CN107356759A - Method and its special ELISA reagent kit a kind of while that detect rice aspergin A and rice aspergin B - Google Patents
Method and its special ELISA reagent kit a kind of while that detect rice aspergin A and rice aspergin B Download PDFInfo
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- G—PHYSICS
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- G01N33/532—Production of labelled immunochemicals
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Abstract
The invention discloses a kind of while detect rice aspergin A and rice aspergin B method and its special ELISA reagent kit.Monoclonal antibody used in method and kit is that mouse hybridoma cell system 4C4F11 is CGMCC No.14309 in the numbering of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center as caused by mouse hybridoma cell system 4C4F11 secretions.Monoclonal antibody provided by the present invention can identify rice aspergin A and rice aspergin B simultaneously, the enzyme linked immunological kit being prepared using the monoclonal antibody can be used for qualitative or quantitative while detect sample semilate rice aspergin A and rice aspergin B, linear detection range has the advantages of quick, sensitive in 2~76ng/mL.The present invention has broad application prospects in agricultural, animal husbandry and food industry, it is contemplated that can create larger economic benefit.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of while method that detects rice aspergin A and rice aspergin B
And its special ELISA reagent kit.
Background technology
False smut (Rice false smut) is that a kind of fungal disease of rice is seriously endangered in current world wide,
Sick, the pseudo- smut of good harvest, green smut etc. are, cardinal symptom is to form rice curve in Rice Panicle.False smut cause of disease is that rice is green
Pyrenomycetes, Perfect stage scientific name are Villosiclava virens (Nakata) Tanaka＆Tanaka, and Invisible element scientific name is
Ustilaginoidea virens(Cooke)Takahashi.The disease not only causes rice sterile grain rate substantially to rise so that rice
The underproduction, rice curve Polluted Paddy can be also formed, so as to reduce the yield and quality of paddy, more seriously produce has to people and animals
Harmful toxin, has a strong impact on grain and food security.Report the main fungi poison of two classes in rice curve and ustilaginoidea virens be present
The green pyrenomycetes of element, i.e. rice aspergin (Ustiloxins) and rice is plain (Ustilaginoidins).Rice aspergin is water-soluble cyclic peptide,
With extensive toxicity, to rice seed germination, seedling and the toxic effect of callus growth；Growth of animals or poultry and life can be caused
Grow decline and the internal organs lesions of ability；To the assembling of eukaryotic tubulin and cytoskeleton formed with inhibitory action.
Based on the extensive toxicity of rice aspergin, it is quick, accurate understand rice curve, ustilaginoidea virens and straw and paddy and
The species and content of its product semilate rice aspergin are just particularly important.In 5 kinds of rice aspergins of structure have been illustrated, rice is bent
Rhzomorph A (Ustiloxin A, abbreviation UA) and B (Ustiloxin B, abbreviation UB) is the primary toxins being present in sample, they
Toxicity also highest.The current existing determination method on rice aspergin can use mainly for rice aspergin A or B
High performance liquid chromatography (HPLC) method, LC-MS (LC-MS) method and indirect competitive enzyme-linked immunosorbent detection (icELISA) method are divided
Analysis.Because immunoassay has the characteristics that high flux, quick and high sensitivity, obtained in the context of detection of mycotoxin
Obtained and be widely applied.Although rice aspergin A antiserum and monoclonal antibody has been related to, and rice aspergin B Dan Ke
The document report of grand antibody and its special ELISA reagent kit, but all can only single detection rice aspergin A or rice aspergin B.
Generally it is present in simultaneously in actual sample based on rice aspergin A and rice aspergin B, and accounts for more than the 95% of rice aspergin total amount,
So a kind of enzyme-linked immune detection method of the monoclonal antibody that can identify rice aspergin A and rice aspergin B simultaneously of exploitation and its
Special ELISA reagent kit is very convenient and efficient by the quick detection for causing main rice aspergin in sample, thus with non-
Often important meaning.
The content of the invention
It is an object of the invention to provide a kind of while detect rice aspergin A and rice aspergin B method and its special-purpose enzyme-linked
Immune reagent kit.
Mouse hybridoma cell system 4C4F11 provided by the invention, it is preserved in China Microbiological on June 28th, 2017
Culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, postcode 100101), numbering of registering on the books is CGMCC No.14309.
The monoclonal antibody as caused by mouse hybridoma cell system 4C4F11 secretions falls within protection scope of the present invention.
The monoclonal antibody can be used for detection or auxiliary detection rice aspergin, is particularly suitable for detecting or aids in detection to plant
Rice aspergin in thing sample.
Mouse hybridoma cell system 4C4F11 provided by the present invention secretes production by mouse hybridoma cell system 4C4F11
Raw monoclonal antibody can be used for the reagent or kit for preparing detection or auxiliary detection rice aspergin, particularly be used to prepare inspection
Survey or aid in the reagent or kit of the rice aspergin in detection plant sample.
The present invention also protects a kind of enzyme linked immunological kit for detecting or aiding in detection rice aspergin, contains in the kit
There is the monoclonal antibody as caused by mouse hybridoma cell system 4C4F11 secretions of independent packaging.
Also contain rice aspergin A standard items and/or rice aspergin B standard product in the kit.
The rice aspergin A standard items can be that rice aspergin A or a series of differences being configured to by the rice aspergin A are dense
The rice aspergin solution A of degree；A series of concentration of the rice aspergin solution A of various concentrations is concretely： 200ng/mL、
100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 3.125ng/mL.The rice aspergin solution A
Concretely water, dilution can use sample diluting liquid to solvent.
