CN105838680A - Estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof - Google Patents
Estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof Download PDFInfo
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- CN105838680A CN105838680A CN201610324645.XA CN201610324645A CN105838680A CN 105838680 A CN105838680 A CN 105838680A CN 201610324645 A CN201610324645 A CN 201610324645A CN 105838680 A CN105838680 A CN 105838680A
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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
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Abstract
The invention provides an estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 and application thereof and belongs to the technical field of food safety immune detection. The estriol-specificity-resistant monoclonal antibody hybridoma cell strain NaN-2 is preserved in General Microbiology Center, Microbial Preservation Management Committee, China, and the preservation number is CGMCC No. 12016. An estriol-specificity-resistant monoclonal antibody is generated by secretion of the hybridoma cell strain NaN-2. The monoclonal antibody secreted by the cell strain has good specificity to estriol (the IC 50 value is 0.25 ng/mL) and can be used for estriol specificity detection in food safety. The obtained estriol-resistant monoclonal antibody cell strain has good detection sensitivity and appetency to estriol, a novel method for synthesizing estriol immunogen is further provided, the synthesis step of hapten is more simplified and effective, and a thought and method for synthesizing immunogen are provided for people to conduct research in the future.
Description
Technical field
The present invention relates to a strain anti-estriol monoclonal antibody specific hybridoma cell strain NaN-2 and application thereof, belong to
Food safety technical field of immunoassay.
Background technology
Estriol (E3) it is a kind of Endogenous steroids, it is estradiol (E2), estrone (E1) metabolite, in anemia of pregnant woman's body
E3Being mainly derived from Placenta Hominis, it is the important Testing index of late pregnancy fetal development situation and placental function, is also to make up
The common index of estrogen detection in product.Estriol is used for promoting domestic animal growth promoter in animal husbandry, and the improper of estriol makes
With may result in aquatic products, meat, eggs and the estriol residual contamination of breast apoplexy due to endogenous wind.China the most clearly forbids at present edible
The raising of animal adds estriol.
At present estriol detection uses high performance liquid chromatography, liquid chromatograph and MS, gas chromatography mass spectrometry method more, puts
Penetrate immunization, euzymelinked immunosorbent assay (ELISA), Chemiluminescence immunoassay, high performance capillary electrophoresis etc..Domestic general employing GB/T 21981-2008
" hormone residues detection method in animal-derived food: liquid chromatograph one mass spectrum one mass spectrography " detects, need to be through homogenizing, enzyme
The steps such as solution, extraction, solid phase extraction concentration purification, Instrument measuring, interior scalar quantity.Mainly use radioimmunology detection
Estriol, there is radiosiotope waste disposal problem and the shortcoming such as label storage life is short in the method.Above-mentioned detection method
Quantitative analysis can be carried out and there is relatively low detection limit, but typically requiring the instrument of costliness and complicated operation, pre-treatment and
The detection time is long, seriously constrains the popularization of these detection methods.And immune analysis method has low cost, high flux, Gao Ling
Quick, to features such as technical staff's relative requirement are low, be therefore applicable to the rapid screening of a large amount of sample.It is an object of the invention to carry
Preparation method for a kind of monoclonal antibody hybridoma cell strain that estriol is had higher affinity and detection sensitivity.For
The research and development of indirect competitive ELISA test kit and colloidal gold strip are promoted and are laid a good foundation.
Summary of the invention
It is an object of the invention to provide a strain anti-estriol monoclonal antibody specific hybridoma cell strain, by this cell strain
The antibody of preparation has preferable affinity and detection sensitivity to estriol, can be used to set up estriol enzyme linked immunosorbent detection
Method, or set up colloidal gold immunochromatographimethod technology method for quick.
Technical scheme, a strain anti-estriol monoclonal antibody specific hybridoma cell strain NaN-2, preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, being called for short CGMCC, deposit number is CGMCC
No.12016。
Anti-estriol monoclonal antibody specific, it is special by the anti-estriol that described deposit number is CGMCC No.12016
Specific monoclonal antibodies hybridoma cell strain NaN-2 secretes generation.
