CN108359642A - The hybridoma of one seed shrimp tropomyosin monoclonal antibody and its application - Google Patents
The hybridoma of one seed shrimp tropomyosin monoclonal antibody and its application Download PDFInfo
- Publication number
- CN108359642A CN108359642A CN201810302087.6A CN201810302087A CN108359642A CN 108359642 A CN108359642 A CN 108359642A CN 201810302087 A CN201810302087 A CN 201810302087A CN 108359642 A CN108359642 A CN 108359642A
- Authority
- CN
- China
- Prior art keywords
- antibody
- shrimp tropomyosin
- tropomyosin
- shrimp
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43508—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from crustaceans
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the hybridoma of a seed shrimp tropomyosin monoclonal antibody and its applications, belong to food security field of immunodetection.The hybridoma cell strain FB12 1 of one plant of shrimp tropomyosin monoclonal antibody of the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 5th in September in 2017, deposit number is CGMCC No.14690, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.The antibody 7H6 secreted using cell strain FB12 1 is coated antibody, it is enzyme labelled antibody (7H6 HRP) after antibody 7H6 labels HRP, the double antibody sandwich enzyme immunologic detection method of detection shrimp tropomyosin is established using shrimp tropomyosin as standard items, application in detecting food allergen has actual application value.
Description
Technical field
The present invention relates to the hybridoma of a seed shrimp tropomyosin monoclonal antibody and its applications, belong to food security
Technical field of immunoassay.
Background technology
In recent years, food hypersenstivity has become the food-safety problem of a public character.Food hypersenstivity is common one of the mankind
Kind immunity disease, is mainly caused by protein in food, the type Ⅰ hypersensitivity mediated by IgE, and clinical symptoms are main
There are oedema, dermatitis, asthma and bowel syndrome etc., shock even threat to life can be caused when serious, in complicated foodstuff samples
The detection of minute amounts of allergen and quantitatively become the task of top priority.Shellfish is the easy mistake of 8 major class that FAO (Food and Agriculture Organization of the United Nation) proposes
One of quick food, shellfish main allergen are the shrimp tropomyosin that molecular mass is about 36-38kDa.It is external at present
There are immunological method, protein science method, PCR methods, chemiluminescence immunoassay method for the detection method of minute amounts of allergen.Although
There are many current method in relation to Allergic skin test, but all there are respective different problems, as detection speed is slow, of high cost, detection
Specific analytical instrument etc. is needed, the development of Allergic skin test method in food is constrained.Therefore accurate, safety, warp are realized
Ji, vitro detection technical method quickly, high-throughput, highly sensitive are with realistic meaning.
Compared with other Allergic skin test methods, enzyme-linked immunization (ELISA) is a kind of extremely efficient, sensitive, quick
Detection method, when detection, be not high and easy to operate to the purity requirement of sample, is suitable for the scene quickly inspection of great amount of samples
It surveys.Efficient immunological detection method is established, the monoclonal monomer for screening high specific is important prerequisite.
Invention content
The object of the present invention is to provide the hybridoma cell strain of shrimp tropomyosin monoclonal antibody and its applications, establish inspection
Survey the double antibody sandwich ELISA of shrimp tropomyosin.
The first purpose of the invention is to provide a seed shrimp tropomyosin monoclonal cell strains, in September in 2017 5 days
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, Classification And Nomenclature is monoclonal cell strain, preservation
Number is CGMCC No.14690, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute.
Second object of the present invention is to provide a seed shrimp tropomyosin monoclonal antibody, is CGMCC by deposit number
The monoclonal cell strain secretion of No.14690 generates.
Third object of the present invention is to provide the applications of shrimp tropomyosin monoclonal antibody.
In one embodiment of the invention, the application is the analysis detection for food Prawn tropomyosin.
In one embodiment of the invention, the application is to be used to prepare ELISA competition laws detection shrimp tropomyosin
White reagent.
In one embodiment of the invention, it is packet that the application, which is with the shrimp tropomyosin monoclonal antibody,
By antibody, antibody detects the double antibody sandwich ELISA of shrimp tropomyosin, uses this after marking HRP for enzyme labelled antibody, foundation
The double-antibody method of foundation detects food Prawn tropomyosin, and detection is limited to 0.9ng/ml, has actual application value.