The rice aspergin B standard product can be that rice aspergin B or a series of differences being configured to by the rice aspergin B are dense
The rice aspergin B solution of degree；A series of concentration of the rice aspergin B solution of various concentrations is concretely：200 ng/mL、
100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 3.125ng/mL.The rice aspergin B solution
Concretely water, dilution can use sample diluting liquid to solvent.
Also contain rice aspergin A enzyme markers UA-HRP in the kit.The rice aspergin A enzyme markers UA-
Rice aspergin A is marked to obtain by HRP using horseradish peroxidase HRP.
The preparation method of the rice aspergin A enzyme markers UA-HRP comprises the following steps：(1) by horseradish peroxidase
HRP solution mixes with rice aspergin solution A, stirring, obtains mixed solution；(2) added in the mixed solution obtained to step (1)
NaBH4The aqueous solution, stirring；(3) by the solution of step (2) with PBS (4 times), obtained solution mixes with isometric glycerine,
Obtain rice aspergin A enzyme markers UA-HRP.The preparation method of the horseradish peroxidase HRP solution comprises the following steps：
Horseradish peroxidase HRP is dissolved in coating buffer solution, adds 0.1M NaIO4The aqueous solution, 20min is stirred at room temperature,
Then with 0.2M Acetic acid-sodium acetate buffer solution dialysis (twice, each 6h)；Acetic acid-the vinegar for the solution 0.2M that dialysis obtains
Sour sodium buffer solution dilutes 200 times, obtains horseradish peroxidase HRP solution.The horseradish peroxidase HRP, coating buffering
Liquid and NaIO4The proportion relation of the aqueous solution is 4mg：1mL：200μL.The preparation method of the rice aspergin solution A includes as follows
Step：Rice aspergin A is dissolved in the water, obtains rice aspergin solution A.The proportion relation of the rice aspergin A and water is
3mg：350μL.The horseradish peroxidase HRP solution and the proportion relation of rice aspergin solution A are 1mL：350μL.It is described
The mixed solution pH=9.5 that step (1) obtains.In the step (1), 2h is concretely stirred at room temperature in stirring.The step (2)
In, the NaBH4NaBH in the aqueous solution4Concentration be 4mg/mL.
Sheep anti-mouse igg in the kit also containing independent packaging (is purchased from Jackson companies, cat. no is
115-005-003)。
Coating buffer solution in the kit also containing independent packaging.
The also cleaning solution containing independent packaging in the kit.
The also sample diluting liquid containing independent packaging in the kit.
The also substrate buffer solution containing independent packaging in the kit.
The also stop buffer containing independent packaging in the kit.
The solvent of sample diluting liquid described in any of the above is water, solute Na2HPO4、KH2PO4With NaCl, Tween-20 and
Gelatin；Solute Na2HPO4、KH2PO4With concentration of the NaCl in the sample diluting liquid be respectively 0.02M, 0.0015M and
0.14M；The percentage composition of the Tween-20 and gelatin in the sample diluting liquid is 0.1% (percentage by volume) and 0.5%
(0.5g/100mL).The pH of the sample diluting liquid is 7.5.
The solvent that buffer solution is coated with described in any of the above is water, solute Na2CO3And NaHCO3；Solute Na2CO3With
NaHCO3Concentration in buffer solution is coated with is respectively 0.01M and 0.04M.The coating pH of buffer is 9.6.
The solvent of cleaning solution described in any of the above is water, solute Na2HPO4、KH2PO4, NaCl and Tween-20；Solute
Na2HPO4、KH2PO4It is respectively 0.02M, 0.0015M and 0.14M with concentration of the NaCl in cleaning solution；Tween-20 is in cleaning solution
In volumn concentration be 0.1%.The pH value of the cleaning solution is 7.5.
Substrate buffer solution described in any of the above is mixed to get in equal volume by A liquid and B liquid；A liquid solvent is water, and solute is tetramethyl
Base benzidine (TMB) and monohydrate potassium, the concentration of solute TMB and monohydrate potassium in A liquid be respectively 0.003M and
0.05M；The solvent of B liquid is water, and solute is trisodium citrate and Na2HPO4, solute trisodium citrate and Na2HPO4In B liquid
Concentration is respectively 0.01M and 0.03M.The pH values of the substrate buffer solution are 3.5.
Stop buffer described in any of the above is the aqueous hydrochloric acid solution that concentration is 1M.
Kit described in any of the above can be used for quantitatively detecting rice aspergin, be particularly suitable in quantitative detection plant sample
Rice aspergin.
The present invention also protects a kind of method for detecting or aiding in detection rice aspergin, including the use of the examination described in any of the above
The step of agent box detects to testing sample.
Methods described comprises the following steps (1)~(7)：
(1) testing sample extracts through water, and extract solution directly detects or adds appropriate amount of sample diluted into sample liquid；
(2) it is coated with：96 hole elisa Plates are taken, are coated with using sheep anti-mouse igg solution, 200 μ L/ holes；Sheep anti-mouse igg
Concentration is 1000ng/mL, and solvent is coating buffer solution；37 DEG C of coating 3h, are washed 4 times with cleaning solution；
(3) product are loaded：100 μ L sample liquid are added per hole；
(4) rice aspergin A enzyme markers and monoclonal antibody are added：50 μ L rice aspergin A enzyme markers are sequentially added per hole
The dilution (rice aspergin A enzyme marker UA-HRP solution is diluted 5000 times with sample diluting liquid) of UA-HRP solution and 50 μ L
The monoclonal antibody solution of mouse hybridoma cell system 4C4F11 secretions (concentration is 250 ng/mL)；Every kind of dilution sets three
Individual multiple holes；Put in wet box 30min, board-washing 4 times under the conditions of 37 DEG C；
(5) develop the color：10mL substrate buffer solutions are taken, add 10 μ L 30% (mass fraction) H thereto2O2, it is molten to obtain substrate
Liquid, substrate solution is added in ELISA Plate, per the μ L of hole 200.Lucifuge colour developing 15min；
(6) terminate：50 μ L stop buffers are added per hole, with the OD values that each hole is determined at ELIASA 450nm.
