CN108456661A - One plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its application - Google Patents
One plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its application Download PDFInfo
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- CN108456661A CN108456661A CN201711442565.5A CN201711442565A CN108456661A CN 108456661 A CN108456661 A CN 108456661A CN 201711442565 A CN201711442565 A CN 201711442565A CN 108456661 A CN108456661 A CN 108456661A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention discloses one plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its applications, belong to food security technical field of immunological detection.The anti-BaP monoclonal antibody specific hybridoma cell strain of the present invention, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.14693.Anti- BaP monoclonal antibody specific, by the deposit number be CGMCC No.14693 hybridoma cell strain secreted by generate.The monoclonal antibody of this cell strain secretion has preferable specificity (IC to BaP5022 μ g/L of value), it can be used for the specific detection of BaP in food security.
Description
Technical field
The present invention relates to one plant of anti-BaP monoclonal antibody specific hybridoma cell strain and its applications, belong to food peace
Panimmunity detection technique field.
Background technology
BaP (Benzopyrene, BAP) is a kind of condensed-nuclei aromatics containing 5 phenyl ring, is one of strong carcinogen,
Chemical formula is C20O12, molecular weight about 252.32, BaP chemical property relatively stablizes in most cases, meets open fire and when by high fever
Decomposable asymmetric choice net simultaneously releases poisonous gas.It is widely present in the environment, coal in industrial production and life process, oil and natural
The exhaust gas and food that the non-complete combustion of fuel such as gas generate can all produce in the high temperature cooking process such as smoking, toasting and frying
It is raw.
Since BaP derives from a wealth of sources, harmfulness is again bigger, collects to the research of BaP detection both at home and abroad at present more
In in the food such as the environment such as air, water body and grain and oil, meat, due to detecting the difference of object, enrichment, extraction method not yet
Together, therefore analysis and the taken method of detection are also different.BaP detection at present mostly uses high performance liquid chromatography
(HPLC), liquid chromatogram and Mass Spectrometry (LC-MS), gas chromatography mass spectrometry method (GC-MS) etc..Above-mentioned detection method can be quantified
It analyzes and there is lower detection to limit, but usually require expensive instrument and complicated operation, pre-treatment and detection time are long,
The popularization for seriously constraining these detection methods is unable to reach the requirement that live high-volume quickly detects.And immunoassay method
Have the characteristics that inexpensive, high-throughput, highly sensitive, low to technical staff's relative requirement therefore quick suitable for a large amount of samples
Screening.Have the monoclonal antibody of higher affinity and detection sensitivity miscellaneous BaP the purpose of the present invention is to provide a kind of
The preparation method for handing over tumor cell strain lays the foundation for the research and development popularization of indirect competitive ELISA kit and colloidal gold strip.
Invention content
The object of the present invention is to provide a kind of anti-BaP monoclonal antibody specific hybridoma cell strains, by the cell strain
The antibody of preparation has preferable affinity and detection sensitivity to BaP, can be used for establishing BaP enzyme linked immunosorbent detection
Method, or establish colloidal gold immunochromatographimethod technology rapid detection method.
The first purpose of the invention is to provide one plant of anti-BaP monoclonal antibody specific hybridoma cell strains, in
On September 5th, 2017 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preservation address is Beijing
The institute 3 of city Chaoyang District North Star West Road 1, deposit number are CGMCC No.14693.
Second object of the present invention is to provide a kind of method of detection detection BaP, and the method is wanted using right
The monoclonal cell strain described in 1 is asked to secrete the anti-BaP monoclonal antibody specific generated.
Third object of the present invention is to provide a kind of anti-BaP monoclonal antibody specific, the monoclonal antibody by
The anti-BaP monoclonal antibody specific hybridoma cell strain secretion that the deposit number is CGMCC No.14693 generates.
Fourth object of the present invention is to provide the application of above-mentioned anti-BaP monoclonal antibody specific, is used for food
The remaining analysis detection of BaP in safety detection.
Fifth object of the present invention is to provide a kind of kit, the kit contains the monoclonal cell strain
Or the anti-BaP monoclonal antibody specific.
In one embodiment of the invention, the kit is for detecting BaP.
Sixth object of the present invention is to provide application of the kit in detecting BaP.
The preparation basic step of cell strain provided by the invention is：
(1) preparation and identification of immunogene：Using the benzo pyrene derivatives with carboxyl as initial reactant, pass through carbonization two
Imines method is connected with protein carrier, the small haptens for detaching comlete antigen by dialysis later and not being coupled, comlete antigen
It is identified by UV absorption scan method；
(2) mouse is immune：After immunogene and freund adjuvant emulsification completely, BALB/c is immunized by subcutaneous multi-point injection
Mouse.First immunisation uses Freund's complete adjuvant, booster immunization to use freund 's incomplete adjuvant, and immunizing dose is when spurt is immune
The half of a preceding immunizing dose is directly injected intraperitoneally after mixing with physiological saline；Each secondary immunization interval is three weeks.