Fourth object of the present invention is to provide the method for preparing the shrimp tropomyosin monoclonal cell strain, is to use shrimp
Animal is immunized in tropomyosin, and polyclonal antibody is detached out of animal body.
In one embodiment of the invention, the preparation method of the shrimp tropomyosin hybridoma cell strain walks substantially
Suddenly it is：
(1) preparation of shrimp tropomyosin anaphylactogen：Prepare shrimp protein leaching liquor, saturated ammonium sulfate fractional precipitation, SDS-
PAGE identifies shrimp tropomyosin, the isolated high purity protein of gel permeation chromatography.
(2) animal immune and titration：Using low dose of short cycle scheme immune health BALB/c female mices, for the first time
It is immunized with being subcutaneously injected after 100 μ g shrimps tropomyosin and equivalent Freund's complete adjuvant mixing, interval is after 3 weeks, then uses
100 μ g shrimps tropomyosin and equivalent incomplete Freund's adjuvant booster immunization, hereafter measured shrimp tropomyosin every 3 weeks with half
Booster immunization is primary；Spurt immunizing dose halves, and peritoneal immunity is used after being mixed with isometric physiological saline, with shrimp original flesh
Globulin detects serum titer as envelope antigen, by Salmonella method；
Its specific ELISA program is as follows：
1) it is coated with：By envelope antigen 0.05M pH9.6 carbonate buffer solution gradient dilutions, 100 holes μ L/, 37 DEG C of incubations
2h；
2) it washs：Solution in plate is inclined, 200 μ L PBST solution are injected per hole, is placed on shaking table and vibrates 3min, is dried,
Washing 3 times；Following washing methods is identical；
3) it closes：After patting dry, 200 holes μ L/ confining liquids are added, 37 DEG C of incubation 2h are dried for standby after washing；
4) it is loaded：By antiserum from 1:1000 start gradient dilution, 100 holes μ L/, 37 DEG C of incubation 30min；Fully washing
Afterwards, it is added 1:3000 diluted mouse secondary antibodies, 100 holes μ L/, 37 DEG C of incubation 30min are patted dry after washing；
5) it develops the color：ELISA Plate is taken out, fully after washing, developing solution (TMB and the substrate solution volume of 100 μ L are added per hole
Ratio 1:5), it is protected from light 15min for 37 DEG C；
6) it terminates and measures：ELISA Plate is taken out, 50 μ L terminate liquids (sulfuric acid of 2mol/L) are added per hole and terminate reaction, then
The light absorption value OD in each hole is measured with microplate reader450nm。
(3) cell fusion and screening：After impact is three days immune, according to conventional PEG (polyethylene glycol, molecular weight 1450)
Method carries out cell fusion, is as follows：
A, sterile to take mouse spleen, it grinds and obtains splenocyte suspension by 200 mesh cell screen clothes, and carry out cytometer
Number；
B, SP2/0 cells are collected, are suspended in RPMI-1640 basic culture solutions, cell count is carried out；
C, by splenocyte and SP2/0 cells according to 10:The ratio of 1 (quantity ratio) mixes, and is merged with 50%PEG after centrifugation,
Time 1min is added RPMI-1640 basic culture solutions, is suspended in after centrifugation containing 20% tire ox blood later according to from slowly to fast
Clearly, in the RPMI-1640 screening and culturing liquid of 2% 50 × HAT, 96 porocyte culture plates is added to, 37 DEG C, 5%CO are placed in2Training
It supports and is cultivated in case.RPMI-1640 screening and culturing liquid is carried out to fused cell in the third day of cell fusion and partly changes liquid, the 6th day use
Containing 20% fetal calf serum, 1%100 × HT RPMI-1640 transition culture solutions progress change liquid entirely, taken at the 9th day cell conditioned medium into
Row screening.
Screening：Use shrimp tropomyosin as envelope antigen, be detected with Salmonella, choose cell mass number it is few and
Positive high hole, is subcloned using limiting dilution assay, is detected with same method.In triplicate to get steady to energy
Surely the cell strain of shrimp tropomyosin monoclonal antibody is secreted.
