying cell species according to claim 1, which is characterized in that: the sample is treated by cell lysis solution, and is dripped on the sample pad after being diluted by PBS.
8. The kit for identifying cell species according to claim 1, which is characterized in that: the cell lysate is composed of NP40 with the mass fraction of 0.2-2%, Tris-HCL with the final concentration of 10-100mmol/L, sodium chloride with the mass fraction of 0.8%, PMSF with the final concentration of 0.05-0.5mmol/L, pepsin inhibitor with the final concentration of 0.2-2mmol/L, leupeptin with the final concentration of 0.2-2mmol/L and aprotinin with the final concentration of 0.2-2mmol/L, and the solvent is deionized water.
9. The method for preparing a cell species identification kit according to claim 1, wherein the cell species identification kit comprises: the method comprises the following steps:
s1, diluting the colloidal gold-labeled internal reference general antibody A by 5 times with a gold-labeled antibody storage solution, adding a trehalose aqueous solution with the mass fraction of 5%, and coating the gold-labeled antibody on a gold-labeled pad according to the amount of 10 mu L/cm to prepare the gold-labeled pad containing the colloidal gold-labeled internal reference general antibody A;
s2, coating an internal reference specificity monoclonal antibody B and a secondary goat anti mouse IgG on the detection line and the quality control line of the NC membrane respectively to prepare a coated nitrocellulose membrane;
and S3, sequentially adhering the water absorption pad, the NC film, the gold label pad and the sample pad from top to bottom by taking the lining plate as a bottom plate to finish assembly.
10. The method for preparing a cell species identification kit according to claim 9 based on the colloidal gold method, wherein: the gold-labeled antibody storage solution comprises the following components in parts by mass: 1% trehalose, 0.5% Tween-20 and 0.02% NaN3The pH value is 8.2, and the solvent is deionized water.
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