(7) standard curve is done simultaneously on same plate, containing for sample semilate rice aspergin is calculated according to standard curve
Amount.
Rice aspergin described in any of the above includes rice aspergin A and rice aspergin B.
Monoclonal antibody provided by the invention has good binding characteristic to rice aspergin A and rice aspergin B, i.e., to rice
Aspergin A and rice aspergin B have universality.
When therefore using monoclonal antibody qualitative detection rice aspergin provided by the invention, due to the non-intellectual of sample to be tested
And it is uncertain, if result had not both contained rice aspergin A or do not contained rice aspergin B for negative, sample to be tested, if knot
Fruit for positive, sample to be tested may contain rice aspergin A may also contain rice aspergin B may also both containing rice aspergin A or
Contain rice aspergin B.
When therefore quantitatively detecting rice aspergin using monoclonal antibody provided by the invention, due to the non-intellectual of sample to be tested
And it is uncertain, the rice aspergillus cellulose content detected (may comprise only rice aspergillus for rice aspergin A content in sample to be tested
Plain A does not contain rice aspergin B), it is also possible to for rice aspergin B content (rice aspergin B is comprised only in sample to be tested and does not contain rice
Aspergin A), it is also possible to (not only contained rice aspergin A in sample to be tested for rice aspergin A and B total content but also contained rice aspergin
B)。
The present invention also protection will monoclonal antibody and the solid phase as caused by mouse hybridoma cell system 4C4F11 secretions
Carrier is mutually coupled obtained immune affinity sorbent.
The present invention also protects the immune affinity chromatographic column using the immune affinity sorbent as filler.
The present invention also kit of the protection containing the immune affinity sorbent.
The present invention also kit of the protection containing the immune affinity chromatographic column.
The present invention also protects application of the immune affinity sorbent in rice aspergin is isolated and purified.
The present invention also protects application of the immune affinity chromatographic column in rice aspergin is isolated and purified.
The present invention also kit of the protection containing the immune affinity sorbent contains the immune affinity chromatographic column
Application of the kit in rice aspergin is isolated and purified.
The rice aspergin includes rice aspergin A and rice aspergin B.
Monoclonal antibody that is provided by the present invention while identifying rice aspergin A and rice aspergin B is with rice aspergin B-
OVA (conjugate of rice aspergin B and oralbumin) is immunogene, by immune mouse, cell fusion and thin to hybridoma
Born of the same parents are screened what is obtained；The competitive ELISA kit being prepared using the monoclonal antibody, for qualitative or
Quantitatively detect the rice aspergin A and rice aspergin B in plant sample, linear detection range in 2~72 ng/mL, have it is quick,
The advantages of sensitive and efficient.The present invention has broad application prospects in agricultural, animal husbandry and food industry, it is contemplated that can create compared with
Big economic benefit.
Brief description of the drawings
Fig. 1 is that enzyme linked immunological kit detects rice aspergin A and the standard curve of rice aspergin B concentration.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
Freund's complete adjuvant：Sigma companies, catalogue numbering is F5881.
Incomplete Freund's adjuvant：Sigma companies, catalogue numbering is F5506.
Rice aspergin A (standard items) and rice aspergin B (standard items) preparation method can be delivered with specific reference to inventor
Following paper：
Shan T,Sun W,Liu H,Gao S,Lu S,Wang M,Chen Z,Wang S,Zhou
L.Determination and analysis of ustiloxins A and B by LC-ESI-MS and HPLC in
false smut balls of rice. International Journal of Molecular Sciences,2012,13
(9):11275-11287.
Shan T,Sun W,Wang X,Fu X,Sun W,Zhou L.Purification of ustiloxins A
and B from rice false smut balls by macroporous resins.Molecules,2013,18(7):
8181-8199.
Rice aspergin A (standard items) and rice aspergin B (standard items) preparation method also can be with specific reference to patents：Detect rice
Aspergin B method and its special ELISA reagent kit (Authorization Notice No.：CN 104569416B).
It is coated with buffer solution：Solvent is water, solute Na2CO3And NaHCO3, pH value 9.6；Solute Na2CO3And NaHCO3
The concentration being coated with buffer solution is respectively 0.01M and 0.04M.
Cleaning solution：Solvent is water, solute Na2HPO4、KH2PO4, NaCl and Tween-20, pH value 7.5；Solute
Na2HPO4、KH2PO4It is respectively 0.02M, 0.0015M and 0.14M with concentration of the NaCl in cleaning solution；Tween-20 is in cleaning solution
In volumn concentration be 0.1%.
Sample diluting liquid：Solvent is water, solute Na2HPO4、KH2PO4With NaCl, Tween-20 and gelatin, pH value is
7.5；Solute Na2HPO4、KH2PO4With concentration of the NaCl in the sample diluting liquid be respectively 0.02M, 0.0015M and
0.14M；The percentage composition of the Tween-20 and gelatin in the sample diluting liquid is 0.1% (percentage by volume) and 0.5%
(0.5g/100mL)。
Substrate buffer solution is mixed to get in equal volume by A liquid and B liquid, pH value 3.5；A liquid solvent is water, and solute is tetramethyl
Benzidine (TMB) and monohydrate potassium, the concentration of solute TMB and monohydrate potassium in A liquid be respectively 0.003M and
0.05M；The solvent of B liquid is water, and solute is trisodium citrate and Na2HPO4, solute trisodium citrate and Na2HPO4In B liquid
Concentration is respectively 0.01M and 0.03M.
Stop buffer：Concentration is 1M aqueous hydrochloric acid solution.