(4) preparation and identification of monoclonal antibody：8-10 week old BALB/c mouses, every mouse peritoneal is taken to inject paraffin oil
1 mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma is collected ascites since the 7th day, ascites is passed through pungent
Acid-ammonium sulfate method purifying, the monoclonal antibody of acquisition are placed in -20 DEG C of preservations.
The present invention also provides the compositions containing the shrimp tropomyosin monoclonal antibody.
The present invention also provides the kits detected for shrimp tropomyosin, are carried containing the monoclonal antibody, solid phase
Body, the antibody for being coated with the solid phase carrier can be with the enzyme labelled antibody of detection antigen binding, chromogenic substrate, cleaning solution and envelope
Close liquid；The antibody for being coated with the solid phase carrier is that the monoclonal of hybridoma CGMCC No.14690 secretions is anti-
Body.
Beneficial effects of the present invention：Present invention obtains the hybridoma that can secrete shrimp tropomyosin monoclonal antibody is thin
Born of the same parents' strain, establishes the double antibody sandwich ELISA of detection shrimp tropomyosin, and the application in detecting food allergen has
Actual application value.
Biomaterial preservation
It is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms on 5th in September in 2017 for monoclonal cell strain
Bio-Centers, deposit number are CGMCC No.14690, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology of the academy of sciences of state.
Description of the drawings
Fig. 1 is the shrimp tropomyosin detection curve equation y=0.22904+0.53319x (R that the present invention obtains2=
0.9937) schematic diagram.
Specific implementation mode
The present invention passes through cell fusion, HAT selective medium cultures, by straight by the way that mouse is immunized in comlete antigen
ELISA screening cell conditioned mediums are connect, the high shrimp tropomyosin cell strain of the potency that screening is obtained expands culture, and passes through abdomen
Water prepares and purifying, is used in combination double antibody sandwich method to match two-by-two, finally establishes the double-antibody sandwich of shrimp tropomyosin
ELISA detection method.
It is prepared by the monoclonal antibody of 1 shrimp tropomyosin of embodiment
1, the preparation of shrimp tropomyosin anaphylactogen：
A, prepared by shrimp protein leaching liquor：Peeled shrimp 20g is weighed, tissue mashing machine handles to obtain shrimp mud.Shrimp mud presses 1:10(W/
V degreasing in petroleum ether (60~90)) is immersed, is stayed overnight under 4 DEG C of magnetic agitations, 6000r/mim centrifuges 15min, abandons supernatant, precipitates
Being placed in draught cupboard makes petroleum ether volatilize to obtain degreasing shrimp.1 is pressed immediately:20 (W/V) immerse extracted in 0.05M pH7.4PBS
Night, leaching liquor centrifuge 20min with 7000r/min at 4 DEG C, discard precipitation, supernatant is shrimp protein leaching liquor.
B, ammonium sulfate precipitation：Saturated ammonium sulfate solid is slowly added in shrimp protein leaching liquor, it is stirring while adding, directly
It is 30% to saturation degree, 4 DEG C of standings 1h, 8000r/min centrifuge 20min, collect precipitation and redissolve in 0.05M pH7.4PBS,
Obtain 30% saturation degree component.Saturated ammonium sulfate solid is continuously added in supernatant until saturation degree is 50%, by above-mentioned 30% saturation
Degree component preparation method obtains 50% saturation degree component.Equally continue the addition ammonium sulfate into supernatant and obtains 70% saturation degree group
Point.Above three rank groups are dialysed for 24 hours in 0.05M pH7.4PBS respectively.
C, SDS-PAGE is identified：Above three is classified component its protein content (Coomassie Brilliant Blue) of ultraviolet determination, so
The protein ingredient of each component is identified with SDS-PAGE afterwards.SDS-PAGE deposition conditions are：10% separation gel, 5% concentration glue are permanent
Press 100V electrophoresis 80min.
