CN105505885A - Specific anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 and application thereof - Google Patents
Specific anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 and application thereof Download PDFInfo
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- CN105505885A CN105505885A CN201610056165.XA CN201610056165A CN105505885A CN 105505885 A CN105505885 A CN 105505885A CN 201610056165 A CN201610056165 A CN 201610056165A CN 105505885 A CN105505885 A CN 105505885A
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- acrylamide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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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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- 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 discloses a specific anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 and the application thereof and belongs to the technical field of food safety immunodetection. The monoclonal cell strain 2F6 is preserved in the China General Microbiological Culture Collection Center, CGMCC for short, with the preservation number of CGMCC No.10874. The crude drug acrylamide and 4-mercaptobenzoic acid are adopted to prepare hapten through chemical derivatization and addition, the hapten and BSA are coupled to prepare immunogen, and the hapten and OVA are coupled to prepare coating antigen. The specific anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 is obtained after a mouse is immunized. The monoclonal antibody secreted by the cell strain 2F6 only aims at derivative acrylamide specifically, and the crossing-over rates between the monoclonal antibody and acrylamide and between the monoclonal antibody and 4-mercaptobenzoic acid are 2.19% and 3.87% respectively. The anti-ceftiofur monoclonal antibody hybridoma cell strain has high detection sensitivity and compatibility, and specific detection of acrylamide can be achieved. The invention provides a new acrylamide derivatization method, and hapten synthesis is easy and efficient, so that a good specific monoclonal antibody cell strain is obtained.
Description
Technical field
The present invention relates to a strain specificity anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 and application thereof, belong to food safety technical field of immunoassay.
Background technology
Acrylamide (AA), English name is Acrylamide, and molecular formula is C
3h
5nO, molecular weight is 71.08, CAS accession number is 79-06-1.Starch food products easily produces acrylamide under high temperature (>120 DEG C) cooking.Research shows, human body is by number of ways contact acrylamides such as digestive tube, respiratory tract, skin mucosa.Acrylamide enters in body and is absorbed by the body by number of ways again, wherein absorbs the fastest through digestive tube.Enter acrylamide about 90% in human body by metabolism, to discharge through urine with original shape only on a small quantity.Acrylamide enters after in body, can be combined formation binding substances by the guanine in vivo on DNA, cause the damage of genetic materials such as transgenation.IARC has carried out evaluating and being classified as two class carcinogenss, i.e. mankind's suspect carcinogen to the carinogenicity of acrylamide.In the investigation of acrylamide crowd, the professional population and adventitious exposure that contact acrylamide are shown that acrylamide also has neurotoxic effect.In April, 2002 Swedish National Food management board and Stockholm University researchist take the lead in report, fried at some and roast starch food products, as detected acrylamide in fried potato, chrisps etc., and content exceedes in drinking-water more than 500 times that allows maximum limitation.So be necessary the method for quick setting up acrylamide.
Detection method mainly instrument detection method and the immunoassay detection method of current acrylamide, instrument detection method instrument cost is high, complicated operation, needs full-time staff to operate.And immune analysis method have low cost, high-throughput, highly sensitive, to features such as technician's relative requirement are low, be therefore applicable to the rapid screening of a large amount of sample.
Summary of the invention
The object of this invention is to provide the strain of a strain specificity anti-acrylamide monoclonal antibody hybridoma cell, the antibody prepared by this cell strain has good avidity and detection sensitivity to acrylamide, can be used for setting up acrylamide enzyme-linked immune detection method, or set up colloidal gold immunochromatographimethod technology method for quick.
Technical scheme of the present invention, a strain specificity anti-acrylamide monoclonal antibody hybridoma cell strain 2F6, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and be called for short CGMCC, deposit number is CGMCCNo.10874.
Anti-acrylamide monoclonal antibody, the specificity anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 that it is CGMCCNo.10874 by described deposit number secretes and produces.
The application of described anti-acrylamide monoclonal antibody, the application during its Determination of Residual Acrylamide in food safety detects.
