CN108359642B - Hybridoma cell of shrimp tropomyosin monoclonal antibody and application thereof - Google Patents
Hybridoma cell of shrimp tropomyosin monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention discloses a hybridoma cell of a shrimp tropomyosin monoclonal antibody and application thereof, belonging to the field of food safety immunodetection. The hybridoma cell strain FB12-1 of the shrimp tropomyosin monoclonal antibody is preserved in the China general microbiological culture Collection center of the culture Collection management Committee for microorganisms at 9.5.2017, the preservation number is CGMCC No.14690, the preservation address is No. 3 of the Beijing university Hokko No.1 of the morning area, and the institute of microbiology of the Chinese academy of sciences. The antibody 7H6 secreted by the cell strain FB12-1 is used as a coating antibody, the antibody 7H6 is used as an enzyme-labeled antibody (7H6-HRP) after being labeled with HRP, the shrimp tropomyosin is used as a standard substance to establish a double-antibody sandwich enzyme-linked immunoassay method for detecting the shrimp tropomyosin, and the method has practical application value in detecting food allergens.
Description
Technical Field
The invention relates to a hybridoma cell of a shrimp tropomyosin monoclonal antibody and application thereof, belonging to the technical field of food safety immunodetection.
Background
In recent years, food allergy has become a public food safety issue. Food allergy is a common immunological disease for human beings, is mainly caused by protein in food, is caused by type I hypersensitivity mediated by IgE, has clinical symptoms such as edema, dermatitis, asthma, intestinal syndrome and the like, can cause shock and even endanger life in severe cases, and becomes an urgent priority for detecting and quantifying trace allergen in complex food samples. The crustacean is one of 8 kinds of easily allergic food proposed by food and agriculture organization of the United nations, and the main allergen of the crustacean is shrimp tropomyosin with the molecular mass of about 36-38 kDa. At present, the in vitro detection methods aiming at the trace allergen comprise an immunological method, a proteomics method, a PCR method and a chemiluminescence immune method. Although there are many methods related to allergen detection at present, there are different problems, such as slow detection speed, high cost, and the need for specific analytical instruments for detection, which restricts the development of allergen detection methods in food. Therefore, the method has practical significance for realizing accurate, safe, economic, rapid, high-throughput and high-sensitivity in-vitro detection technology.
Compared with other allergen detection methods, the enzyme-linked immunosorbent assay (ELISA) is an extremely high-efficiency, sensitive and rapid detection method, has low requirement on the purity of a sample during detection, is simple and convenient to operate, and is suitable for rapid field detection of a large number of samples. Establishing an efficient immunological detection method, and screening monoclonal monomers with high specificity is an important prerequisite.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain of a shrimp tropomyosin monoclonal antibody and application thereof, and establishes a double-antibody sandwich ELISA method for detecting the shrimp tropomyosin.
The invention aims to provide a shrimp tropomyosin monoclonal cell strain which is deposited in the common microorganism center of China general microbiological culture Collection center at 9 and 5 months in 2017, is classified and named as a monoclonal cell strain, and has the deposition number of CGMCC No.14690, the deposition address of Beijing university Hokkaido No.1 of the morning-West Lu, China academy of sciences microbial research institute.
The second purpose of the invention is to provide a shrimp tropomyosin monoclonal antibody which is secreted and produced by a monoclonal cell strain with the preservation number of CGMCCNo.14690.
The third purpose of the invention is to provide the application of the shrimp tropomyosin monoclonal antibody.
In one embodiment of the invention, the use is for the analytical detection of shrimp tropomyosin in food products.
In one embodiment of the invention, the application is to prepare a reagent for detecting shrimp tropomyosin by an ELISA competition method.
In one embodiment of the invention, the application is to establish a double-antibody sandwich ELISA method for detecting shrimp tropomyosin by using the shrimp tropomyosin monoclonal antibody as a coating antibody and using an enzyme-labeled antibody after the antibody is labeled with HRP, wherein the detection limit of the established double-antibody sandwich method for detecting the shrimp tropomyosin in food is 0.9ng/ml, and the application has practical application value.