D, gel permeation chromatography detaches：After being identified by SDS-PAGE, the ammonium sulfate precipitation containing tropomyosin is selected
Component carries out gel permeation chromatography.Chromatographic column is 75 10/300GL solvent resistant columns of Superdex.Chromatography condition：Eluent
It is collected automatically at interval for 0.05M pH7.4PBS, flow velocity 0.35mL/min, 2mL, the monitoring of 280nm UV absorptions.Collect institute
Peak sample, ultra-pure water is needed to dialyse for 24 hours (4 DEG C).Shrimp tropomyosin prepared by being after sample freeze-drying.
2, animal immune：Using low dose of short cycle scheme immune health BALB/c female mices, 100 μ g of first immunisation
It is subcutaneously injected after shrimp tropomyosin and equivalent Freund's complete adjuvant mixing, interval is after 3 weeks, then with 100 μ g shrimp original flesh balls
Albumen and equivalent incomplete Freund's adjuvant booster immunization, it is hereafter primary with half amount shrimp tropomyosin booster immunization every 3 weeks；
Spurt immunizing dose halves, and peritoneal immunity is used after being mixed with isometric physiological saline, uses shrimp tropomyosin as coating
Antigen detects serum titer by Salmonella method；
Its specific ELISA program is as follows：
(1) it is coated with：By envelope antigen 0.05M pH9.6 carbonate buffer solution gradient dilutions, 100 holes μ L/, 37 DEG C of incubations
2h；
(2) it washs：Solution in plate is inclined, 200 μ L PBST solution are injected per hole, is placed on shaking table and vibrates 3min, get rid of
It is dry, it washs 3 times；Following washing methods is identical；
(3) it closes：After patting dry, 200 holes μ L/ confining liquids are added, 37 DEG C of incubation 2h are dried for standby after washing；
(4) it is loaded：By antiserum from 1:1000 start gradient dilution, 100 holes μ L/, 37 DEG C of incubation 30min；Fully washing
Afterwards, it is added 1:3000 diluted mouse secondary antibodies, 100 holes μ L/, 37 DEG C of incubation 30min are patted dry after washing；
(5) it develops the color：ELISA Plate is taken out, fully after washing, developing solution (TMB and the substrate liquid of 100 μ L are added per hole
Product ratio 1:5), it is protected from light 15min for 37 DEG C；
(6) it terminates and measures：ELISA Plate is taken out, 50 μ L terminate liquids (sulfuric acid of 2mol/L) are added per hole and terminate reaction, so
The light absorption value OD in each hole is measured with microplate reader afterwards450nm。
3, cell fusion：After immune three days of impact, according to conventional PEG (polyethylene glycol, molecular weight 1450) methods into
Row cell fusion, is as follows：
(1) sterile to take mouse spleen, it grinds and obtains splenocyte suspension by 200 mesh cell screen clothes, and carry out cytometer
Number；
(2) SP2/0 cells are collected, are suspended in RPMI-1640 basic culture solutions, cell count is carried out；
(3) by splenocyte and SP2/0 cells according to 10:The ratio of 1 (quantity ratio) mixes, and is merged with 50%PEG after centrifugation,
Time 1min is added RPMI-1640 basic culture solutions, is suspended in after centrifugation containing 20% tire ox blood later according to from slowly to fast
Clearly, in the RPMI-1640 screening and culturing liquid of 2% 50 × HAT, 96 porocyte culture plates is added to, 37 DEG C, 5%CO are placed in2Training
It supports and is cultivated in case.
4, cell screening and cell strain are established：RPMI-1640 screenings are carried out to fused cell in the third day of cell fusion
Culture solution partly changes liquid, is changed entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1% within the 6th day
Liquid took cell conditioned medium to be screened at the 9th day.
Screening：Use shrimp tropomyosin as envelope antigen, be detected with Salmonella, choose cell mass number it is few and
Positive high hole, is subcloned using limiting dilution assay, is detected with same method.In triplicate, you can obtain energy
The cell strain of stably excreting shrimp tropomyosin monoclonal antibody.
5, the preparation and identification of monoclonal antibody：8-10 week old BALB/c mouses, every mouse peritoneal is taken to inject paraffin oil
1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma is collected ascites since the 7th day, ascites is passed through pungent
Acid-ammonium sulfate method purifying, the monoclonal antibody of acquisition are placed in -20 DEG C of preservations.