After third time is immune, it is spaced one week blood sampling detection serum titer and inhibition；
(3) cell fusion is established with cell strain：Mouse boosting cell and bone marrow cells in mice is set to merge by polyethylene glycol method,
Culture detection positive cell hole, and the inhibition of positive cell hole is measured, by limiting dilution assay to there is the sun preferably inhibited
Property cell hole is subcloned three times, finally screens acquisition hybridoma cell strain；
(4) identification of hybridoma cell strain property：It is set with and is surveyed with ELIAS secondary antibody using mouse monoclonal Ig classes/subgroup identification
It is fixed；IC50The measurement of value and affinity passes through ELISA method.
In one embodiment of the invention, the polyethylene glycol is using PEG 2000.
In one embodiment of the invention, the culture is using HAT culture mediums.
In one embodiment of the invention, positive cell hole is detected using indirect ELISA method, positive cell
The inhibition in hole is detected using indirect competitive ELISA method.
Beneficial effects of the present invention：The anti-BaP cell strain of monoclonal antibody that the present invention obtains, there is preferably BaP
Detection sensitivity and affinity, additionally provide a kind of method of new synthesis BaP immunogene, the synthesis step of haptens
More simplify, effectively, the thinking and method of synthetic immunogen is provided for the research of people from now on.Monoclonal antibody of the present invention
IC50For 22 μ g/L, linear ranging from 6.0-87.0 μ g/L are detected, lowest detection limit value (LOD) is 3.0 μ g/L.
Biomaterial preservation
It is general to be preserved in China Committee for Culture Collection of Microorganisms on 5th in September in 2017 for one plant of monoclonal cell strain
Logical microorganism center, deposit number are CGMCC No.14693, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Description of the drawings：
The ultra-violet absorption spectrum of Fig. 1 immunogenes characterizes；
Standard suppression curve of Fig. 2 monoclonal antibodies to BaP；
Fig. 3 BaP hapten synthesis routes.
Specific implementation mode
The present invention by BaP comlete antigen by being immunized mouse, by cell fusion, HAT selective medium cultures,
Cell conditioned medium is screened by indirect ELISA and indirect competitive ELISA, finally obtained has preferable affinity and sensitive to BaP
The monoclonal antibody hybridoma cell strain of degree.
The configuration of solution：
Carbonate buffer solution (CBS)：Weigh Na2CO31.59g NaHCO32.93g is mixed after being dissolved in a small amount of distilled water respectively
It closes, adds distilled water to about 800mL mixings, adjust pH value to 9.6, add distilled water to be settled to 1000mL, 4 DEG C of storages are spare；
Phosphate buffer (PBS)：8.00g NaCl, 0.2g KCl, 0.24g KH2PO4, 3.62g Na2HPO4·
12H2O is dissolved in 800mL pure water, with NaOH or HCl tune pH to 7.2~7.4, is settled to 1000mL；
PBST：PBS containing 0.05%Tween20；
TMB developing solutions：A liquid：Na2HPO4.12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000mL；B liquid：
60mg TMB are dissolved in 100mL ethylene glycol.A, B liquid 1 ︰ 5 mixing by volume is TMB developing solutions, current existing mixed.
Embodiment 1：The preparation of hybridoma cell strain
(1) synthesis of comlete antigen：
5mL DMF are added into 3mmol BaPs 1,4mmol POCl are then added3, it is heated to 90 degree and reacts 24 hours,
It is added in 5mL water after being cooled to room temperature, filtering washes solid, is dried in vacuo to obtain product BaP aldehyde 2.By 10mmol 3- bromine fourths
Sour methyl esters is added in 100mL flasks, and 30mL benzene and 10mmol triphenylphosphines is then added, is heated to reflux 24 hours, is cooled to
It is filtered after room temperature, is dried in vacuo to obtain product quaternary alkylphosphonium salt 3.The above-mentioned quaternary alkylphosphonium salts of 5mmol 3 are added in 50mL flasks, are then added
30mLTHF and 6mmol potassium tert-butoxides add 5mol BaPs aldehyde 2 after reaction being stirred at room temperature 2 hours.The reaction was continued 12 for room temperature
Hour, it is spin-dried for solvent, 20mL water is then added, ethyl acetate extracts 3 times, merges organic phase, dry, is spin-dried for solvent, column chromatography
Detach to obtain Product olefins 4.The above-mentioned alkene of 3mmol is added in 50mL flasks, 20mgPd/C is then added, replaces nitrogen three
It is secondary, then hydrogen is replaced, reaction 12 hours is stirred at room temperature, then filters, is spin-dried for solvent and obtains product ester 5.By above-mentioned product ester 3mmol
It is added in 50mL flasks, 15mL methanol and 15mL30%KOH solvents is then added, reaction 6 hours is stirred at room temperature, is spin-dried for molten
Then the HCl of 10mL1M is added in agent, ethyl acetate extracts 3 times, merges organic phase, is spin-dried for, and pillar layer separation obtains final product 6,
Route map is as shown in Figure 3.