The synthesis of embodiment 1, rice aspergin B-OVA immunogenes
1st, weigh 1.1mg rice aspergins B to be dissolved in 0.5mL (dimethylformamide) DMF, obtain solution A.
2nd, 8mg (ovalbumin) OVA 1mL PBS are weighed to dissolve, obtain solution B.
3rd, the solution A for obtaining step 1 is added in the solution B that step 2 obtains, and is stirred, and adds 6.8 μ L 5%
(percentage by volume) glutaraldehyde, 4 DEG C are coupled overnight, are dialysed 3 days in PBS, obtain rice aspergin B-OVA immunogen solutions.
The synthesis of embodiment 2, rice aspergin A enzyme markers UA-HRP
1st, weigh 4mg horseradish peroxidases HRP to be dissolved in 1mL coating buffer solutions, add 200 μ L 0.1M's
NaIO4Solution, 20min is stirred at room temperature.
2nd, the solution that step 1 obtains is dialysed twice with 0.2M Acetic acid-sodium acetate buffer solution, each 6h；Dialysis obtains
Solution dilute 200 times with 0.2M Acetic acid-sodium acetate buffer solution, obtain HRP solution.
3rd, the rice aspergin A powder for weighing 3mg is dissolved in 350 μ L distilled water, is obtained the rice aspergin A aqueous solution；
The rice aspergin A aqueous solution is added in the HRP solution that 1mL steps 2 obtain, adjusts pH=9.5 with 0.1M NaOH, at room temperature
Stir 2h.
4th, toward the NaBH of addition 0.1mL 4mg/mL in the solution of 3 gained4The aqueous solution, 2h is stirred at 4 DEG C.
5th, by 4 resulting solutions PBS 4 times, obtain solution and add isometric glycerine mixing, obtain rice aspergin A enzymes
Label UA-HRP solution, packing, -20 DEG C of preservations.
Embodiment 3, the acquisition of hybridoma cell strain and the monoclonal antibody for identifying rice aspergin A and rice aspergin B simultaneously
Preparation
Bal b/C small white mouses：8-10 week old female mices, purchased from Military Medical Science Institute's Experimental Animal Center.
SP2/0 myeloma cell：Purchased from China Veterinery Drug Inspection Office.
First, animal immune
1st, experimental animal is used as using the female Bal b/C small white mouses of 8-10 week old.
2nd, fundamental immunity：Take rice aspergin B-OVA immunogen solutions prepared by 1mL embodiments 1 (concentration 1mg/mL, it is molten
Agent is PBS), isometric Freund's complete adjuvant is added, emulsification is sufficiently stirred with magnetic stirring apparatus, until instilling indiffusion in water.
Abdominal cavity and dorsal sc multi-point injection Bal b/C small white mouses are used with the immunogene emulsified, injection dosage is that every mouse is noted
0.1mg rice aspergin B-OVA immunogenes are penetrated, wherein 0.05mg is injected intraperitoneally, 2 points of dorsal sc injection, 0.025mg/ points.
3rd, booster immunization：After fundamental immunity 2 weeks, the rice aspergin B-OVA immunogen solutions for taking 1mL embodiments 1 to prepare are (dense
Spend for 1mg/mL, solvent PBS), 1mL incomplete Freund's adjuvants are added, emulsification is sufficiently stirred with magnetic stirring apparatus, until instilling
Indiffusion in water.The immunogene emulsified is used into abdominal cavity and dorsal sc multi-point injection Bal b/C mouse, injection dosage is every
Mouse injection 0.1mg rice aspergin B-OVA immunogenes, wherein 0.05mg is injected intraperitoneally, 2 points of dorsal sc injection,
0.025mg/ points.
2nd, cell fusion and cloning
Booster immunization since third time booster immunization, the 3rd day after being immunized every time, is adopted every once every 2 weeks from mouse orbit
Blood, antibody titer is determined, the definition of potency is serum diluting multiple when OD values are 1.Treat that potency is more than 1:8000 (i.e. OD values
For 1, extension rate 8000) after, select the optimal mouse of serum titer, extracting spleen cell, by 9:1 (quantitative proportion) ratio with
SP2/0 myeloma cell is merged；Using the good monoclonal of the method screening secretory antibody potency and specificity of direct competive ELISA
Cell line.
The step of above-mentioned direct competive ELISA method, is specific as follows：
(1) it is coated with：96 hole elisa Plates are taken, the sheep anti-mouse igg that 200 μ L are added in every hole (is purchased from Jackson companies, commodity
Catalog number (Cat.No.) is 115-005-003；Concentration is 1000ng/mL, and solvent is coating buffer solution), 37 DEG C are coated with 3 hours, use cleaning solution
Washing 4 times.
(2) product are loaded：Suppress hole to add the pre-configured rice aspergin B standard product solution of 100 μ L per hole (concentration is
200ng/mL, solvent are sample diluting liquid), blank well adds 100 μ L sample diluting liquid per hole.
(3) rice aspergin A enzyme markers and antibody are added：50 μ L rice aspergin A enzyme markers UA-HRP are sequentially added per hole
Dilution (the rice aspergin A enzyme marker UA-HRP solution dilution 5000 for being obtained embodiment 2 with sample diluting liquid of solution
Times) and 50 μ L Hybridoma Cell Culture liquid, put in wet box 30min, board-washing 4 times under the conditions of 37 DEG C.
(4) develop the color：10mL substrate buffer solutions are taken, add 10 μ L 30% (mass fraction) H thereto2O2, it is molten to obtain substrate
Liquid, substrate solution is added in ELISA Plate, per the μ L of hole 200.Lucifuge colour developing 15min.