The foundation of 2 double-antibody sandwich elisa of embodiment：
1) it is coated with：The antibody coated elisa plate for being FB12-1 with the cell strain number of a concentration of 4 μ g/mL, 100 holes μ L/, 4
DEG C overnight；
2) it washs：Three times with PBST washing reactions plate, each 3min, 200 holes μ L/, then dries reaction plate；
3) it closes：200 holes μ L/ confining liquids, 37 DEG C of incubation 2h are added；
4) it washs：With 2)；
5) sample：Shrimp tropomyosin is diluted to 1 μ g/mL with PBS, carries out gradient dilution again repeatedly；Separately set a PBS
Blank control.100 μ L samples are added per hole, 1h is incubated in 37 DEG C；
6) it washs：With 2)；
7) enzyme labeling antibody (7H6-HRP, 2 μ g/mL), 100 holes μ L/, 37 DEG C of reaction 1h；
8) it washs：With 2)；
9) it develops the color：Add 100 holes μ L/ substrate TMB, develop the color 12min；
10) it terminates：Add 50 holes μ L/ of terminate liquid；
11) it measures：OD is detected with microplate reader450nm.Shown in result figure 1, shrimp tropomyosin detection curve equation is y=
0.2290+0.5332x(R2=0.9937), lowest detection is limited to 0.63ng/ml.
Embodiment 3
By taking shrimp sample as an example, allergen shrimp tropomyosin therein is detected
1) it is coated with：The anti-shrimp tropomyosin monoclonal antibody of mouse is diluted to peridium concentration with coating buffer, enzyme is added with 100 holes μ L/
In target, 4 DEG C overnight；
2) it washs：The liquid in ELISA Plate hole is discarded, three times with PBST washing reactions plate, each 3min, 200 holes μ L/,
Then reaction plate is dried；
3) it closes：200 holes μ L/ confining liquids, 37 DEG C of incubation 2h are added；
4) it washs：With 2)；
5) sample：Shrimp tropomyosin is made into gradient dilution with sample diluting liquid, sample to be tested makees appropriate dilution, per hole
100 μ L incubate 1h if positive control and negative control in 37 DEG C；
6) it washs：With 2)；
7) enzyme labelled antibody is added：Enzyme is marked into the anti-shrimp tropomyosin antibody of mouse, is diluted to working concentration, 100 holes μ L/,
37 DEG C of reaction 1h；
8) it washs：With 2)；
9) it develops the color：Add 100 holes μ L/ substrate TMB, develop the color 12min；
10) it terminates：Add 50 holes μ L/ of terminate liquid；
11) it measures：OD is detected with microplate reader450nm.As a result the quantitative detection of display is limited to 0.9ng/mL.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection of the present invention without departing from the spirit and scope of the present invention
Range should be subject to what claims were defined.
Claims (10)
1. a seed shrimp tropomyosin monoclonal cell strain is preserved in Chinese microorganism strain preservation pipe on 5th in September in 2017
Reason committee common micro-organisms center, deposit number are CGMCC No.14690, and preservation address is Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica.
2. a seed shrimp tropomyosin monoclonal antibody, which is characterized in that by deposit number described in claim 1 be CGMCC
The monoclonal cell strain secretion of No.14690 generates.
3. the application of the shrimp tropomyosin monoclonal antibody described in claim 2.
4. application according to claim 3, which is characterized in that the analysis for food Prawn tropomyosin detects.
5. application according to claim 3, which is characterized in that be used to prepare ELISA competition laws detection shrimp tropomyosin
Reagent.
6. the application described in claim 3, it is characterised in that：Using the antibody described in claim 2 as coated antibody, antibody label
It is enzyme labelled antibody after HRP, establishes the double antibody sandwich ELISA of detection shrimp tropomyosin.
7. the method for preparing monoclonal cell strain described in claim 1, which is characterized in that animal is immunized with shrimp tropomyosin,
Polyclonal antibody is detached out of animal body.