The above-mentioned haptens of 4.5mg is taken, 2.0mg EDC (1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt is added
Hydrochlorate) and 1.0mg NHS (n-hydroxysuccinimide), it is dissolved, is stirred at room temperature using DMF (n,N-Dimethylformamide), it is living
Change 4h；Another CB (carbonate buffer solution) solution for taking 5mg KLH (keyhole limpet hemocyanin) to be dissolved in 2mL, 0.05M, pH9.6
In, above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, 4 DEG C are dialysed three days, and -20 DEG C of packing are protected
It deposits.
(2) animal immune：The Balb/C mouse of 6~8 week old of health are selected to be immunized.Take BaP comlete antigen
BALB/c mouse, every 100 μ L are immunized by subcutaneous multi-point injection with after the emulsification uniformly of equivalent freund adjuvant in (1mg/mL).It is first
It is secondary it is immune use Freund's complete adjuvant, booster immunization to use freund 's incomplete adjuvant, immunizing dose is preceding primary when spurt is immune
The half of immunizing dose is directly injected intraperitoneally after mixing with physiological saline；Each secondary immunization interval is three weeks.For the third time
After immune, it is spaced one week blood sampling detection serum titer and inhibition；Selection inhibits best mouse, makes a spurt within 21 days after exempting from five and exempts from
Epidemic disease prepares fusion.
(3) cell fusion：After immune three days of spurt, according to conventional PEG (polyethylene glycol, molecular weight 2000) methods into
Row 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 counting ratio 5-10:1 ratio mixing, is merged after centrifugation with PEG, the time
1min is added RPMI-1640 basic culture solutions, is suspended in after centrifugation containing 20% fetal calf serum, 2% later according to from slowly to fast
50 × HAT RPMI-1640 screening and culturing liquid in, be added to 96 porocyte culture plates, be placed in 37 DEG C, 5%CO2Incubator
Middle culture.
(4) cell screening is established with cell strain：RPMI-1640 screenings are carried out to fused cell in the third day of cell fusion
Culture solution partly changes liquid, carries out within the 5th day being carried out with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1%
Liquid is changed entirely, took cell conditioned medium to be screened at the 7th day.Screening is in two steps：The first step first filters out positive cell with indirect ELISA
Hole, it is standard items that second step, which selects BaP, and inhibition measurement is carried out to positive cell with indirect competitive ELISA.Selection pair
BaP has the cell hole preferably inhibited, is subcloned using limiting dilution assay, is detected with same method.Repeat three
It is secondary, obtain cell strain.
(5) 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 collects ascites since the 7th day, ascites is passed through octanoic acid
Saturated ammonium sulfate method purifies, and the monoclonal antibody of acquisition is placed in -20 DEG C of preservations.
It is sub- that the monoclonal antibody obtained to ascites purifying using mouse monoclonal subtype identification kit carries out immunoglobulin
Type identifies that hypotype is IgG2a types.
Embodiment 2：The foundation of enzyme linked immuno sorbent assay
Hybridoma cell strain is adsorbed by monoclonal antibody prepared by internal ascites applied to indirect competitive enzyme-linked immunosorbent
The foundation of method method, is as follows：
1, use the 0.1 μ g/mL that carbonate buffer solution (CBS) has diluted as coating 96 hole elisa Plates of primordial covering, per hole 100
After μ L, 37 DEG C of coating 2h, three times with PBST washing lotions board-washing, patted dry every time per 200 μ L of hole, each 3min；
2, it is closed with the CBS containing 0.2% gelatin, per hole 200 μ L, 37 DEG C of closing 2h, three times with PBST washing lotions board-washing,
It is patted dry every time per 200 μ L of hole, each 3min；
3, the BaP standard that 0,2,5,10,20,50,100,200 μ g/L are respectively configured with phosphate buffer (PBS) is molten
Liquid.Standard solution is added separately in the ELISA Plate closed, per 50 μ L of hole, each standard solution repeats 3 holes, then
50 μ L, 1 ︰, 32000 diluted anti-BaP monoclonal antibodies are added per hole, after 37 DEG C are reacted 0.5h, board-washing pats dry；
4, the sheep anti-mouse igg secondary antibody that 100 μ L use 1 ︰ of PBS, the 3000 diluted HRP labels containing 0.1% gelatin is added per hole,
After 37 DEG C of reaction 0.5h, board-washing pats dry；
5, per hole be added 100 μ L TMB developing solutions, 37 DEG C colour developing 15min after, per hole be added 50 μ L 2M H2SO4Terminate liquid,
450nm surveys light absorption value；
6, Specification Curve of Increasing：Standard suppression curve is drawn with origin 8.5.