The application of described anti-estriol monoclonal antibody specific: it is answering in estriol residue detection in food safety
With.
The preparation basic step of the NaN-2 cell strain that the present invention provides is:
(1) immunogenic preparation and qualification: estriol and 6-bromocaproic acid react preparation hapten Han carboxyl, pass through carbodiimides
Method is connected with the amino of protein carrier, after reaction terminates, separates complete antigen and the small haptens of non-coupling by dialysis,
Complete antigen is identified by uv absorption scan method；
(2) immunity of mice: after complete to immunogen and freund adjuvant emulsifying, little by subcutaneous multi-point injection immunity BALB/c
Mus.First immunisation uses Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, and during spurt immunity, immunizing dose is front
The half of primary immune response dosage, directly carries out lumbar injection with normal saline after mixing homogeneously；Each time immunization interval is three weeks.The
After three immunity, it is spaced one week blood sampling detection serum titer and suppression；
(3) cell merges with cell strain foundation: by Polyethylene Glycol (PEG 4000) method, make mouse boosting cell and mouse myeloma
Cell merges, and by HAT culture medium culturing, utilizes indirect ELISA to detect positive cell hole, and further with indirect competition
ELISA method measures the inhibition in positive cell hole, by limiting dilution assay to there being the positive cell hole preferably suppressed to carry out three
Secondary sub-clone, final screening obtains hybridoma cell strain NaN-2；
(4) qualification of hybridoma cell strain character: use mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody suit to measure；
IC50The mensuration of value, cross reacting rate and affinity passes through ELISA method.
Beneficial effects of the present invention: the anti-estriol cell strain of monoclonal antibody that the present invention obtains, has preferably estriol
Detection sensitivity and affinity, additionally provide a kind of new immunogenic method of synthesis estriol, haptenic synthesis step
More simplify, effectively, provide thinking and the method for synthetic immunogen for the research of people from now on.
Biological material specimens preservation: monoclonal cell strain NaN-2, has been preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, is called for short CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microorganism
Institute, deposit number is CGMCC No.12016, preservation date on January 20th, 2016.
Accompanying drawing explanation
The immunogenic ultra-violet absorption spectrum of Fig. 1 characterizes.
The standard of estriol is suppressed curve by Fig. 2 NaN-2 monoclonal antibody.
Detailed description of the invention
The following examples of the present invention are only used as further illustrating of present invention, it is impossible to as in the restriction of the present invention
Perhaps scope.Below by embodiment, the invention will be further described.
The present invention is by by estriol complete antigen immune mouse, merging by cell, and HAT selective medium is cultivated,
Screen cell conditioned medium by indirect ELISA and indirect competitive ELISA, finally given and estriol is had preferable affinity and detection
The monoclonal antibody hybridoma cell strain of sensitivity.
The preparation of embodiment 1 hybridoma cell strain NaN-2
(1) synthesis of complete antigen: 300mg(1.10 mmol) E3It is dissolved in the dimethyl sulfoxide that 6mL is dried, adds 1g KOH powder
End.300mg bromocaproic acid is added after stirring 5min.Continue stirring, after reaction 2h, add 50mL frozen water.Ethyl acetate extraction and recovery
Unreacted E3.Aqueous phase 2mol/L HCl is acidified, and white precipitate occurs.Sand core funnel filters, and precipitate distilled water is washed till
Neutrality, vacuum drying.Methanol-chloroform recrystallization, obtains clear crystal, is estriol hapten.Take 4.5mg above-mentioned half to resist
Former, add 2.0mg EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) and 1.0mg NHS(N-hydroxyl
Butanimide), use DMF(N, dinethylformamide) dissolve, it is stirred at room temperature, activates 4h；Separately take 5mg BSA(Ox blood serum
Albumin) it is dissolved in the CB(carbonate buffer solution of 2mL, 0.05M, pH9.6) in solution, above-mentioned activating solution is added dropwise over
In BSA solution, after reaction overnight is stirred at room temperature, dialysing three days for 4 DEG C ,-20 DEG C of subpackages preserve.