8. the method according to the description of claim 7 is characterized in that being as follows：
(1) animal immune and titration：First shrimp tropomyosin is used to be carried out subcutaneously with after equivalent Freund's complete adjuvant mixing
Injection, interval is after 2~3 weeks, then with shrimp tropomyosin and equivalent incomplete Freund's adjuvant booster immunization, hereafter every 2~3 weeks
It is primary with half amount shrimp tropomyosin booster immunization；Spurt immunizing dose halves, and is used after being mixed with isometric physiological saline
Peritoneal immunity, uses shrimp tropomyosin as envelope antigen, and serum titer is detected by Salmonella method；
(2) cell fusion and screening：After impact is three days immune, cell fusion is carried out, is used in combination shrimp tropomyosin as coating
Antigen is detected with Salmonella, obtains the cell strain of energy stably excreting shrimp tropomyosin monoclonal antibody.
9. the composition containing shrimp tropomyosin monoclonal antibody described in claim 2.
10. a seed shrimp tropomyosin detection kit, which is characterized in that contain the monoclonal antibody, solid described in claim 2
Phase carrier, the antibody for being coated with the solid phase carrier can be with the enzyme labelled antibody of detection antigen binding, chromogenic substrate, cleaning solution
And confining liquid；The antibody for being coated with the solid phase carrier is the monoclonal of hybridoma CGMCC No.14690 secretions
Antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810302087.6A CN108359642B (en) | 2018-04-04 | 2018-04-04 | Hybridoma cell of shrimp tropomyosin monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810302087.6A CN108359642B (en) | 2018-04-04 | 2018-04-04 | Hybridoma cell of shrimp tropomyosin monoclonal antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108359642A true CN108359642A (en) | 2018-08-03 |
| CN108359642B CN108359642B (en) | 2020-09-04 |
Family
ID=63002185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810302087.6A Active CN108359642B (en) | 2018-04-04 | 2018-04-04 | Hybridoma cell of shrimp tropomyosin monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108359642B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951696A (en) * | 2019-12-19 | 2020-04-03 | 浙江工商大学 | Hybridoma cell strain and anti-shrimp arginine kinase monoclonal antibody secreted by same |
| CN111073860A (en) * | 2019-12-19 | 2020-04-28 | 浙江工商大学 | Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881769A (en) * | 2010-06-08 | 2010-11-10 | 江南大学 | Colloidal gold immunochromatographic test strip for detecting shrimp allergen and preparation method thereof |
| US20120219545A1 (en) * | 2009-07-31 | 2012-08-30 | Mount Sinai School Of Medicine | Materials and methods for diagnosing and treating shellfish allergy |
| WO2012145925A1 (en) * | 2011-04-29 | 2012-11-01 | 深圳大学 | Uses of food allergens and antibodies thereof in iga nephropathy |
| CN104262476A (en) * | 2014-09-29 | 2015-01-07 | 天津商业大学 | Purifying method of shrimp tropomyosin |
| CN105388296A (en) * | 2015-10-28 | 2016-03-09 | 中国海洋大学 | Method for detecting tropomyosin by means of homologous epitope peptide antibody |
| CN107267465A (en) * | 2017-07-13 | 2017-10-20 | 江南大学 | One plant of Procalcitonin monoclonal antibody hybridoma cell strain CS12 1 and its application |
-
2018
- 2018-04-04 CN CN201810302087.6A patent/CN108359642B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120219545A1 (en) * | 2009-07-31 | 2012-08-30 | Mount Sinai School Of Medicine | Materials and methods for diagnosing and treating shellfish allergy |
| CN101881769A (en) * | 2010-06-08 | 2010-11-10 | 江南大学 | Colloidal gold immunochromatographic test strip for detecting shrimp allergen and preparation method thereof |
| WO2012145925A1 (en) * | 2011-04-29 | 2012-11-01 | 深圳大学 | Uses of food allergens and antibodies thereof in iga nephropathy |
| CN104262476A (en) * | 2014-09-29 | 2015-01-07 | 天津商业大学 | Purifying method of shrimp tropomyosin |