The preparation basic step of hybridoma cell strain 2F6 cell strain provided by the invention is:
(1) immunogenic preparation and qualification: by former medicine acrylamide with adopt chemically derived additive process to prepare haptens to Thiosalicylic acid (4-MBA), derived products is identified by LC-MS, use active ester method coupling protein afterwards, be separated the small haptens of complete antigen and non-coupling by dialysis, complete antigen is identified by uv-absorbing scan method;
(2) immunity of mouse: after antigen and freund adjuvant emulsification completely, by subcutaneous multi-point injection immunity BALB/c mouse.First immunisation adopts Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, and during spurt immunity, immunizing dose is the half of a front immunizing dose, mixes directly to carry out abdominal injection afterwards with physiological saline; Each time immunization interval is three weeks.After third time immunity, interval blood sampling in a week detects serum titer and suppression;
(3) cytogamy and cell strain are set up: by polyoxyethylene glycol (PEG4000) method, mouse boosting cell and murine myeloma cell are merged, by HAT culture medium culturing, indirect ELISA is utilized to detect positive cell hole, and utilize indirect competitive ELISA method to measure the inhibition in positive cell hole further, carry out three subclones by limiting dilution assay to there being the positive cell hole preferably suppressed, final screening obtains hybridoma cell strain 2F6;
(4) qualification of hybridoma cell strain character: adopt mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody suit to measure; IC
50the mensuration of value, cross reacting rate and avidity passes through ELISA method.
Beneficial effect of the present invention: the anti-acrylamide monoclonal antibody hybridoma cell strain that the present invention obtains, not only has good detection sensitivity and avidity, can reach the object of specific detection acrylamide.The invention provides a kind of new acrylamide derivatization method, haptenic synthesis step is simple, efficient, in order to obtain good monoclonal antibody specific cell strain.
Biological material specimens preservation: the anti-acrylamide monoclonal cell strain 2F6 of a strain specificity, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date on May 19th, 2015, deposit number is CGMCCNo.10874.
Accompanying drawing explanation
Fig. 1 is the haptenic LC-MS qualification of acrylamide.
Fig. 2 is immunogenic ultra-violet absorption spectrum.1、BSA，2、AA-4-MBA，3、AA-4-MBA-BSA。
Fig. 3 is the canonical plotting of 2F6 monoclonal antibody to acrylamide AA.
Embodiment
The following examples of the present invention further illustrating only as content of the present invention, perhaps scope in not as limiting to the invention.Below by embodiment, the invention will be further described.
The present invention passes through acrylamide complete antigen immune mouse, pass through cytogamy, HAT selective medium is cultivated, and by indirect ELISA and indirect competitive ELISA screening cell conditioned medium, finally obtains monoclonal antibody hybridoma cell strain acrylamide being had to better avidity and sensitivity.
Embodiment 1: the preparation of hybridoma cell strain 2F6
(1) synthesis of complete antigen: by 15mg acrylamide, 20mg puts into reaction flask to Thiosalicylic acid and 12mg sodium bicarbonate, adds 8mL water dissolution, 50 DEG C of reaction 1h, hydrochloric acid adjusts pH=5, separates out white solid, filter, washing filter cake, dry product, i.e. acrylamide haptens.Get 5.2mg acrylamide haptens, add 2.5mgEDC and 2.0mgNHS, use DMF to dissolve, stirring at room temperature, activation 4h; Separately getting 5mgBSA is dissolved in the CB solution of 2mL, 0.05M, pH9.6, is dropwise added at a slow speed in the CB solution of BSA by the acrylamide haptens solution after above-mentioned activation, and after stirring at room temperature reaction is spent the night, dialyse three days, obtain immunogen for 4 DEG C ,-20 DEG C of packing are preserved.Acrylamide haptens LC-MS identifies as shown in Figure 1, and immunogenic ultra-violet absorption spectrum characterizes as shown in Figure 2.
(2) animal immune: select the BALB/c mouse in 6 ~ 8 week healthy age to carry out immunity.After getting acrylamide complete antigen (1mg/mL) and equivalent freund adjuvant emulsification evenly, by subcutaneous multi-point injection immunity BALB/c mouse, every only 100 μ L.First immunisation adopts Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, and during spurt immunity, immunizing dose is the half of a front immunizing dose, mixes directly to carry out abdominal injection afterwards with physiological saline; Each time immunization interval is three weeks.After third time immunity, interval blood sampling in a week detects serum titer and suppression; Select to suppress best mouse, after exempting from five, spurt immunity in 21 days, prepares to merge.
