CN108277206A - A kind of wheat gliadin monoclonal antibody hybridoma cell strain and its application - Google Patents
A kind of wheat gliadin monoclonal antibody hybridoma cell strain and its application Download PDFInfo
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- CN108277206A CN108277206A CN201810299600.0A CN201810299600A CN108277206A CN 108277206 A CN108277206 A CN 108277206A CN 201810299600 A CN201810299600 A CN 201810299600A CN 108277206 A CN108277206 A CN 108277206A
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of wheat gliadin monoclonal antibody hybridoma cell strain and its applications, belong to food security technical field of immunoassay.The hybridoma cell strain CS6 1 of one plant of wheat gliadin monoclonal antibody of the invention, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC NO.14687.Present invention obtains the hybridoma cell strains that can secrete wheat gliadin only clonal antibody, the antibody 4F2 secreted using cell strain CS6 1 is coated antibody, it is enzyme labelled antibody (4F2 HRP) after antibody 4F2 labels HRP, the double antibody sandwich enzyme immunologic detection method of detection wheat gliadin is established using wheat gliadin as standard items, application in detecting food allergen has actual application value.
Description
Technical field
The present invention relates to a kind of wheat gliadin monoclonal antibody hybridoma cell strain and its applications, belong to food security
Technical field of immunoassay.
Background technology
Wheat gliadin is the monomeric protein that molecular weight is 22-58kDa, is the main component of wheat gluten.Alcohol is molten
Albumen is the main allergen of the cereals such as wheat, and Wheat Dood Allergy can influence the health of skin, internal organ, respiratory tract, cause to move
Excite allergy, professional asthma, rhinitis, contact nettle disease, chylous diarrhea enteritis, leprosy skin disease etc..Chylous diarrhea enteritis is to lose
It passes after susceptible patient takes in gluten and is not resistant to caused chronic intestinal malabsorption syndrome, and it is now recognized that alcohol is molten
Albumen is the main inducing of celiac patients.Food hypersenstivity has become the food-safety problem of a public character.Food hypersenstivity is
A kind of common immunity disease of the mankind, is mainly caused by protein in food, and the type Ⅰ hypersensitivity mediated by IgE faces
Bed symptom mainly has oedema, dermatitis, asthma and bowel syndrome etc., can cause shock even threat to life when serious, complicated
In foodstuff samples the detection of minute amounts of allergen and quantitatively become the task of top priority.The external detection method for being directed to minute amounts of allergen at present
There are immunological method, protein science method, PCR methods, chemiluminescence immunoassay method.Although the current method in relation to Allergic skin test is very
It is more, but all there are respective different problems, such as detection speed is slow, of high cost, detection needs specific analytical instrument, constrains
The development of Allergic skin test method in food.Therefore accurate, safety, economy, quick, high-throughput, highly sensitive external inspection are realized
Survey technology method has realistic meaning.
Invention content
The first purpose of the invention is to provide the hybridoma cell strains of wheat gliadin monoclonal antibody, in 2017
On September is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC for 5
No.14687, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Second object of the present invention is to provide wheat gliadin monoclonal antibody, it is by the deposit number
The monoclonal cell strain secretion of CGMCC No.14687 generates.
Third object of the present invention is to provide the applications of the wheat gliadin monoclonal antibody.
In one embodiment of the invention, the application is the analysis detection for wheat gliadin in food.
In one embodiment of the invention, the application prepares ELISA competition laws detection wheat gliadin
Reagent.
Fourth object of the present invention is to provide the preparation method of the monoclonal cell strain, and the method is to use wheat alcohol
Molten protein immune animal, detaches polyclonal antibody out of animal body.
In one embodiment of the invention, the wheat gliadin hybridoma cell strain is prepared as follows
's：
(1) animal immune and titration：Using low dose of short cycle scheme immune health BALB/c female mices, for the first time
It is immune with being subcutaneously injected after 100 μ g wheat gliadins and equivalent Freund's complete adjuvant mixing, after being spaced 3 weeks, then with 100
Hereafter μ g wheat gliadins and equivalent incomplete Freund's adjuvant booster immunization were reinforced every 3 weeks with half amount wheat gliadin
It is immune primary；Spurt immunizing dose halves, and peritoneal immunity is used after being mixed with isometric physiological saline, uses wheat gliadin
As envelope antigen, serum titer is detected 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。
(2) 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,
RPMI-1640 basic culture solutions are added later according to from slowly to fast in time 1min, be suspended in after centrifugation containing 20% fetal calf serum,
In the RPMI-1640 screening and culturing liquid of 2% 50 × HAT, 96 porocyte culture plates are added to, are placed in 37 DEG C, 5%CO2Culture
It 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 with containing
The RPMI-1640 transition culture solutions progress of 20% fetal calf serum, 1%100 × HT changes liquid entirely, takes cell conditioned medium to carry out at the 9th day
Screening.
Screening：Use wheat gliadin 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 wheat gliadin monoclonal antibody is secreted.
(3) 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.
