CN104448002A - Preparation method of acrylamide monoclonal antibody - Google Patents
Preparation method of acrylamide monoclonal antibody Download PDFInfo
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- CN104448002A CN104448002A CN201410696602.5A CN201410696602A CN104448002A CN 104448002 A CN104448002 A CN 104448002A CN 201410696602 A CN201410696602 A CN 201410696602A CN 104448002 A CN104448002 A CN 104448002A
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Abstract
The invention discloses a preparation method of an acrylamide monoclonal antibody, and belongs to the technical field of preparation of antibodies. The preparation method comprises preparation of an acrylamide antigen and preparation of the acrylamide monoclonal antibody, wherein the antigen comprises a semiantigen, an immunogen and a coating antigen; the preparation of the acrylamide monoclonal antibody comprises the following steps: animal immunization, cell fusion and cloning, ascites preparation and purification of the monoclonal antibody. The preparation method is simple, and the immune effect is good; the acrylamide monoclonal antibody can be secreted efficiently and stably; the acrylamide monoclonal antibody has the characteristics of high titer, strong specificity and low cost.
Description
Technical field
The present invention relates to a kind of preparation method of antibody, the particularly preparation of the immunodetection monoclonal antibody specific of suspect carcinogen acrylamide in a kind of fried food product.
Background technology
Acrylamide is commonly called as " the third poison ", is a kind of white crystal, molecular formula CH
2cHCONH
2odorlessness, fusing point is 84.5 DEG C, in very easily water-soluble, ethanol, methyl alcohol, acetone, dme and trichloromethane, be slightly soluble in benzene, toluene, containing amido and double-strand in acrylamide molecules, chemical property is quite active, Hofmann reaction, hydrolysis reaction, Michael-type addition reaction and polyreaction etc. can occur, and are the raw materials of synthesis polyacrylamide.In April, 2002, Swedish National Food management board holds a press conference, and discloses in its official website in starch food products that is fried at some or that bake and detect high level acrylamide, and content exceedes in drinking-water 500 times that allow maximum limitation.A large amount of animal experiment shows, acrylamide actuatable thing multi viscera tumour, comprises mammary gland, Tiroidina, testis, suprarenal gland, nervus centralis, oral cavity, uterus, pituitary body etc.The carinogenicity of IARC to acrylamide is evaluated, and is classified as two class carcinogenss, i.e. mankind's suspect carcinogen.
Because acrylamide has potential neurotoxicity, genetoxic and carinogenicity, cause the very big concern of global researchist.And the inspection of Acrylamide in Foods is classified as " 15 " national major scientific and technological project " food safety gordian technique " problem by China, Ministry of Health's food safety plan also using the foundation of Acrylamide in Foods detection method and China's food acrylamide Morbidity investigation as an integral part.
The detection method of current acrylamide mainly contains high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), LC-MS analysis method (LC-MS), liquid chromatography-tandem mass spectrometry analytical method (LC-MS-MS) etc.Although these method sensitivity are high, also exist sample pre-treatments loaded down with trivial details, detect length consuming time, complicated operation, and high in cost of production shortcoming, be difficult to the needs meeting Rapid identification diagnosis.
In recent years, there is high specificity, enzyme-linked immunosorbent assay (ELISA) that is simple and quick, sensitivity advantages of higher is in widespread attention, in food safety rapid detection, show the advantage of its uniqueness.And the research report of its immunodetection aspect of acrylamide is less in fried food product, patent does not have, and therefore, is necessary exploitation acrylamide rapid screening immunoassay technology.Because acrylamide is small-molecule substance, itself does not possess immunogenicity, immunology belongs to haptens, antibody can not be produced by stimulating animal body, only have and become complete antigen that animal body just can be impelled to produce immune response with macromolecular substance coupling, thus obtain antibody, therefore, the synthesis complete antigen that also purifying immune performance is good researches and develops the primary committed step of its immunoassay technology and technological difficulties.What current acrylamide immunoassay technology only rested on the development of polyclonal antibody and detection method sets up aspect, and specificity is poor.
Summary of the invention
The present invention be directed to the deficiency of existing acrylamide detection technique, provide a kind of synthesis of acrylamide antigen and the preparation method of monoclonal antibody, make it have that antibody titer is high, high specificity, feature with low cost.