(3) cytogamy: in spurt immunity after three days, conveniently PEG(polyoxyethylene glycol, molecular weight is 4000) method carries out cytogamy, and concrete steps are as follows:
A, asepticly get mouse spleen, grinding also obtains splenocyte suspension by 200 order cell screen clothes, and carries out cell counting;
B, collection SP2/0 cell, be suspended in RPMI-1640 basic culture solution, carry out cell counting;
C, splenocyte and SP2/0 cell to be mixed according to the counting ratio of 5 ~ 10:1, centrifugal rear PEG merges, time 1min, afterwards according to from slowly to soon, add RPMI-1640 basic culture solution, be suspended in after centrifugal containing volume percent be 20% foetal calf serum, 2% 50 × HAT RPMI-1640 screening and culturing liquid in, be added to 96 porocyte culture plates, be placed in 37 DEG C, volumetric concentration is the CO of 5%
2incubator in cultivate.
(4) cell screening and cell strain are set up: carrying out RPMI-1640 screening and culturing liquid to fused cell on the 3rd day and partly change liquid in cytogamy, within 5th day, carry out with containing volume percent be 20% foetal calf serum, 1% the RPMI-1640 transition nutrient solution of 100 × HT entirely change liquid, got cell conditioned medium at the 7th day and screen.Screen in two steps: the first step first filters out positive cell hole with indirect ELISA, and second step selects AA, 4-MBA, AA-4-MBA to be standard substance, carries out inhibition mensuration with indirect competitive ELISA to positive cell.Select there is better suppression to AA-4-MBA, adopt limiting dilution assay to carry out subclone, use the same method and detect.In triplicate, cell strain 2F6 is obtained.
The preparation of embodiment 2 monoclonal antibody and qualification:
Get 8-10 BALB/c mouse in age in week, every mouse peritoneal injection paraffin oil 1mL; Every mouse peritoneal injection 1 × 10 afterwards in 7 days
6hybridoma 2F6, collected ascites from the 7th day, and by ascites by sad-saturated ammonium sulphate method purifying, the monoclonal antibody of acquisition is placed in-20 DEG C of preservations.
Use mouse monoclonal hypotype identification kit to carry out the qualification of immunoglobulin (Ig) hypotype to the monoclonal antibody that ascites purifying obtains, specifically as shown in table 1, its hypotype is IgG2a type.
Table 1
Use indirect competitive ELISA and indirect ELISA, measure monoclonal antibody to the IC of AA-4-MBA, AA and 4-MBA
50be respectively 31ng/mL, 1415.52ng/mL and 801.03ng/mL, cross reacting rate is all less than 10%, specifically as shown in table 2.Can be used for the specificity rapid detection of acrylamide.Monoclonal antibody to the typical curve of AA as shown in Figure 3.