(5) terminate：50 μ L stop buffers are added per hole, with the OD values that each hole is determined at ELIASA 450nm, partial results
It is shown in Table 1.In table 1,2B2,2E2,3H1,4C4 and 5C7 are represented in the Tissue Culture Plate difference hole added in screening process respectively
Hybridoma, inhibiting rate (%)=[(blank well OD values-suppress hole OD values)/blank well OD values.
The direct competive ELISA method of table 1 screens the result of hybridoma
Light absorption value is bigger, illustrates that antibody is higher to the affinity of antigen；Inhibiting rate is higher, illustrates that the specificity of antibody is got over
It is good.It is maximum that 2E2 light absorption value is can be seen that from the data of table 1, illustrates affinity highests of the 2E2 to envelope antigen, but it suppresses
Rate only has 7.5%, shows 2E2 poor specificity；2B2 and 5C7 inhibiting rate respectively up to 81.2% and 70.5%, but with coating
The affinity of antigen is very weak；In 5 plants of positive cell strains that fusion obtains, the affinity of only 4C4 and envelope antigen is higher, suppression
Rate processed is also preferable (68.8%).Therefore 4C4 hybridomas are chosen to continue to screen, finally obtain secretory antibody specificity
Mouse hybridoma cell system 4C4F11 good, affinity is high.Mouse hybridoma cell system 4C4F11 protects on June 28th, 2017
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), numbering of registering on the books is CGMCC
No.14309。
3rd, cell cryopreservation and recovery
Mouse hybridoma cell system 4C4F11 is made 1 × 10 with frozen stock solution6Individual/mL cell suspension, it is long in liquid nitrogen
Phase preserves.During recovery, cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, centrifugation is moved into blake bottle after removing frozen stock solution
Culture.
4th, the preparation and purification of monoclonal antibody
1st, increment culture
The preparation method of cell culture medium：Calf serum is added into DMEM culture mediums and (is purchased from GIBCOBRL, catalogue
Number be 26170-043) and sodium acid carbonate, final concentration of 20% (volumn concentration) of calf serum, the end of sodium acid carbonate it is dense
Spend for 0.2% (weight/mass percentage composition), pH 7.4.
Mouse hybridoma cell system 4C4F11 is placed in above-mentioned cell culture medium, 37 DEG C of cultures, during which observed daily, and
Timely amplification cultivation.
2nd, prepared by ascites
Balb/C mouse peritoneal injections sterilizing paraffin oil (0.3mL/ is only).7 days pneumoretroperitoneum injection mouse hybridoma cell systems
4C4F11 (about 106Individual/only).Ascites is gathered after 7 days, is purified with octanoic acid-saturated ammonium sulfate method, ascites after purification is (i.e.
The monoclonal antibody solution of mouse hybridoma cell system 4C4F11 secretions) -20 DEG C preserve.
5th, the identification of monoclonal antibody
1st, the monoclonal antibody solution that the mouse hybridoma cell system 4C4F11 obtained the 2 of step 4 secretes uses Dan Ke
The hypotype of grand Antibody types detection kit (being purchased from Sigma, production code member ISO2-1KT) detection monoclonal antibody, specific behaviour
Make referring to kit specification.
As a result show, the immunoglobulin subclass of the monoclonal antibody of mouse hybridoma cell system 4C4F11 secretions is
IgG1 types.
2nd, the sensitivity and cross reaction of monoclonal antibody are determined using direct competive ELISA method
(1) it is coated with：96 hole elisa Plates are taken, adding 200 μ L sheep anti-mouse iggs in every hole (is purchased from Jackson companies, commodity mesh
Record number is 115-005-003；Concentration is 1000ng/mL, and solvent is coating buffer solution), 37 DEG C are coated with 3 hours, are washed with cleaning solution
Wash 4 times.
(2) product are loaded：Per hole, (solvent is sample for rice aspergin A pre-configured 100 μ L of addition or B standard product solution
Dilution), rice aspergin solution A concentration is：200ng/mL、100ng/mL、50ng/mL、25ng/mL、 12.5ng/mL、
6.25ng/mL and 3.125ng/mL；Rice aspergin B solution concentration is：200ng/mL、100ng/mL、 50ng/mL、25ng/mL、
12.5ng/mL, 6.25ng/mL and 3.125ng/mL；Control wells add 100 μ L sample diluting liquid per hole.
(3) rice aspergin A enzyme markers and antibody are added：50 μ L rice aspergin A enzyme markers UA-HRP are sequentially added per hole
Dilution (the rice aspergin A enzyme marker UA-HRP solution dilution 5000 for being obtained embodiment 2 with sample diluting liquid of solution
Times) and four the step of 50 μ L in 2 obtained monoclonal antibody solutions (concentration 250ng/mL)；Every kind of dilution sets three
Multiple holes；Put in wet box 30min, board-washing 4 times under the conditions of 37 DEG C.
(4) develop the color：10mL substrate buffer solutions are taken, add 10 μ L 30% (mass fraction) H thereto2O2, it is molten to obtain substrate
Liquid, substrate solution is added in ELISA Plate, per the μ L of hole 200.Lucifuge colour developing 15min.
(5) terminate：50 μ L stop buffers are added per hole, with the OD values that each hole is determined at ELIASA 450nm.
Using testing compound solution concentration as abscissa, B/B0(B represents light absorption value during containing testing compound；B0Represent
Do not contain light absorption value during testing compound, i.e. control wells absorbance) it is ordinate, distinguish with the softwares of OriginPro 8
Calculate rice aspergin A and rice aspergin B IC50Value, cross reacting rate is calculated by following equation：
Cross reacting rate (%)=[IC50(rice aspergin A)/IC50(rice aspergin to be measured)] × 100%
It the results are shown in Table 2.As a result show, obtained monoclonal antibody is to rice aspergin A and rice aspergin B cross reacting rate
Respectively 100% and 105%, illustrate recognition capability basic phase of the gained monoclonal antibody to rice aspergin A and rice aspergin B
When.