It is a fourth object of the present invention to provide a method for preparing the shrimp tropomyosin monoclonal cell line by immunizing an animal with shrimp tropomyosin and isolating polyclonal antibodies from the animal.
In one embodiment of the present invention, the preparation method of the shrimp tropomyosin hybridoma cell strain comprises the following basic steps:
(1) preparation of shrimp tropomyosin allergen: preparing shrimp protein leaching liquor, carrying out fractional precipitation by using saturated ammonium sulfate, identifying shrimp tropomyosin by using SDS-PAGE, and carrying out gel filtration chromatography separation to obtain high-purity protein.
(2) Animal immunization and potency determination: immunizing a healthy BALB/c female mouse by adopting a small-dose short-cycle scheme, uniformly mixing 100 mu g of shrimp tropomyosin and an equivalent amount of Freund's complete adjuvant for the first immunization, performing subcutaneous injection, boosting the immunization by using 100 mu g of shrimp tropomyosin and an equivalent amount of Freund's incomplete adjuvant after 3 weeks, and boosting the immunization by using half amount of shrimp tropomyosin every 3 weeks; reducing the amount of the spurt immune agent by half, mixing the spurt immune agent with physiological saline with the same volume, then adopting abdominal immunity, using shrimp tropomyosin as a coating antigen, and detecting the serum titer by a direct ELISA method;
the specific ELISA procedure was as follows:
1) coating: diluting the coated antigen with 0.05M carbonate buffer solution (pH9.6), incubating at 37 deg.C for 2 hr, and adding 100 μ L/well buffer solution;
2) washing: pouring off the solution in the plate, injecting 200 μ L of PBST solution into each hole, placing on a shaking table, oscillating for 3min, spin-drying, and washing for 3 times; the following washing methods were the same;
3) and (3) sealing: after patting dry, adding 200 mu L/hole sealing liquid, incubating for 2h at 37 ℃, washing and drying for later use;
4) sample adding: the antiserum is diluted from 1:1000 in a gradient manner, 100 mu L/hole, and incubated at 37 ℃ for 30 min; after fully washing, adding a mouse secondary antibody diluted by 1:3000, incubating for 30min at 37 ℃, and then drying after washing;
5) color development: taking out the ELISA plate, fully washing, adding 100 μ L of color development liquid (the ratio of TMB to substrate liquid is 1:5) into each hole, and reacting at 37 deg.C in dark for 15 min;
6) termination and measurement: taking out the ELISA plate, adding 50 μ L stop solution (2mol/L sulfuric acid) into each well to stop reaction, and measuring the light absorption OD of each well with ELISA reader450nm。
(3) Cell fusion and screening: three days after the impact immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1450) method, and the specific steps were as follows:
a. taking a spleen of a mouse aseptically, grinding the spleen, passing through a 200-mesh cell screen to obtain a spleen cell suspension, and counting cells;
b. collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and counting cells;
c. mixing splenocytes and SP2/0 cells at a ratio of 10:1 (number ratio), centrifuging, fusing with 50% PEG for 1min, adding RPMI-1640 basic culture medium from slow to fast, centrifuging, suspending in RPMI-1640 screening culture medium containing 20% fetal calf serum and 2% 50 × HAT, adding to 96-well cell culture plate, standing at 37 deg.C and 5% CO2The fused cells were subjected to half-exchange of RPMI-1640 screening medium on the third day of cell fusion, to full-exchange with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 × HT on the 6 th day, and to screening with cell supernatant on the 9 th day.
Screening: the shrimp tropomyosin was used as a coating antigen, and detection was performed by direct ELISA, and wells with a small number of cell clusters and high positive values were selected, subcloned by the limiting dilution method, and detected by the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the shrimp tropomyosin monoclonal antibody.
(4) The monoclonal antibody is prepared by injecting 10 mL paraffin oil into the abdominal cavity of BALB/c mouse of 8-10 weeks old, and injecting 1 × 10 into the abdominal cavity of each mouse 7 days later6And (3) hybridoma cells, collecting ascites from the seventh day, purifying the ascites by an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.