| CN105388296A (en) * | 2015-10-28 | 2016-03-09 | 中国海洋大学 | Method for detecting tropomyosin by means of homologous epitope peptide antibody |
| CN107267465A (en) * | 2017-07-13 | 2017-10-20 | 江南大学 | One plant of Procalcitonin monoclonal antibody hybridoma cell strain CS12 1 and its application |
Non-Patent Citations (2)
| Title |
|---|
| 方蕾等: "一种以虾原肌球蛋白为致敏原的Th2反应小鼠模型的建立", 《中国免疫学杂志》 * |
| 黄建芳等: "凡纳滨对虾主要过敏原-原肌球蛋白单克隆抗体的制备及其过敏原表位分析", 《免疫学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951696A (en) * | 2019-12-19 | 2020-04-03 | 浙江工商大学 | Hybridoma cell strain and anti-shrimp arginine kinase monoclonal antibody secreted by same |
| CN111073860A (en) * | 2019-12-19 | 2020-04-28 | 浙江工商大学 | Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection |
| CN110951696B (en) * | 2019-12-19 | 2021-08-10 | 浙江工商大学 | Hybridoma cell strain and anti-shrimp arginine kinase monoclonal antibody secreted by same |
| CN111073860B (en) * | 2019-12-19 | 2021-08-24 | 浙江工商大学 | Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108359642B (en) | 2020-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103558388B (en) | Double-antibody sandwich method for detecting salmonella typhimurium in food based on monoclonal antibodies | |
| CN103134931B (en) | Double antibody sandwich method of detecting staphylococcus aureus enterotoxin A of food | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN103713104B (en) | Double-antibody sandwich method for detecting enterobacter sakazakii in food | |
| CN101271107A (en) | Fast immunology detecting method for goat milk and its milk product doped with cow's milk | |
| CN108912090B (en) | Test strip for rapidly detecting total amount of alternariol and alternariol monomethyl ether | |
| CN106282125A (en) | Hybridoma cell strain NaN 1 and the application thereof of anti-sulfa antibiotics monoclonal antibody is secreted in one strain | |
| CN104004717A (en) | Anti-aflatoxin general type monoclonal antibody hybridoma cell line and application thereof | |
| CN108359642A (en) | The hybridoma of one seed shrimp tropomyosin monoclonal antibody and its application | |
| CN108517317A (en) | A kind of anti-Clorprenaline monoclonal antibody hybridoma cell strain and its application | |
| CN109868261A (en) | The preparation of mass selection monoclonal antibody and its application of anti-eight kinds of triazines | |
| CN103197073A (en) | Enzyme-linked immunosorbent assay for detecting escherichia coli in food | |
| CN105087498B (en) | One plant of benzene thiophene cyanogen monoclonal antibody hybridoma cell strain and its application | |
| US20220010029A1 (en) | Hybridoma cell strain that secrets anti-dinitolmide monoclonal antibodies and the application of hybridoma cell strain | |
| CN104178458B (en) | One plant of Listeria monocytogenes monoclonal antibody hybridoma cell strain and its application | |
| CN107523554A (en) | One plant of hybridoma cell strain SS0708 for secreting Madumycin monoclonal antibody specific and its application | |
| CN102998457A (en) | Competitive inhibition enzyme-linked immunosorbent assay (ELISA) method for grass carp tumor necrosis factors alpha | |
| CN103513035A (en) | Test strip and method for detecting aflatoxin M1 | |
| CN107267465A (en) | One plant of Procalcitonin monoclonal antibody hybridoma cell strain CS12 1 and its application | |
| Zhou et al. | Development of a novel antibody probe useful for domoic acid detection | |
| CN104017774A (en) | Anti-chlorothalonil monoclonal antibody hybridoma cell strain and application thereof | |
| CN110590949A (en) | Method for preparing myoglobin pairing monoclonal antibody and pairing monoclonal antibody prepared by method | |
| CN108277206A (en) | A kind of wheat gliadin monoclonal antibody hybridoma cell strain and its application | |
| CN107121552B (en) | One plant of hybridoma cell strain ZY-2G5-3 for secreting 3,4- methylene benzylene chloride propylamine monoclonal antibodies and its application | |
| US10808041B2 (en) | Lincosamides universal monoclonal antibody hybridoma cell strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |