CN108866010B - One plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain and its application - Google Patents
One plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain and its application Download PDFInfo
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- CN108866010B CN108866010B CN201810934315.1A CN201810934315A CN108866010B CN 108866010 B CN108866010 B CN 108866010B CN 201810934315 A CN201810934315 A CN 201810934315A CN 108866010 B CN108866010 B CN 108866010B
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- C—CHEMISTRY; METALLURGY
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- 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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- 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
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
One plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716 and its application, belong to food safety technical field of immunoassay.Anti- furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716 prepared by the present invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.14686, and the deposit date is on September 5th, 2017.Anti- furans benzene olefin(e) acid sodium monoclonal antibody is generated as secreted by the hybridoma cell strain SS0716 that the deposit number is CGMCC No.14686.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity (IC to furans benzene olefin(e) acid sodium (NSOD)50Value is 0.38 μ g/L), it can be used for the detection of furans benzene olefin(e) acid sodium in food safety.
Description
Technical field
The present invention relates to one plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain and its applications, belong to food peace
Panimmunity detection technique field.
Background technique
Furans benzene olefin(e) acid sodium (abbreviation NSOD) belongs to Nitrofuran metabolites, is a kind of artificial synthesized broad spectrum antibiotic
Object, preventing and treating to the bacterium of animal and fish has good result.But the World Health Organization and FAO's connection
Closing report Nitrofuran metabolites has potential mutagenesis and carcinogenicity.For the health for protecting consumer, countries in the world are numerous and confused
Forbid use of the Nitrofuran metabolites on food animal.However, the illegal of these drugs is still had using phenomenon.
Nitrofuran metabolites are classified as A class disabling medicine by European Union, and detection, which must not be limited to, in animal derived food examines
Out.The Ministry of Agriculture, China is using partial nitro nitrofurans as the project that must not be detected in animal-derived food, furans benzene olefin(e) acid
Sodium is disabled in 2002 by China.
Currently, chromatography mainly uses HPLC-UV as detection means furans benzene olefin(e) acid sodium residue detection both at home and abroad, and
HPLC method is not able to satisfy in foreign trade various countries to furans benzene alkene since detection sensitivity is low, pre-treatment is cumbersome time-consuming
Sour sodium residue limits requirement.It is time-consuming moreover, instrumental method is there are cumbersome, it the disadvantages of expense is somewhat expensive, can not achieve a large amount of
The quick detection of sample, therefore establish the fast and convenient furans benzene olefin(e) acid sodium method for detecting residue of one kind and be of great significance.
Enzyme-linked immunization (ELISA) is a kind of extremely efficient, sensitive, quick detection method, easy to operate, is suitable for big
Measure the field quick detection of sample.However before the monoclonal monomer for obtaining high specific and high sensitivity is immunology detection
It mentions, so, it is most important to prepare a kind of highly sensitive, specificity hybridoma.
Summary of the invention
It is an object of the present invention to overcome the above deficiencies, and it is thin to provide anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma
Born of the same parents' strain and its application, the antibody prepared by the cell strain have preferably specificity to furans benzene olefin(e) acid sodium NSOD and detect sensitive
Degree can be used to establish the immunological detection method of furans benzene olefin(e) acid sodium NSOD.
According to technical solution provided by the invention, one plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain
SS0716 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC
No.14686, the deposit date is on September 5th, 2017.
Anti- furans benzene olefin(e) acid sodium monoclonal antibody, the anti-furans benzene olefin(e) acid sodium for being CGMCC No.14686 by deposit number
Monoclonal antibody hybridoma cell strain SS0716 secretion generates.
The application of the anti-furans benzene olefin(e) acid sodium monoclonal antibody, is applied the furans benzene olefin(e) acid sodium in food safety residual
It stays in analysis detection.
The preparation step of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716 are as follows:
(1) preparation and identification of immunogene: using furans benzene olefin(e) acid sodium as initial reactant, passed through using the carboxyl of its own
Carbodiimide method is connected with the amino of protein carrier, synthetic immunogen, and coating antigen is also to use carbodiimide method with NSOD
It is coupled to obtain with OVA, after reaction, by small haptens dialysis separation comlete antigen and be not coupled, by ultraviolet
Absorb scan method identification；
(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, and booster immunization uses 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, interval blood sampling in one week detection serum titer and inhibition；
(3) cell fusion and cell strain are established: by polyethylene glycol (PEG 2000) method, making mouse boosting cell and Mouse Bone
Myeloma cells fusion detects positive cell hole using indirect ELISA by HAT culture medium culture, and further using indirectly competing
Strive ELISA method measurement positive cell hole inhibitory effect, by limiting dilution assay to have the positive cell hole preferably inhibited progress
It is subcloned three times, finally screens acquisition hybridoma cell strain；
Beneficial effects of the present invention: anti-furans benzene olefin(e) acid sodium (NSOD) monoclonal antibody hybridoma cell that the present invention obtains
Strain SS0716, has preferable detection sensitivity and specificity (IC to NSOD50Value is 0.38 μ g/L).
Biological material specimens preservation: one plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716, it is described
Hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Beijing
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.14686, preservation day
Phase is on September 5th, 2017, and classification naming is monoclonal cell strain.
Detailed description of the invention
Fig. 1 is the inhibition standard curve of anti-furans benzene olefin(e) acid sodium monoclonal antibody.
Specific embodiment
The following examples of the present invention are only used as the further explanation of the content of present invention, not as a limitation of the present invention interior
Perhaps range.Below by embodiment, the invention will be further described.
The present invention is by being immunized mouse for NSOD comlete antigen, and by cell fusion, HAT selective medium culture is led to
Indirect ELISA and indirect competitive ELISA screening cell conditioned medium are crossed, having finally obtained has preferably specificity and sensitivity to NSOD
Monoclonal antibody hybridoma cell strain.
The preparation of 1 hybridoma cell strain of embodiment
(1) preparation of comlete antigen: taking 5mg furans benzene olefin(e) acid sodium (NSOD), adds 2.5mg EDC(1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochloride) and 1.5mg NHS(N- HOSu NHS), use DMF(N, N- dimethyl
Formamide) dissolution, it is stirred at room temperature, activates 4h；Separately take 6mg KLH(keyhole limpet hemocyanin) it is dissolved in 2mL, 0.05M, pH9.6
CBS(carbonate buffer solution) in solution, above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, obtains
To comlete antigen, 4 DEG C are dialysed three days, and -20 DEG C of packing save.
(2) animal immune: the BALB/c mouse of 6~8 week old of health is selected to be immunized.Take NSOD comlete antigen
After the emulsification uniformly of (1mg/mL) and equivalent freund adjuvant, BALB/c mouse, every 100 μ L are immunized by subcutaneous multi-point injection.It is first
It is secondary it is immune use Freund's complete adjuvant, booster immunization uses freund 's incomplete adjuvant, and 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, interval blood sampling in one week detection serum titer and inhibition；Selection inhibits best mouse, and spurt in 21 days is exempted from after exempting from five
Epidemic disease prepares fusion.
(3) cell fusion: after spurt immune three days, according to conventional PEG(polyethylene glycol, molecular weight 2000) method into
Row cell fusion, the specific steps are 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 cell is collected, is suspended in RPMI-1640 basic culture solution, cell count is carried out；
C, splenocyte and SP2/0 cell are mixed according to the ratio counted than 1:10, is merged after centrifugation with 50% PEG, when
Between 1 min later according to from slowly to fast RPMI-1640 basic culture solution is added, 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% CO2Incubator
Middle culture.
(4) cell screening and cell strain are established: carrying out RPMI-1640 screening to fused cell within the 3rd day in cell fusion
Culture solution partly changes liquid, carries out within the 5th day being carried out entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1%
Liquid is changed, took cell conditioned medium to be screened at the 7th day.Screen in two steps: the first step first filters out positive cell with indirect ELISA
Hole, it is standard items that second step, which selects NSOD, carries out inhibitory effect measurement to positive cell with indirect competitive ELISA.Selection pair
NSOD standard items have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are detected with same method.
In triplicate, anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716 is obtained.
The preparation and identification of the anti-furans benzene olefin(e) acid sodium monoclonal antibody of embodiment 2
8-10 week old BALB/c mouse is taken, every mouse peritoneal injects 1 mL of paraffin oil；Every mouse peritoneal injection after 7 days
1×106Hybridoma collected ascites since the 7th day, ascites was purified by caprylic acid-ammonium, the monoclonal antibody of acquisition
It is placed in -20 DEG C of preservations.
Using indirect competitive ELISA, the IC of monoclonal antibody is measured50It is respectively as follows: 0.38 μ g/L, illustrates have to NSOD very well
Sensitivity, can be used for furans benzene olefin(e) acid sodium (NSOD) residual immunoassay detection.