Using the light absorption value of enzyme linked immuno sorbent assay detection as ordinate, established with a concentration of abscissa of BaP
Anti- BaP monoclonal antibody is to the standard suppression curve of BaP, as shown in Figure 2.This can be calculated by painted standard curve
The IC of monoclonal antibody5022 μ g/L, detect linear ranging from 6.0-87.0 μ g/L, and lowest detection limit value (LOD) is 3.0 μ g/L.
Embodiment 3：Monoclonal antibody application
Sample pre-treatments and addition recovery experiment：
To the addition recovery experiment of actual sample, tested by taking edible oil as an example.First, it is edible that two parts of 2.0g are weighed
Oil, is separately added into 80ng, 200ng and 400ng BaP marks, mixing, be added 10mL 0.1M Tris-HCl (pH 8.4) with
10% methanol solution, high speed homogenization 2min extractions, after standing 5min, after taking 200 μ L supernatants PBS to dilute 4 times, as
ELISA sample extracting solutions are added recovery test using indirect competitive ELISA, and the rate of recovery is respectively 89%, 87%,
91%.
The subtype identification of 1. monoclonal antibody of table
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 (7)
1. it is common to be preserved in China Committee for Culture Collection of Microorganisms on 5th in September in 2017 for one plant of monoclonal cell strain
Microorganism center, deposit number are CGMCC No.14693, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. a kind of method of detection BaP, which is characterized in that the method is to utilize monoclonal cell described in claim 1
The anti-BaP monoclonal antibody specific that strain secretion generates.
3. a kind of anti-BaP monoclonal antibody specific, which is characterized in that divided by hybridoma described in claim 1
Secrete generation.
4. the application of the anti-BaP monoclonal antibody specific described in claim 3, which is characterized in that examined for food security
The remaining analysis detection of BaP in survey.
5. a kind of kit, which is characterized in that described in monoclonal cell strain described in claim 1 or claim 3
Anti- BaP monoclonal antibody specific.
6. a kind of kit according to claim 5, which is characterized in that the kit is for detecting BaP.
7. application of the kit described in claim 5 or 6 in detecting BaP.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022367A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | The anti-sibutramine monoclonal antibody specific hybridoma cell strain of one plant of secretion and its application |
| CN115028732A (en) * | 2022-07-11 | 2022-09-09 | 华中农业大学 | Anti-benzo [ a ] pyrene monoclonal antibody and application thereof |
| CN115028733A (en) * | 2022-07-11 | 2022-09-09 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting pyrene and benzo [ a ] pyrene |
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| CN103288965A (en) * | 2013-06-20 | 2013-09-11 | 重庆理工大学 | Polychlorobiphenyl monoclonal antibody preparation method |
| CN103852581A (en) * | 2014-03-11 | 2014-06-11 | 河南工业大学 | 3,4-benzopyrene enzyme-linked immune detection kit |
| CN104312978A (en) * | 2014-09-29 | 2015-01-28 | 江南大学 | Tobramycin monoclonal antibody as well as preparation method and application of tobramycin monoclonal antibody |
| CN105087498A (en) * | 2015-09-07 | 2015-11-25 | 江南大学 | TCMTB monoclonal antibody hybridoma cell strain and application thereof |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103288965A (en) * | 2013-06-20 | 2013-09-11 | 重庆理工大学 | Polychlorobiphenyl monoclonal antibody preparation method |
| CN103852581A (en) * | 2014-03-11 | 2014-06-11 | 河南工业大学 | 3,4-benzopyrene enzyme-linked immune detection kit |
| CN104312978A (en) * | 2014-09-29 | 2015-01-28 | 江南大学 | Tobramycin monoclonal antibody as well as preparation method and application of tobramycin monoclonal antibody |
| CN105087498A (en) * | 2015-09-07 | 2015-11-25 | 江南大学 | TCMTB monoclonal antibody hybridoma cell strain and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022367A (en) * | 2018-08-16 | 2018-12-18 | 江南大学 | The anti-sibutramine monoclonal antibody specific hybridoma cell strain of one plant of secretion and its application |
| CN115028732A (en) * | 2022-07-11 | 2022-09-09 | 华中农业大学 | Anti-benzo [ a ] pyrene monoclonal antibody and application thereof |
| CN115028733A (en) * | 2022-07-11 | 2022-09-09 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting pyrene and benzo [ a ] pyrene |
| CN115028732B (en) * | 2022-07-11 | 2023-09-08 | 华中农业大学 | Anti-benzo [ a ] pyrene monoclonal antibody and application thereof |
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Application publication date: 20180828 |