Table 2
Embodiment 3 antibody is applied
Hybridoma cell strain 2F6 is applied to acrylamide ELISA by monoclonal antibody prepared by ascites in body and adds recovery test, concrete steps are as follows:
A, with carbonate buffer solution (CBS) the 0.5 μ g/mLAA-4-MBA-OVA that dilute as coating antigen bag by 96 hole enzyme plates, every hole 100 μ L, 37 DEG C of bags, by after 2h, wash plate three times by PBST washing lotion, at every turn every hole 250 μ L, and each 3min, pats dry;
B, use are closed containing the CBS of 0.2% gelatin, and every hole 200 μ L, 37 DEG C of closed 2h, wash plate three times by PBST washing lotion, each every hole 250 μ L, and each 3min, pats dry;
C, configure the acrylamide standardized solution of 0,0.82,2.46,7.4,22.22,66.67,200,600ng/mL respectively with phosphate buffered saline buffer (PBS).By standardized solution and detected sample extracting solution, join in the enzyme plate closed respectively, every hole 50 μ L, each sample repeats 3 holes, the anti-acrylamide monoclonal antibody that 1 ︰ 16000 that every hole adds 50 μ L again dilutes, after 37 DEG C of reaction 0.5h, washes plate and pats dry;
D, every hole add the 100 μ L sheep anti-mouse igg two that the HRP of PBS1 ︰ 3000 dilution containing mass concentration being 0.1% gelatin marks and resist, and after 37 DEG C of reaction 0.5h, wash plate and pat dry;
E, every hole add 100 μ LTMB nitrite ions, and after 37 DEG C of colour developing 15min, every hole adds 50 μ L, 2MH
2sO
4stop buffer, 450nm surveys light absorption value;
F, interpolation are reclaimed and sample pre-treatments: weigh 1g powdered milk sample as in 15mL tetrafluoroethylene centrifuge tube, add 300ng, 600ng and 1200ngAA respectively.Add extracting solution (1000mL0.2M Sodium phosphate dibasic and the mixing of 625mL0.1M citric acid) 10mL.The centrifugal 10min of 5000r/min, get and reset and add excessive in Thiosalicylic acid and sodium bicarbonate, 50 DEG C of reaction 1h adjust pH to be about 7 to be measured.After diluting 5 times with sample diluting liquid (0.01MPBS), as ELISA sample extracting solution, adopt indirect competitive ELISA to carry out interpolation recovery test, its rate of recovery is respectively 95.73%, and 85.32%, 76.34%.
The configuration of solution:
Carbonate buffer solution (CBS): take Na
2cO
31.59g, NaHCO
32.93g, mixes after being dissolved in a small amount of distilled water respectively, and add distilled water and mix to about 800mL, adjust pH to 9.6, add distilled water and be settled to 1000mL, 4 DEG C of storages are for subsequent use;
Phosphate buffered saline buffer (PBS): 8.00gNaCl, 0.2gKCl, 0.24gKH
2pO
4, 3.62gNa
2hPO
412H
2o, is dissolved in 800mL pure water, adjusts pH to 7.2 ~ 7.4, be settled to 1000mL with NaOH or HCl;
PBST: the PBS containing volumetric concentration being the Tween20 of 0.05%;
TMB nitrite ion: A liquid: Na
2hPO
412H
2o18.43g, citric acid 9.33g, pure water is settled to 1000mL; B liquid: 60mgTMB is dissolved in 100mL ethylene glycol.A, B liquid by volume 1 ︰ 5 mixing is TMB nitrite ion, existing with existing mixed.
Be only preferred embodiment of the present invention in sum, be not used for limiting practical range of the present invention.Namely all equivalences done according to the content of the present patent application scope change and modify, and all should be technology category of the present invention.
Claims (3)
1. a strain specificity anti-acrylamide monoclonal antibody hybridoma cell strain 2F6, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and be called for short CGMCC, deposit number is CGMCCNo.10874.
2. anti-acrylamide monoclonal antibody, is characterized in that: the specificity anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 that it is CGMCCNo.10874 by described deposit number secretes and produces.
3. the application of anti-acrylamide monoclonal antibody described in claim 2, is characterized in that: the application during its Determination of Residual Acrylamide in food safety detects.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110018308A (en) * | 2019-03-29 | 2019-07-16 | 江苏大学 | A kind of method that carbon quantum dot fluorescent immune method quickly detects acrylamide |
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| CN101285836A (en) * | 2007-04-10 | 2008-10-15 | 北京大学 | Acrylic amide complete antigen preparation and its ELISA quantitative determination method |
| CN104448002A (en) * | 2014-11-26 | 2015-03-25 | 北京农业职业学院 | Preparation method of acrylamide monoclonal antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285836A (en) * | 2007-04-10 | 2008-10-15 | 北京大学 | Acrylic amide complete antigen preparation and its ELISA quantitative determination method |
| CN104448002A (en) * | 2014-11-26 | 2015-03-25 | 北京农业职业学院 | Preparation method of acrylamide monoclonal antibody |
Non-Patent Citations (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110018308A (en) * | 2019-03-29 | 2019-07-16 | 江苏大学 | A kind of method that carbon quantum dot fluorescent immune method quickly detects acrylamide |
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