The application of the anti-furans benzene olefin(e) acid sodium monoclonal antibody of embodiment 3
The monoclonal that anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716 is prepared by internal ascites
Antibody adds recovery test applied to the ELISA of NSOD, the specific steps are as follows:
(1) the 0.3 μ g/mL for using carbonate buffer solution (CBS) to dilute is coated with 96 hole elisa Plates as primary concentration of envelope, often
After hole 100 μ L, 37 DEG C of 2 h of coating, three times with PBST washing lotion board-washing, 200 μ L of every hole, each 3min are patted dry every time；
(2) it is closed with the CBS containing 0.2% gelatin, every hole 200 μ L, 37 DEG C of closing 2h, three times with PBST washing lotion board-washing,
200 μ L of every hole, each 3min are patted dry every time；
(3) it is respectively configured 0,0.0625,0.125,0.25,0.5,1,2,4 μ g/L's with phosphate buffer (PBS)
NSOD standard solution.By standard solution and sample to be tested extracting solution, it is added separately in the ELISA Plate closed, often
50 μ L of hole, each sample repeat 3 holes, then the diluted anti-NSOD monoclonal antibody of 50 μ L, 1 ︰ 32000 is added in every hole, and 37 DEG C anti-
After answering 0.5h, board-washing is patted dry；
(4) sheep anti-mouse igg two that 100 μ L use the diluted HRP label of 1 ︰ of PBS 3000 containing 0.1% gelatin is added in every hole
Anti-, after 37 DEG C of reaction 0.5h, board-washing is patted dry；
(5) every hole is added 100 μ L TMB developing solutions, after 37 DEG C of colour developing 15min, 50 μ L 2M H of every hole addition2SO4It terminates
Liquid, 450 nm survey light absorption value；
(6) addition recycling and sample pre-treatments: 5 ± 0.01 g of the flesh of fish after weighing three parts of homogeneous in 50mL centrifuge tube,
20mL methanol and water volume ratio 1:1 mixed solution is added, vibrates 10min, 4000r/min is centrifuged 5min, discards liquid, residue
Middle addition 10mL 0.2mol/L hydrochloric acid, with homogenizer with 10000r/min homogeneous 1min after, then be separately added into 1ppb, 5ppb,
20ppb NSOD after whirling motion mixes 30s, then vibrates 30min.0.5% 40 DEG C of ultrasonic extraction 1h of formic acid acetonitrile 20mL are added,
4000 r/min are centrifuged 10min, and supernatant is transferred in 50mL centrifuge tube, and residue is repeated to extract 1 with 0.5% formic acid acetonitrile 20mL
It is secondary, merge supernatant, ammonium hydroxide 1mL is added, whirlpool mixes, and 4000r/min is centrifuged 10min, and supernatant is transferred to 100 mL brown pears
In shape bottle, 50 DEG C of rotary evaporations are done to close, with 0.1% formic acid-acetonitrile solution dissolved residue, n-hexane 3mL are added, whirlpool mixes
1 min, 4000r/min are centrifuged 10min, discard n-hexane layer, water phase is used as ELISA sample extracting solution.Using indirect competition
ELISA is added recovery test, and the rate of recovery is respectively 84%, 87%, 93%.
The configuration of solution:
Carbonate buffer solution (CBS): Na is weighed2CO31.59g NaHCO32.93g is mixed after being dissolved in a small amount of distilled water respectively
It closes, adds distilled water to mix to about 800mL, adjust pH value to 9.6, add distilled water to be settled to 1000 mL, 4 DEG C of storages are spare；
Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, 2.9 g Na2HPO4·12
H2O is dissolved in 800 mL pure water, with NaOH or HCl tune pH to 7.2~7.4, is settled to 1000 mL；
PBST: the PBS containing 0.05% polysorbas20；
TMB developing solution: A liquid: Na2HPO4·12H218.43 g of O, 9.33 g of citric acid, pure water are settled to 1000 mL；B
Liquid: 60 mg TMB are dissolved in 100 mL ethylene glycol.A, B liquid 1:5 mixing by volume is TMB developing solution, current existing mixed.
It is in summary only presently preferred embodiments of the present invention, practical range not for the purpose of limiting the invention.It is i.e. all
Equivalent changes and modifications made by content according to the present patent application range all should be technology scope of the invention.
Claims (3)
1. one plant of anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716, is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number are CGMCC No.14686, and the deposit date is on September 5th, 2017.
2. anti-furans benzene olefin(e) acid sodium monoclonal antibody, it is characterised in that: its deposit number as described in claim 1 is CGMCC
The anti-furans benzene olefin(e) acid sodium monoclonal antibody hybridoma cell strain SS0716 of No.14686, which secretes, to be generated.
3. the application of anti-furans benzene olefin(e) acid sodium monoclonal antibody described in claim 2, it is characterized in that: being applied in food safety
In middle furans benzene olefin(e) acid sodium retention analysis detection.
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