The invention also provides a composition containing the shrimp tropomyosin monoclonal antibody.
The invention also provides a kit for detecting the shrimp tropomyosin, which contains the monoclonal antibody, a solid phase carrier, an antibody for coating the solid phase carrier, an enzyme-labeled antibody capable of being combined with a detection antigen, a chromogenic substrate, a washing solution and a confining liquid; the antibody used for coating the solid phase carrier is a monoclonal antibody secreted by hybridoma cell CGMCC No. 14690.
The invention has the beneficial effects that: the hybridoma cell strain capable of secreting the shrimp tropomyosin monoclonal antibody is obtained, a double-antibody sandwich ELISA method for detecting the shrimp tropomyosin is established, and the application of the hybridoma cell strain in detecting the food allergen has practical application value.
Biological material preservation
The monoclonal cell strain has been preserved in China general microbiological culture Collection center (CGMCC) at 9.5.2017 with the preservation number of CGMCC No.14690, and the preservation address of No. 3, the institute of microbiology, China academy of sciences, of North Xilu No.1, the south China Korean district, Beijing.
Drawings
FIG. 1 shows the shrimp tropomyosin detection curve equation y ═ 0.22904+0.53319x (R)20.9937).
Detailed Description
The method comprises the steps of immunizing a mouse with complete antigen, carrying out cell fusion, culturing in HAT selective culture medium, screening cell supernatant through direct ELISA, carrying out amplification culture on the shrimp tropomyosin cell strain with high titer obtained by screening, preparing and purifying ascites, pairing each two by using a double-antibody sandwich method, and finally establishing the double-antibody sandwich ELISA detection method for the shrimp tropomyosin.
EXAMPLE 1 monoclonal antibody preparation of shrimp tropomyosin
1. Preparation of shrimp tropomyosin allergen:
a. preparing a shrimp protein leaching solution: weighing 20g of shelled shrimps, and processing by a tissue triturator to obtain mashed shrimp. Immersing the shrimp paste into petroleum ether (60-90) according to a ratio of 1:10(W/V) for degreasing, stirring overnight under magnetic force at 4 ℃, centrifuging for 15min at 6000r/mim, discarding supernatant, and placing precipitate in a fume hood to volatilize the petroleum ether to obtain degreased shrimp meat. Soaking in 0.05M PBS (pH7.4PBS) at a ratio of 1:20(W/V) for leaching overnight, centrifuging the leaching solution at 4 deg.C at 7000r/min for 20min, and removing precipitate to obtain supernatant as shrimp protein leaching solution.
b. Ammonium sulfate fractional precipitation: slowly adding saturated ammonium sulfate solid into shrimp protein leaching liquor while stirring until the saturation degree is 30%, standing for 1h at 4 ℃, centrifuging for 20min at 8000r/min, collecting precipitate, and dissolving in 0.05M PBS (pH7.4PBS) to obtain 30% saturation component. Saturated ammonium sulfate solid was continuously added to the supernatant until the saturation was 50%, and the 50% saturation fraction was obtained according to the above 30% saturation fraction obtaining method. Ammonium sulfate was also added to the supernatant to give a 70% saturated fraction. The three fractions were dialyzed for 24h against 0.05M PBS pH7.4M.
c. SDS-PAGE identification: the protein content of the three fractions was determined by UV (Coomassie blue) and the protein content of each fraction was then identified by SDS-PAGE. SDS-PAGE conditions were: 10% separation gel, 5% concentration gel, and performing electrophoresis at constant pressure of 100V for 80 min.
d. Gel filtration chromatography separation: after identification by SDS-PAGE, the ammonium sulfate precipitate fraction containing tropomyosin was selected for gel filtration chromatography. The chromatographic column is Superdex 7510/300 GL gel filtration column. Chromatography conditions are as follows: the eluent is 0.05M PBS (pH7.4PBS), the flow rate is 0.35mL/min, the automatic collection is carried out at the interval of 2mL, and the ultraviolet absorption monitoring at 280nm is carried out. The desired peak sample was collected and dialyzed against ultrapure water for 24h (4 ℃). And freezing and drying the sample to obtain the prepared shrimp tropomyosin.