Acrylamide monoclonal antibody of the present invention is achieved by the following technical programs:
1, the preparation of acrylamide antigen
(1) haptenic synthesis
The concrete synthetic method of haptens is as follows:
Utilize Michael reaction, acrylamide and a Thiosalicylic acid are obtained by reacting adduct under the catalysis of borax in water, and mass spectrum (mass) qualification result is correct.Haptenic structure is:
be called for short AM.
(2) immunogenic synthesis
The synthetic method that immunogen is concrete is as follows:
Haptenic carboxyl is by NHS (N-hydroxy-succinamide), EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) activation amino condensation that is rear and KLH (Keyhole Limpet Hemocyanin) forms amido linkage, thus forms stable artificial antigen---immunogen.
(3) synthesis of coating antigen, concrete synthetic method is as follows:
Haptenic carboxyl forms amido linkage by NHS, EDC activation amino condensation that is rear and BSA, thus forms stable coating antigen.
2, the preparation of acrylamide monoclonal antibody
(1) animal immune
Carry out three minor ticks immunity with the Balb/c mouse of acrylamide immunogen to 8-10 age in week, after the 6th immunity, carry out a booster immunization again, swash intravital lymphocyte, produce acrylamide specific antibody.
(2) cytogamy and clone
The splenocyte and myeloma cell SP2/0 volume that produce the Balb/c mouse of acrylamide specific antibody are merged in 5:1 ratio, 37 DEG C, carbonic acid gas volumetric concentration be 5% incubator cultivate 10 days after, adopt direct competive ELISA to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is adopted to carry out subclone by positive cell after sensing 2 days, until obtain the stable hybridoma cell strain can secreting acrylamide monoclonal antibody.
(3) ascites of monoclonal antibody is prepared and purifying
With whiteruss sensitization Balb/c mouse, clean collecting the hybridoma cell strain DMEM (Dulbecco'sModified Eagle Medium) obtained, and be injected into the abdominal cavity of mouse, the inactive and abdominal cavity enlargement of visible mouse state after a week.The ascites collected, with sad-thick purifying of saturated ammonium sulphate method, carries out purifying (being enriched the specific monoclonal antibody of acrylamide in ascites) with affinity chromatography, and the ascites after purifying is put into-20 DEG C of environment and preserved.Ascites is dialysed under 4 DEG C of environment through 0.01mol/L NaCl before use.
The invention has the advantages that:
(1) structure of acrylamide is transformed by the present invention, obtains haptens, then has synthesized immunogen with carrier protein couplet, and the method is simple, good immune effect;
(2) the present invention obtains hybridoma cell strain by screening, can efficiently, the anti-acrylamide monoclonal antibody of stably excreting, this antibody has height of tiring, high specificity, feature with low cost.
Accompanying drawing explanation
Fig. 1 is haptens mass spectrum in the present invention;
Fig. 2 is immunogen UV scanning collection of illustrative plates in the present invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.Following examples only for illustration of the present invention, but are not used for limiting the scope of the invention.
embodiment 1:
The first step, the preparation of acrylamide antigen
(1) the haptenic synthesis of acrylamide
The NaHCO of 92.4mg is added in the flask of 10ml
3, the water of 4ml, adds borax 38.14mg, a Thiosalicylic acid 154.19mg, acrylamide 84.1mg after dissolving completely, and 40 DEG C are reacted 2 hours; 1M hydrochloric acid adjusts pH=5 ~ 6, separates out white solid, filters, washing filter cake, dry 200mg product, i.e. acrylamide haptens.Mass spectrum mass identifies product ,-M=223.8 (target product MW=225 is M-1 signal), and haptens structure is correct, as Fig. 1.
(2) immunogenic synthesis
Take acrylamide haptens 6.6mg and be dissolved in 2ml DMF (N, dinethylformamide), add 10.2mg NHS (N-hydroxy-succinamide) and 11.4mg EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride), stirring at room temperature 2h; 50mg KLH (Keyhole Limpet Hemocyanin), 66mg sodium bicarbonate is dissolved in 6ml water, in cooling bath, pharmacological activation (NHS, EDC) is dropwise added albumen, stirring at room temperature 24h, reaction product is loaded 1 clean dialysis tubing of distilled water flushing (15cm), the PBS (phosphate buffered saline(PBS)) of 1L pH7.2 dialyses 3 days, be placed on 4 DEG C of stirring (100rpm) dialysis, change liquid every day 3 times (morning, noon and afternoon respectively once), change liquid altogether 9 times, to be dialysed the centrifugal 6min of product 4500rpm, obtain immunogen (AM-KLH).The packing of 1ml/ pipe, immunogen numbered ,-20 DEG C save backup.