(2) animal immune: select the BALB/c mouse of 6～8 healthy week old to carry out immunity.Take estriol complete antigen
(1mg/mL) after uniform with equivalent freund adjuvant emulsifying, by subcutaneous multi-point injection immunity BALB/c mouse, every 100 μ L.First
Secondary immunity use Freund's complete adjuvant, booster immunization use freund 's incomplete adjuvant, spurt immunity time immunizing dose be front once
The half of immunizing dose, directly carries out lumbar injection with normal saline after mixing homogeneously；Each time immunization interval is three weeks.For the third time
After immunity, it is spaced one week blood sampling detection serum titer and suppression；Selecting the mice that suppression is best, after exempting from five, spurt in 21 days is exempted from
Epidemic disease, prepares to merge.
(3) cell merges: after spurt immunity three days, according to conventional PEG(Polyethylene Glycol, molecular weight is 4000) method enters
Row cell merges, and specifically comprises the following steps that
A, aseptic take mouse spleen, grind and obtain splenocyte suspension by 200 mesh cell screen clothes, and carrying out cell counting；
B, collection SP2/0 cell, be suspended in RPMI-1640 basic culture solution, carry out cell counting；
C, by splenocyte and SP2/0 cell according to the counting ratio mixing than 5-10:1, centrifugal after merge with PEG, time 1min,
Afterwards according to from slowly to soon, add RPMI-1640 basic culture solution, centrifugal after be suspended in containing 20% hyclone, 2% 50 ×
In the RPMI-1640 screening and culturing liquid of HAT, it is added to 96 porocyte culture plates, is placed in 37 DEG C, 5%CO2Incubator in cultivate.
(4) cell screening is set up with cell strain: fused cell was carried out RPMI-1640 screening in the 3rd day what cell merged
Culture fluid partly changes liquid, within the 5th day, carries out carrying out entirely with the RPMI-1640 transition culture fluid containing 20% hyclone, the 100 × HT of 1%
Change liquid, took cell conditioned medium at the 7th day and screen.Screen in two steps: the first step first filters out positive cell with indirect ELISA
Hole, second step is selected estriol to be standard substance, with indirect competitive ELISA, positive cell hole is carried out inhibition mensuration.Select
Estriol is had the cell hole of preferably suppression, uses limiting dilution assay to carry out sub-clone, detect by same method.Repeat
Three times, it is thus achieved that cell strain NaN-2.
(5) preparation of monoclonal antibody and qualification: take 8-10 week old BALB/c mouse, every mouse peritoneal injection paraffin oil
1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma, started to collect ascites from the 7th day, by ascites by pungent
Acid-saturated ammonium sulfate method purification, it is thus achieved that monoclonal antibody be placed in-20 DEG C of preservations.
Use mouse monoclonal hypotype identification kit that the monoclonal antibody that ascites purification obtains carries out immunoglobulin sub-
Type is identified, its hypotype is IgG1 type.
Use indirect competitive ELISA method, measure the monoclonal antibody IC to estriol50It is 0.25 μ g/L, can be used for female three
The specificity of alcohol quickly detects.
(6) antibody application
Hybridoma cell strain NaN-2 is applied to estriol ELISA by monoclonal antibody prepared by internal ascites and adds recovery
Test, specifically comprises the following steps that
6.1 the 0.1 μ g/mL diluted with carbonate buffer solution (CBS) is coated 96 hole ELISA Plate as coating antigen, every hole 100 μ L,
After 37 DEG C are coated 2 h, wash plate three times by PBST washing liquid, the most every hole 250 μ L, each 3 min, pat dry；
6.2 close with the CBS containing 0.2% gelatin, every hole 200 μ L, close 2 h, wash plate three times by PBST washing liquid, often for 37 DEG C
Secondary every hole 250 μ L, each 3 min, pat dry；
6.3 are respectively configured 0 with phosphate buffer (PBS), the estriol standard solution of 0.2,0.5,1,2,5,10,20ng/mL.