2. Animal immunization: immunizing a healthy BALB/c female mouse by adopting a small-dose short-cycle scheme, uniformly mixing 100 mu g of shrimp tropomyosin and an equivalent amount of Freund's complete adjuvant for the first immunization, performing subcutaneous injection, boosting the immunization by using 100 mu g of shrimp tropomyosin and an equivalent amount of Freund's incomplete adjuvant after 3 weeks, and boosting the immunization by using half amount of shrimp tropomyosin every 3 weeks; reducing the amount of the spurt immune agent by half, mixing the spurt immune agent with physiological saline with the same volume, then adopting abdominal immunity, using shrimp tropomyosin as a coating antigen, and detecting the serum titer by a direct ELISA method;
the specific ELISA procedure was as follows:
(1) coating: diluting the coated antigen with 0.05M carbonate buffer solution (pH9.6), incubating at 37 deg.C for 2 hr, and adding 100 μ L/well buffer solution;
(2) washing: pouring off the solution in the plate, injecting 200 μ L of PBST solution into each hole, placing on a shaking table, oscillating for 3min, spin-drying, and washing for 3 times; the following washing methods were the same;
(3) and (3) sealing: after patting dry, adding 200 mu L/hole sealing liquid, incubating for 2h at 37 ℃, washing and drying for later use;
(4) sample adding: the antiserum is diluted from 1:1000 in a gradient manner, 100 mu L/hole, and incubated at 37 ℃ for 30 min; after fully washing, adding a mouse secondary antibody diluted by 1:3000, incubating for 30min at 37 ℃, and then drying after washing;
(5) color development: taking out the ELISA plate, fully washing, adding 100 μ L of color development liquid (the ratio of TMB to substrate liquid is 1:5) into each hole, and reacting at 37 deg.C in dark for 15 min;
(6) termination and measurement: taking out the ELISA plate, adding 50 μ L stop solution (2mol/L sulfuric acid) into each well to stop reaction, and measuring the light absorption OD of each well with ELISA reader450nm。
3. Cell fusion: three days after the impact immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 1450) method, and the specific steps were as follows:
(1) taking a spleen of a mouse aseptically, grinding the spleen, passing through a 200-mesh cell screen to obtain a spleen cell suspension, and counting cells;
(2) collecting SP2/0 cells, suspending in RPMI-1640 basic culture solution, and counting cells;
(3) mixing splenocytes and SP2/0 cells at a ratio of 10:1 (number ratio), centrifuging, fusing with 50% PEG for 1min, adding RPMI-1640 basic culture medium from slow to fast, centrifuging, suspending in RPMI-1640 screening culture medium containing 20% fetal calf serum and 2% 50 × HAT, adding to 96-well cell culture plate, standing at 37 deg.C and 5% CO2Cultured in an incubator.
4. Cell screening and cell strain establishment: on the third day of cell fusion, the fused cells were subjected to RPMI-1640 screening medium half-exchange, on the 6 th day, to total-exchange with RPMI-1640 medium containing 20% fetal bovine serum and 1% 100 XHT, and on the 9 th day, cell supernatants were collected for screening.
Screening: the shrimp tropomyosin was used as a coating antigen, and detection was performed by direct ELISA, and wells with a small number of cell clusters and high positive values were selected, subcloned by the limiting dilution method, and detected by the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the shrimp tropomyosin monoclonal antibody.
5. The monoclonal antibody is prepared by injecting 10 mL paraffin oil into the abdominal cavity of BALB/c mouse of 8-10 weeks old, and injecting 1 × 10 into the abdominal cavity of each mouse 7 days later6And (3) hybridoma cells, collecting ascites from the seventh day, purifying the ascites by an octanoic acid-ammonium sulfate method, and storing the obtained monoclonal antibody at-20 ℃.