Scanning spectra shows, and the photoabsorption of albumen at 250nm wavelength place through hapten conjugation increases, and shows the haptens containing benzene ring structure and carrier protein couplet success.It is 7:1 that ultraviolet spectrophotometry measures coupling ratio, as Fig. 2.
(3) synthesis of coating antigen;
Take acrylamide haptens 6.6mg and be dissolved in 2ml DMF, add 10.2mgNHS (first adding) and 11.4mgEDC, stirring at room temperature 2h; 50mg BSA, 66mg sodium bicarbonate is dissolved in 6ml water, in cooling bath, pharmacological activation is dropwise added albumen, stirring at room temperature 24h, reaction product is loaded 1 clean dialysis tubing of distilled water flushing (15cm), 1L PBS (1 ×, pH7.2) dialyse 3 days, be placed on 4 DEG C of stirring (100rpm) dialysis, change liquid every day 3 times (morning, noon and afternoon respectively once), change liquid altogether 9 times, to be dialysed the centrifugal 6min of product 4500rpm, the packing of 1ml/ pipe, numbered by coating antigen ,-20 DEG C save backup.
Second step, the preparation of acrylamide monoclonal antibody
(1) animal immune
With PBS (phosphate buffered saline(PBS)) liquid of aseptic pH7.0, the immunogen (AM-KLH) of synthesis is dissolved, then make oil emulsion vaccine in immunogen and freund's adjuvant volume in the ratio of 1:3 is fully emulsified.First immunisation Freund's complete adjuvant, carries out immunity to Balb/c mouse in 8 ~ 10 week age, neck dorsal sc multi-point injection, and immunizing dose is 112 μ L/.After 14 days, two exempt from, and adopt Freund's incomplete adjuvant, and immunizing dose 56 μ L/ only; Later 4 immunity are exempted from two; 6 exempt from rear booster immunization, do not add adjuvant, directly adopt AM-KLH and physiological saline immunity.Immunization protocol is as shown in table 1 below:
Table 1 Balb/c mouse immunization protocol
(2) cytogamy and cultivation
The splenocyte of the Balb/c mouse producing acrylamide specific antibody is mixed by 5:1 volume ratio with myeloma cell SP2/0, by the cell centrifugation (1500rpm mixed, centrifugal 12min), dry supernatant liquor, sedimentation cell block bullet is become pasty state, put 37 DEG C of water-baths, 1ml fusogen is added in 1min, fusogen is polyoxyethylene glycol (PEG) 0.8mL, mix cell gently, leave standstill 90S, add DMEM (Dulbecco's Modified Eagle Medium) serum free medium termination reaction, adding speed is front 2min 1ml/min, 3min 2ml/min, 4min 5ml/min, now can pour residue DMEM into centrifuge tube, final volume is 25 ~ 30ml, the centrifugal 7min of 800rpm, abandoning supernatant.With 20 ~ 50ml HAT substratum re-suspended cell, cover plant is in containing feeder cell (every hole 100 μ l) 96 porocyte culture plates, every hole 150 μ l.Put 37 DEG C, cultivate in the incubator of carbonic acid gas volumetric concentration 5%.
(3) cell screening and subclone
When cell grows to the 1/2-1/3 of hole floorage, adopt direct competive ELISA to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is adopted to carry out subclone by positive cell after sensing 2 days, until obtain the stable hybridoma cell strain can secreting acrylamide monoclonal antibody.This hybridoma cell strain carried out enlarged culturing and go down to posterity, generation acrylamide monoclonal antibody specific that can be endless.
(4) ascites of monoclonal antibody is prepared and purifying
A. mouse sensitization: whiteruss sensitization Balb/c mouse, 6 ~ 8 weeks, volume injected 500 μ l/ only.Ascites can be prepared after 7 days.
B. cell is injected: collect hybridoma and wash cell twice with DMEM, getting 10
5-10
6individual cell infusion in mouse peritoneal, the inactive and abdominal cavity enlargement of mouse of visible mouse state after a week.
C. ascites collection: injection cell gathered ascites with asepsis injector in mouse peritoneal to the mouse of abdominal cavity enlargement after 5 days, gathered once every to two days, so repeatedly repeatedly gathered until mouse natural death.