Fifth object of the present invention is to provide a kind of detection methods of wheat gliadin, and the method includes walking as follows
Suddenly：
(1) it is coated with：The antibody coated elisa plate for being CS6-1 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/, drying；
(3) it closes：200 holes μ L/ confining liquids, 37 DEG C of incubation 2h are added；
(4) it washs：With (2)；
(5) sample：Wheat gliadin is diluted to 5 μ g/mL with PBS, carries out gradient dilution again repeatedly；Separately set one
PBS blank controls.100 μ L samples are added per hole, 1h is incubated in 37 DEG C；
(6) it washs：With (2)；
(7) enzyme labeling antibody (4F2-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。
Sixth object of the present invention is to provide a kind of wheat gliadin detection kit, the kit contains described
Monoclonal antibody, solid phase carrier, the antibody for being coated with the solid phase carrier can be shown with the enzyme labelled antibody of detection antigen binding
Color substrate, cleaning solution and confining liquid；The antibody for being coated with the solid phase carrier is hybridoma CGMCC No.xxxx
The monoclonal antibody of secretion.
Beneficial effects of the present invention：Present invention obtains the hybridoma that can secrete wheat gliadin monoclonal antibody is thin
Born of the same parents' strain, establishes the double antibody sandwich ELISA of detection wheat gliadin, and this method detection is limited to 9.28ng/ml, is detecting
Application in food allergen has actual application value.
Biomaterial preservation
Monoclonal cell strain, deposit number are CGMCC No.14687, are preserved within 5th the micro- life of China in September in 2017
Object culture presevation administration committee common micro-organisms center, preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of the academy of sciences.
Description of the drawings
Fig. 1 is the wheat gliadin detection curve equation y=0.60495-0.0285x (R that the present invention obtains2=
0.9979) schematic diagram.
Specific implementation mode
The preparation of 1 hybridoma of embodiment
1, 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 wheat gliadin and equivalent Freund's complete adjuvant mixing, after being spaced 3 weeks, then it is molten with 100 μ g wheat alcohol
Albumen and equivalent incomplete Freund's adjuvant booster immunization, it is hereafter primary with half amount wheat gliadin booster immunization every 3 weeks；Punching
Thorn immunizing dose halves, and peritoneal immunity is used after being mixed with isometric physiological saline, uses wheat gliadin anti-as coating
Original 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 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, so
The light absorption value OD in each hole is measured with microplate reader afterwards450nm。
2, 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,
RPMI-1640 basic culture solutions are added later according to from slowly to fast in time 1min, be suspended in after centrifugation containing 20% fetal calf serum,
In the RPMI-1640 screening and culturing liquid of 2% 50 × HAT, 96 porocyte culture plates are added to, are placed in 37 DEG C, 5%CO2Culture
It is cultivated in case.
3, 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 wheat gliadin 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 wheat gliadin monoclonal antibody.
4, 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.
Embodiment 2 detects the foundation of the double-antibodies sandwich ELISA of wheat gliadin
1) it is coated with：The antibody coated elisa plate for being CS6-1 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：Wheat gliadin is diluted to 5 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 (4F2-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.The results are shown in Figure 1, and wheat gliadin detection curve equation is y
=-0.4968+0.8869x, R2=0.9919, lowest detection is limited to 9.28ng/ml.
Embodiment 3
By taking wheat samples as an example, allergen wheat gliadin therein is detected
1) it is coated with：The anti-alcohol soluble protein monoclonal antibody of mouse is diluted to peridium concentration with coating buffer, ELISA Plate is added with 100 holes μ L/
In, 4 DEG C are 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/, so
After dry 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：Wheat gliadin 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-alcohol soluble protein antibody of mouse, is diluted to working concentration, 100 holes μ L/, 37 DEG C
React 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 19ng/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 model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (9)
1. a kind of wheat gliadin 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.14687, and preservation address is Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica.
2. a kind of wheat gliadin monoclonal antibody, which is characterized in that by deposit number described in claim 1 be CGMCC
The monoclonal cell strain secretion of No.14687 generates.
3. the application of the wheat gliadin monoclonal antibody described in claim 2.
4. application according to claim 3, which is characterized in that the analysis detection for wheat gliadin in food.
5. application according to claim 3, which is characterized in that be used to prepare ELISA competition laws detection wheat gliadin
Reagent.
6. the method for preparing monoclonal cell strain described in claim 1, which is characterized in that animal is immunized with wheat gliadin,
Polyclonal antibody is detached out of animal body.
7. according to the method described in claim 6, it is characterized in that, being as follows：
(1) animal immune and titration：First wheat gliadin is used to be carried out subcutaneously with after equivalent Freund's complete adjuvant mixing
Injection, interval is after 2~3 weeks, then with wheat gliadin and equivalent incomplete Freund's adjuvant booster immunization, hereafter every 2~3 weeks
It is primary with half amount wheat gliadin booster immunization；Spurt immunizing dose halves, and is used after being mixed with isometric physiological saline
Peritoneal immunity, uses wheat gliadin 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 wheat gliadin as coating
Antigen is detected with Salmonella, obtains the cell strain of energy stably excreting wheat gliadin monoclonal antibody.
8. the composition containing wheat gliadin monoclonal antibody described in claim 2.
9. a kind of wheat gliadin 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.14687 secretions
Antibody.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998425A (en) * | 2018-09-07 | 2018-12-14 | 江南大学 | One plant of pyridoxol monoclonal antibody hybridoma cell strain and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108998425A (en) * | 2018-09-07 | 2018-12-14 | 江南大学 | One plant of pyridoxol monoclonal antibody hybridoma cell strain and its application |
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Application publication date: 20180713 |