Example 2 establishment of a double antibody sandwich ELISA:
1) coating: coating the ELISA plate with an antibody with the cell strain number of FB12-1 at the concentration of 4 mug/mL, 100 mug/hole and standing overnight at 4 ℃;
2) washing: washing the reaction plate with PBST for three times, each time for 3min, at 200 μ L/well, and then spin-drying the reaction plate;
3) and (3) sealing: adding 200 mu L/hole sealing liquid, and incubating for 2h at 37 ℃;
4) washing: the same as 2);
5) sample preparation: diluting shrimp tropomyosin to 1 mu g/mL by PBS, and performing gradient dilution by three times; an additional PBS blank was set. Add 100. mu.L of sample per well and incubate for 1h at 37 ℃;
6) washing: the same as 2);
7) adding enzyme standard antibody (7H6-HRP, 2. mu.g/mL), 100. mu.L/well, reacting at 37 ℃ for 1H;
8) washing: the same as 2);
9) color development: adding substrate TMB 100 μ L/hole, and developing for 12 min;
10) and (4) terminating: adding 50 mu L of stop solution into the hole;
11) and (3) determination: detection of OD Using microplate reader450nm. Results as shown in fig. 1, the equation for the shrimp tropomyosin detection curve is 0.2290+0.5332x (R)20.9937), the lowest detection limit was 0.63 ng/ml.
Example 3
Taking a shrimp sample as an example, the shrimp tropomyosin as an sensitizing component in the shrimp sample is detected
1) Coating: diluting the anti-shrimp tropomyosin monoclonal antibody to coating concentration by using a coating solution, adding the diluted anti-shrimp tropomyosin monoclonal antibody into an ELISA plate at 100 mu L/hole, and standing overnight at 4 ℃;
2) washing: discarding the liquid in the holes of the enzyme label plate, washing the reaction plate with PBST for three times, 3min each time, 200 mu L/hole, and then drying the reaction plate;
3) and (3) sealing: adding 200 mu L/hole sealing liquid, and incubating for 2h at 37 ℃;
4) washing: the same as 2);
5) sample preparation: diluting shrimp tropomyosin with sample diluent, diluting sample to be tested appropriately, setting positive control and negative control in each well of 100 μ L, and incubating for 1h at 37 deg.C;
6) washing: the same as 2);
7) adding an enzyme-labeled antibody: diluting an enzyme-labeled mouse-anti-shrimp tropomyosin antibody to a working concentration of 100 mu L/hole, and reacting at 37 ℃ for 1 h;
8) washing: the same as 2);
9) color development: adding substrate TMB 100 μ L/hole, and developing for 12 min;
10) and (4) terminating: adding 50 mu L of stop solution into the hole;
11) and (3) determination: detection of OD Using microplate reader450nm. The results showed a limit of quantitative detection of 0.9 ng/mL.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A shrimp tropomyosin monoclonal cell strain is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC No. 14690) at 9.5.2017, and the preservation address is No. 3 of the institute of microbiology, China academy of sciences, North West Lu No.1 of the morning-Yangxi district, Beijing.
2. A shrimp tropomyosin monoclonal antibody produced by secretion from the monoclonal cell line having a collection number of CGMCC No.14690 according to claim 1.
3. The use of a shrimp tropomyosin monoclonal antibody as claimed in claim 2.
4. Use according to claim 3, for the analytical detection of shrimp tropomyosin in food products.
5. The use according to claim 3, characterized in that it is used for preparing reagents for detecting shrimp tropomyosin by ELISA competition method.
6. The use of claim 3, wherein: the antibody of claim 2 is used as a coating antibody, after the antibody is marked with HRP, the antibody is used as an enzyme-labeled antibody, and a double-antibody sandwich ELISA method for detecting shrimp tropomyosin is established.
7. A composition comprising a shrimp tropomyosin monoclonal antibody as claimed in claim 2.
8. A shrimp tropomyosin detection kit, characterized by comprising the monoclonal antibody of claim 2, a solid carrier, an antibody for coating the solid carrier, an enzyme-labeled antibody capable of binding with a detection antigen, a chromogenic substrate, a washing solution and a blocking solution; the antibody used for coating the solid phase carrier is a monoclonal antibody secreted by hybridoma cell CGMCC No. 14690.
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