The monoclonal antibody of table 2 and rice aspergin A and rice aspergin B cross reaction
Embodiment 4, the enzyme linked immunological kit that rice aspergin A and rice aspergin B can be detected simultaneously
First, the preparation of kit
The enzyme linked immunological kit that can detect rice aspergin A and rice aspergin B simultaneously of the application is by sheep anti mouse (coating
It is former), the monoclonal antibody of mouse hybridoma cell system 4C4F11 secretions, rice aspergin A enzyme markers UA-HRP, coating buffering
Liquid, sample diluting liquid, cleaning solution, rice aspergin A standard solutions or rice aspergin B standard product solution, substrate buffer solution and end
Only buffer solution forms.
Sheep anti-mouse igg：Purchased from Jackson companies, cat. no 115-005-003.
The monoclonal antibody of mouse hybridoma cell system 4C4F11 secretions：It is prepared by the 2 of the step 4 of embodiment 3.
Rice aspergin A enzyme markers UA-HRP：It is prepared by embodiment 2.
It is coated with buffer solution：Solvent is water, solute Na2CO3And NaHCO3, pH value 9.6；Solute Na2CO3And NaHCO3
The concentration being coated with buffer solution is respectively 0.01M and 0.04M.
Cleaning solution：Solvent is water, solute Na2HPO4、KH2PO4, NaCl and Tween-20, pH value 7.5；Solute
Na2HPO4、KH2PO4It is respectively 0.02M, 0.0015M and 0.14M with concentration of the NaCl in cleaning solution；Tween-20 is in cleaning solution
In volumn concentration be 0.1%.
Sample diluting liquid：Solvent is water, solute Na2HPO4、KH2PO4With NaCl, Tween-20 and gelatin, pH value is
7.5；Solute Na2HPO4、KH2PO4With concentration of the NaCl in the sample diluting liquid be respectively 0.02M, 0.0015M and
0.14M；The percentage composition of the Tween-20 and gelatin in the sample diluting liquid is 0.1% (percentage by volume) and 0.5%
(0.5g/100mL)。
Rice aspergin A standard solutions：For the rice aspergin solution A of serial various concentrations, concentration is specially：200ng/
ML, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 3.125ng/mL, it is dissolved in sample diluting liquid.
Rice aspergin B standard product solution：For the rice aspergin B solution of serial various concentrations, concentration is specially 200ng/
ML, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL and 3.125ng/mL, it is dissolved in sample diluting liquid.
Substrate buffer solution is mixed to get in equal volume by A liquid and B liquid, pH value 3.5；A liquid solvent is water, and solute is tetramethyl
Benzidine (TMB) and monohydrate potassium, the concentration of solute TMB and monohydrate potassium in A liquid be respectively 0.003M and
0.05M；The solvent of B liquid is water, and solute is trisodium citrate and Na2HPO4, solute trisodium citrate and Na2HPO4In B liquid
Concentration is respectively 0.01M and 0.03M.
Stop buffer：Concentration is 1M aqueous hydrochloric acid solution.
2nd, the application of kit
1st, standard curve is made
(1) it is coated with：96 hole elisa Plates are taken, are coated with using sheep anti-mouse igg solution, 200 μ L/ holes；Sheep anti-mouse igg
Concentration is 1000ng/mL, and solvent is coating buffer solution；37 DEG C of coating 3h, are washed 4 times with cleaning solution.
(2) standard solution is added：Using as the rice aspergin A of the serial various concentrations of standard items or rice aspergin B solution
Different test holes is added separately to, per the μ L of hole 100；Control wells are sample diluting liquid, per the μ L of hole 100.
(3) rice aspergin A enzyme markers and monoclonal antibody are added：50 μ L rice aspergin A enzyme markers are sequentially added per hole
The dilution (rice aspergin A enzyme marker UA-HRP solution is diluted 5000 times with sample diluting liquid) of UA-HRP solution and 50 μ L
The monoclonal antibody solution of mouse hybridoma cell system 4C4F11 secretions (concentration is 250 ng/mL)；Every kind of dilution sets three
Individual multiple holes；Put in wet box 30min, board-washing 4 times under the conditions of 37 DEG C.
(4) develop the color：10mL substrate buffer solutions are taken, add 10 μ L 30% (mass fraction) H thereto2O2, it is molten to obtain substrate
Liquid, substrate solution is added in ELISA Plate, per the μ L of hole 200.Lucifuge colour developing 15min.
(5) terminate：50 μ L stop buffers are added per hole, with the OD values that each hole is determined at ELIASA 450nm.
(6) standard curve is drawn：Using the rice aspergin A or rice aspergin B solution of various concentrations as abscissa, B/B0 (B
Represent light absorption value during containing testing compound；B0Light absorption value when representing not containing testing compound, i.e. control wells absorbance
Value) it is ordinate, with the Software on Drawing standard curves of OriginPro 8.
Experiment sets 3 repetitions, takes the average value of experimental result three times, and obtained standard curve is as shown in Figure 1.
With rice aspergin A do calibration curve equation be Y=1.10058/ [1+ (X/18.10851) ^0.70629]-
0.10058(R2=0.99941)；It is Y=1.06993/ [1+ (X/15.753) ^ to do calibration curve equation with rice aspergin B
0.68999]– 0.07035(R2=0.99681), Y represents B/B0, the concentration (ng/mL) of X expression rice aspergins A in the solution.
(B0-B)/B0For 20%-80% when rice aspergin A concentration range be detection range；(B0-B)/B0For 10% when rice aspergillus
Plain A concentration is test limit.Linear detection range is in 2~72ng/mL when doing standard curve with rice aspergin A；Detection is limited to
0.7ng/mL；IC50It is worth for 14ng/mL.