D. ascites purifying: the ascites collected, with sad-thick purifying of saturated ammonium sulphate method, affinity chromatography carries out purifying, and the ascites after purifying is put into-20 DEG C of environment and preserved, ascites to be dialysed 48h through 0.01mol/LNaCl before use under 4 DEG C of environment, middlely changes liquid once every 6 ~ 8h.
(5) mensuration of antibody titer
On enzyme plate, every hole bag is by the diluted coating antigen of 100 μ l, hatches 2h for 37 DEG C, after washing plate 3 times with washings, every hole adds confining liquid 150 μ L, 37 DEG C of closed 1h, after washing plate 3 times, add 50ul PBS (phosphate buffered saline(PBS)), 50ul supernatant liquor 37 DEG C of lucifuges cultivate 30min; After washing plate 3 times, add the ELIAS secondary antibody 100 μ l by conventional ELIAS secondary antibody diluted 5000 times, 37 DEG C of lucifuges cultivate 30min; After washing plate 3 times, add AB liquid 100 μ l, lucifuge reaction 15min, adds 50 μ L stop buffers and stops; Measure absorbancy by microplate reader, absorbing wavelength is 450/630nm.Described AB liquid is mixed according to the volume ratio of 1:1 by nitrite ion A and nitrite ion B, and nitrite ion A and nitrite ion B directly can buy and obtain.
Claims (3)
1. a preparation method for acrylamide monoclonal antibody, is characterized in that:
The first step, the preparation of acrylamide antigen;
(1.1) haptenic synthesis;
The concrete synthetic method of haptens is as follows:
(1.2) immunogenic synthesis;
Haptenic carboxyl forms amido linkage by NHS, EDC activation amino condensation that is rear and KLH, thus forms stable artificial antigen---immunogen;
(1.3) synthesis of coating antigen, concrete synthetic method is as follows:
Haptenic carboxyl forms amido linkage by NHS, EDC activation amino condensation that is rear and BSA, thus forms stable coating antigen.
Second step, the preparation of acrylamide monoclonal antibody;
(2.1) animal immune;
Carry out three minor ticks immunity with the Balb/c mouse of acrylamide immunogen to 8-10 age in week, after the 6th immunity, carry out a booster immunization again, swash intravital lymphocyte, produce acrylamide specific antibody;
(2.2) cytogamy and clone;
The splenocyte and myeloma cell SP2/0 volume that produce the Balb/c mouse of acrylamide specific antibody are merged in 5:1 ratio, 37 DEG C, carbonic acid gas volumetric concentration be 5% incubator cultivate 10 days after, adopt direct competive ELISA to measure cell conditioned medium liquid, screen positive hole; Limiting dilution assay is adopted to carry out subclone by positive cell after sensing 2 days, until obtain the stable hybridoma cell strain can secreting acrylamide monoclonal antibody;
(2.3) ascites of monoclonal antibody is prepared and purifying;
With whiteruss sensitization Balb/c mouse, clean collecting the hybridoma cell strain DMEM that obtains, and be injected into the abdominal cavity of mouse, the inactive and abdominal cavity enlargement of mouse state as seen after a week; The ascites collected, with sad-thick purifying of saturated ammonium sulphate method, carries out purifying with affinity chromatography, and the ascites after purifying is put into-20 DEG C of environment and preserved.
2. the preparation method of a kind of acrylamide monoclonal antibody according to claim 1, is characterized in that: described in step (2.3), ascites is dialysed under 4 DEG C of environment through 0.01mol/L NaCl before use.