By standard solution and detected sample extracting solution, it is added separately in the ELISA Plate closed, every hole 50 μ L, each sample
Product repeat 3 holes, more every hole adds the anti-estriol monoclonal antibody of 50 μ L 1 16000 dilutions, after 37 DEG C of reaction 0.5 h,
Wash plate to pat dry；
6.4 every holes add the sheep anti-mouse igg two of the HRP labelling that 100 μ L dilute with the PBS 1 3000 containing 0.1% gelatin and resist,
After 37 DEG C of reaction 0.5 h, wash plate and pat dry；
6.5 every holes add 100 μ L TMB nitrite ions, and after 37 DEG C of colour developing 15 min, every hole adds 50 μ L 2M H2SO4Stop buffer,
450 nm survey light absorption value；
6.6 add reclaim and sample pre-treatments: weigh 1g milk sample and insert in 50mL centrifuge tube, add respectively 0.2ng, 0.4
Ng and 1ng estriol.After sample adds 25 mL 80% methanol PBS solution (w/v) concussions uniformly, supersound extraction 15 min,
After standing, supernatant is with 0.45 μm membrane filtration, after filtrate dilutes 4 times with the PBS containing 0.01% gelatin, as ELISA sample extraction
Liquid, uses indirect competitive ELISA to be added recovery test, and its response rate is respectively 83%, and 87%, 84%.
The configuration of solution:
Carbonate buffer solution (CBS): weigh Na2CO31.59g, NaHCO32.93g, mixes after being dissolved in a small amount of distilled water respectively,
Adding distilled water to mix to about 800mL, adjust pH value to 9.6, add distilled water and be settled to 1000mL, 4 DEG C of storages are standby；
Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, 3.62 g Na2HPO4·12
H2O, is dissolved in 800 mL pure water, adjusts pH to 7.2～7.4 with NaOH or HCl, is settled to 1000 mL；
PBST: containing the PBS of 0.05% Tween 20；
TMB nitrite ion: A liquid: Na2HPO4·12H2O 18.43g, citric acid 9.33g, pure water is settled to 1000 mL；B liquid: 60
Mg TMB is dissolved in 100 mL ethylene glycol.15 mixing by volume of A, B liquid are TMB nitrite ion, existing with existing mixed.
The hypotype of table 1.NaN-2 monoclonal antibody is identified
Table 2.NaN-2 monoclonal antibody is to estrone, the IC of estradiol50And cross reacting rate
It is only presently preferred embodiments of the present invention in sum, is not used for limiting the practical range of the present invention.I.e. Fan Yiben
Equivalence change and the modification that the content of patent application the scope of the claims is made, all should be the technology category of the present invention.
Claims (3)
1. a strain anti-estriol monoclonal antibody specific hybridoma cell strain NaN-2, has been preserved in Chinese microorganism strain and has protected
Hiding administration committee's common micro-organisms center, be called for short CGMCC, deposit number is CGMCC No.12016.
The most anti-estriol monoclonal antibody specific, it is characterised in that: it is CGMCC No.12016's by described deposit number
Anti-estriol monoclonal antibody specific hybridoma cell strain NaN-2 secretes generation.
3. the application of anti-estriol monoclonal antibody specific described in claim 2, it is characterised in that: it is female in food safety
Application in triol residue detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106680516A (en) * | 2016-12-05 | 2017-05-17 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay (ELISA) kit for detecting estriol (E3) and application thereof |
| CN116836280A (en) * | 2023-09-01 | 2023-10-03 | 北京纳百生物科技有限公司 | Anti-estriol monoclonal antibody, detection reagent and application thereof |
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| WO2010009514A1 (en) * | 2008-07-25 | 2010-01-28 | Newcastle Innovation Limited | Methods and kits for predicting the onset of labour |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106680516A (en) * | 2016-12-05 | 2017-05-17 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay (ELISA) kit for detecting estriol (E3) and application thereof |
| CN106680516B (en) * | 2016-12-05 | 2018-07-06 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of estriol |
| CN116836280A (en) * | 2023-09-01 | 2023-10-03 | 北京纳百生物科技有限公司 | Anti-estriol monoclonal antibody, detection reagent and application thereof |
| CN116836280B (en) * | 2023-09-01 | 2023-10-31 | 北京纳百生物科技有限公司 | Anti-estriol monoclonal antibody, detection reagent and application thereof |
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