It is Y=1.06993/ [1+ (X/15.753) ^0.68999] -0.07035 to do calibration curve equation with rice aspergin B
(R2=0.99681), Y represents B/B0, the concentration (ng/mL) of X expression rice aspergins B in the solution.(B0-B)/B0For 20%-
Rice aspergin B concentration range is detection range when 80%；(B0-B)/B0For 10% when rice aspergin B concentration be to detect
Limit.Linear detection range is in 2~76ng/mL when doing standard curve with rice aspergin B；Detection is limited to 0.6ng/mL； IC50It is worth and is
13ng/mL。
Result above is shown does standard curve with rice aspergin A or rice aspergillus B, its linear detection range, test limit and
IC50It is worth equal no significant difference, illustrates that the monoclonal antibody is consistent to rice aspergin A or B recognition capability, the direct competitive of foundation
ELISA method can be used for the content for detecting rice aspergin A and rice aspergin B simultaneously, and rice aspergin A or B is equal in actual use
It can be used as the content that standard curve is used to calculate sample semilate rice aspergin A and rice aspergin B.
2nd, in step 1 kit application method
(1) testing sample extracts through water, and extract solution directly detects or adds appropriate amount of sample diluted into sample liquid.
(2) it is coated with：96 hole elisa Plates are taken, are coated with using sheep anti-mouse igg solution, 200 μ L/ holes；Sheep anti-mouse igg
Concentration is 1000ng/mL, and solvent is coating buffer solution；37 DEG C of coating 3h, are washed 4 times with cleaning solution.
(3) product are loaded：100 μ L sample liquid are added per hole.
(4) rice aspergin A enzyme markers and monoclonal antibody are added：50 μ L rice aspergin A enzyme markers are sequentially added per hole
The dilution (rice aspergin A enzyme marker UA-HRP solution is diluted 5000 times with sample diluting liquid) of UA-HRP solution and 50 μ L
The monoclonal antibody solution of mouse hybridoma cell system 4C4F11 secretions (concentration is 250 ng/mL)；Every kind of dilution sets three
Individual multiple holes；Put in wet box 30min, board-washing 4 times under the conditions of 37 DEG C.
(5) develop the color：10mL substrate buffer solutions are taken, add 10 μ L 30% (mass fraction) H thereto2O2, it is molten to obtain substrate
Liquid, substrate solution is added in ELISA Plate, per the μ L of hole 200.Lucifuge colour developing 15min.
(6) terminate：50 μ L stop buffers are added per hole, with the OD values that each hole is determined at ELIASA 450nm.
Do standard curve simultaneously on same plate and (referring to step 1), sample semilate rice song is calculated according to standard curve
Rhzomorph A and rice aspergin B content.
3rd, rice curve sample and rice sample addition recovery experiment
(1) it is respectively 1~12 to weigh 50mg Hunan Hanshou area rice curve (collection of in September, 2015) 12 parts of numberings of powder,
3 repetitions of every part of setting, 1-6 add 0mg/g, 0.2mg/g, 0.5mg/g, 1.0mg/g, 2.0mg/g and 4.0mg/g rice respectively
Aspergin A standard items, 7-12 add 0mg/g, 0.2mg/g, 0.5mg/g, 1.0mg/g, 2.0mg/g and 4.0mg/g rice song respectively
Rhzomorph B standard product.Add the min of 1.5mL distilled water ultrasonic extraction 30 into sample respectively, extract solution is collected by centrifugation, repeat extraction three
It is secondary, merge extract solution three times, be settled to 5mL.Extract solution sample diluting liquid dilutes 800 times and obtains the dilution of rice curve sample extraction
Liquid.
(2) weigh and 17 (in October, 2013 collection) powder, 12 parts of numberings are spent in 1g Beijing areas (non-Infected regions) paddy
Respectively 1 '~12 ', every part setting 3 repetitions, 1 ' -6 ' respectively add 0mg/g, 0.2mg/g, 0.5mg/g, 1.0mg/g,
2.0mg/g and 4.0mg/g rice aspergin A standard items, 7 ' -12 ' respectively add 0mg/g, 0.2mg/g, 0.5mg/g, 1.0mg/g,
2.0mg/g and 4.0mg/g rice aspergin B standard product.Add 6.0mL distilled water ultrasonic extraction 30min, centrifugation into sample respectively
Extract solution is collected, repeats extraction 3 times, merges No. 3 extract solutions, 1.0mL distilled water constant volumes is used after freeze-drying.Concentrate sample
Diluted 50 obtains rice sample extraction dilution again.
(3) each rice sample extraction dilution takes 100 μ L to carry out direct competive ELISA analysis respectively, according to above-mentioned steps 2
In step (2)~(6) carry out.
(4) prepare rice aspergin A respectively with sample diluting liquid or B standard product solution does standard curve (referring to step 1).
It is calculated each rice curve sample and rice sample extraction dilution semilate rice aspergin A's and rice aspergin B according to standard curve
Content.
(5) rate of recovery is calculated.
Rate of recovery calculation formula is：The rate of recovery (%)=[(rice aspergin A and rice aspergin B detection level-do not add
Rice aspergin A or during rice aspergin B standard product rice aspergin A and rice aspergin B detection level)/rice aspergin A or rice it is bent
Rhzomorph B addition content] × 100%.
Wherein, when calculation procedure (1) Hunan Hanshou area rate of recovery, " rice aspergin A and rice aspergin B detection contain
Amount " be number be 2-6 and 8-12 10 parts of testing samples in the rice aspergin A that measures and rice aspergin B content, " do not add
Rice aspergin A or during rice aspergin B standard product rice aspergin A and rice aspergin B detection level " be that numbering is that 1 and 7 (rice is bent
Rhzomorph A or rice aspergin B standard product addition content contain for the rice aspergin A's that is measured in testing sample 0) and rice aspergin B
Amount.