3. the preparation method of a kind of acrylamide monoclonal antibody according to claim 1, is characterized in that:
The first step, the preparation of acrylamide antigen;
(1.1) the haptenic synthesis of acrylamide;
The NaHCO of 92.4mg is added in the flask of 10ml
3, the water of 4ml, adds borax 38.14mg, a Thiosalicylic acid 154.19mg, acrylamide 84.1mg after dissolving completely, and 40 DEG C are reacted 2 hours; 1M hydrochloric acid adjusts pH=5 ~ 6, separates out white solid, filters, washing filter cake, dry 200mg product, i.e. acrylamide haptens;
(1.2) immunogenic synthesis;
Take acrylamide haptens 6.6mg and be dissolved in 2ml DMF, add 10.2mg NHS and 11.4mg EDC, stirring at room temperature 2h; 50mg KLH, 66mg sodium bicarbonate is dissolved in 6ml water, in cooling bath, pharmacological activation is dropwise added albumen, stirring at room temperature 24h, reaction product is loaded 1 clean dialysis tubing of distilled water flushing, the PBS of 1L pH7.2 dialyses 3 days, be placed on 4 DEG C and stir dialysis, change liquid every day 3 times, change liquid altogether 9 times, to be dialysed the centrifugal 6min of product 4500rpm, obtain immunogen;
(1.3) synthesis of coating antigen;
Take acrylamide haptens 6.6mg and be dissolved in 2ml DMF, add 10.2mgNHS and 11.4mg EDC, stirring at room temperature 2h; 50mg BSA, 66mg sodium bicarbonate is dissolved in 6ml water, in cooling bath, pharmacological activation is dropwise added albumen, stirring at room temperature 24h, reaction product is loaded 1 clean dialysis tubing of distilled water flushing, 1L PBS dialyses 3 days, be placed on 4 DEG C and stir dialysis, change liquid every day 3 times, change liquid altogether 9 times, to be dialysed the centrifugal 6min of product 4500rpm, obtain coating antigen;
Second step, the preparation of acrylamide monoclonal antibody;
(2.1) animal immune;
With the PBS liquid of aseptic pH7.0, the immunogen of synthesis is dissolved, then make oil emulsion vaccine in immunogen and freund's adjuvant volume in the ratio of 1:3 is fully emulsified; First immunisation Freund's complete adjuvant, carries out immunity to Balb/c mouse in 8 ~ 10 week age, neck dorsal sc multi-point injection, and immunizing dose is 112 μ L/; After 14 days, two exempt from, and adopt Freund's incomplete adjuvant, and immunizing dose 56 μ L/ only; Later 4 immunity are exempted from two; 6 exempt from rear booster immunization, do not add adjuvant, directly adopt AM-KLH and physiological saline immunity;
(2.2) cytogamy and cultivation;
The splenocyte of the Balb/c mouse producing acrylamide specific antibody is mixed by 5:1 volume ratio with myeloma cell SP2/0, by the cell centrifugation mixed, dry supernatant liquor, sedimentation cell block bullet is become pasty state, put 37 DEG C of water-baths, 1ml fusogen is added in 1min, fusogen is polyoxyethylene glycol 0.8mL, mix cell gently, leave standstill 90S, add DMEM serum free medium termination reaction, adding speed is front 2min 1ml/min, 3min 2ml/min, 4min5ml/min, now can pour residue DMEM into centrifuge tube, final volume is 25 ~ 30ml, the centrifugal 7min of 800rpm, abandoning supernatant, with 20 ~ 50ml HAT substratum re-suspended cell, cover plant is in containing feeder cell 96 porocyte culture plate, every hole 150 μ l, put 37 DEG C, cultivate in the incubator of carbonic acid gas volumetric concentration 5%,
(2.3) cell screening and subclone;
When cell grows to 1/2 ~ 1/3 of hole floorage, adopt direct competive ELISA to measure cell conditioned medium liquid, screen positive hole; Limiting dilution assay is adopted to carry out subclone by positive cell after sensing 2 days, until obtain the stable hybridoma cell strain can secreting acrylamide monoclonal antibody;
(2.4) ascites of monoclonal antibody is prepared and purifying;
A. mouse sensitization: whiteruss sensitization Balb/c mouse, 6 ~ 8 weeks, volume injected 500 μ l/ was only; Ascites is prepared after 7 days;
B. cell is injected: collect hybridoma and wash cell twice with DMEM, getting 10
5~ 10
6individual cell infusion in mouse peritoneal, the inactive and abdominal cavity enlargement of mouse of mouse state after a week;
C. ascites collection: injection cell gathered ascites with asepsis injector in mouse peritoneal to the mouse of abdominal cavity enlargement after 5 days, gathered once every to two days, so repeatedly repeatedly gathered until mouse natural death;
D. ascites purifying: the ascites collected, with sad-thick purifying of saturated ammonium sulphate method, affinity chromatography carries out purifying, and the ascites after purifying is put into-20 DEG C of environment and preserved, ascites to be dialysed 48h through 0.01mol/LNaCl before use under 4 DEG C of environment, middlely changes liquid once every 6 ~ 8h.
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| CN105505885A (en) * | 2016-01-28 | 2016-04-20 | 江南大学 | Specific anti-acrylamide monoclonal antibody hybridoma cell strain 2F6 and application thereof |
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