Wherein, when calculation procedure (2) Beijing area rate of recovery, " rice aspergin A and rice aspergin B detection level " is
The rice aspergin A and rice aspergin B content measured in 10 parts of testing samples that numbering is 2 ' -6 ' and 8 ' -12 ', " does not add rice
Aspergin A or during rice aspergin B standard product rice aspergin A and rice aspergin B detection level " be that numbering is that 1 ' and 7 ' (rice is bent
Rhzomorph A or rice aspergin B standard product addition content contain for the rice aspergin A's that is measured in testing sample 0) and rice aspergin B
Amount.
Hunan Hanshou area rate of recovery statistical result is shown in Table 3.Make standard curve calculating with rice aspergin A or rice aspergin B
Obtained rice aspergin A and B content are basically identical, and rate of recovery scope is 86%~102%.In the Septembers, 2015 detected are adopted
It is 0.54 mg/g to collect from rice the curve sample semilate rice aspergin A and rice aspergin B in Hunan Hanshou area content.
Beijing area rate of recovery statistical result is shown in Table 4.It is calculated with rice aspergin A or rice aspergin B as standard curve
Rice aspergin A and B content it is basically identical, rate of recovery scope is in 84%-104%.Collection (does not fall ill ground from Beijing area
Area) paddy in spend in 17 samples (in October, 2013) to be not detected by rice aspergin A and rice aspergin B presence.
Rice aspergin A or B the addition recovery experiment result of the Hunan Hanshou area rice curve sample of table 3
aEach sample repeats three times；bMake the result of standard curve calculating with rice aspergin A；cStandard is made with rice aspergin B
The result that curve calculates；dAverage value ± SD is detected three times
Rice aspergin A or B the addition recovery experiment result of the Beijing area rice sample of table 4
aEach sample repeats three times；bMake the result of standard curve calculating with rice aspergin A；cStandard is made with rice aspergin B
The result that curve calculates；dND is represented and not detected；eAverage value ± SD is detected three times
4th, instrument confirmatory experiment
The certainly national each regional rice curve sample of collection, 6.0mL distilled water ultrasonic extraction 30min are added into sample, from
The heart collects extract solution, repeats extraction 3 times, merges No. 3 extract solutions, 1.0mL distilled water constant volumes are used after freeze-drying, are concentrated
Liquid, concentrate sample diluting liquid dilute 50 times and obtain rice sample extraction dilution.By rice curve sample extraction dilution point
Into two parts, a direct competive ELISA (dcELISA) detects (step (2)~(6) in step 2), another filtering
(0.22 μm of filter membrane), is detected with HPLC.HPLC analysis systems (Shimadzu, Japan) are defeated by LC-20AT binary high pressures
Send pump, SIL-20AC automatic samplers, SPD-M20A photodiode array detectors, DGU-20A3 automatic deaerating machines, CTO-
10AS column ovens, CBM-20Alite system controllers and Féraud door Hydro-C18 (250mm × 4.6mm, 5 μm) reverse-phase chromatographic column
Form.Chromatographic condition is：Mobile phase：Methanol-water=15:85+0.02%TFA (v/v)；Flow velocity：1mL/min；Ultraviolet detection ripple
It is long：220 nm；Column temperature：30℃；Sample injection volume：50μL；The bulk analysis time：25min.
Rice aspergin A standard liquids are prepared with rice aspergin A standard items (solvent is ultra-pure water)：500μg/mL；250 μg/
mL、125μg/mL、62.5μg/mL、31.25μg/mL、15.625μg/mL、7.8125μg/mL；3.90625 μ g/mL and
1.953125μg/mL。
Carry out detection with HPLC establishes standard curve to testing sample simultaneously：UA, Y=1508667.5516X+
81977.1372(R2=0.9999)；UB, Y=1344310.7866X+38521.9310 (R2=0.9997), Y represents peak area,
X represents the rice aspergin A (UA) detected and rice aspergin B (UB) content (μ g) respectively.
As a result as shown in table 5 and table 6.As a result show, it is same with the direct competive ELISA (dcELISA) and HPLC methods of foundation
When detect rice curve extract solution results relevance it is preferable, Y=0.969X-0.0049 (R2=0.9932), Y represents HPLC detections
Concentration (mg/g), X represent dcELISA detectable concentrations (mg/g), and the dcELISA methods that the present invention is established are accurately and reliably.
The rice curve sample instrument confirmatory experiment result of 5 national different regions of table
aAverage value ± SD is detected three times
Table 6 picks up from the rice sample instrument confirmatory experiment result of national different regions
aND is represented and not detected；bAverage value ± SD is detected three times.
Claims (10)
1. mouse hybridoma cell system 4C4F11, in stepping on for China Committee for Culture Collection of Microorganisms's common micro-organisms center
It is CGMCC No.14309 to charge to volume numbering.
2. the monoclonal antibody as caused by the mouse hybridoma cell system 4C4F11 secretions described in claim 1.
3. application of the monoclonal antibody in detecting or aiding in detection rice aspergin described in claim 2.
4. prepared by the monoclonal antibody described in mouse hybridoma cell system 4C4F11 or claim 2 described in claim 1
Application in the reagent or kit of detection or auxiliary detection rice aspergin.
A kind of 5. enzyme linked immunological kit for detecting or aiding in detection rice aspergin, it is characterised in that：Contain in the kit
Monoclonal antibody described in the claim 2 of independent packaging.
A kind of 6. method for detecting or aiding in detection rice aspergin, including the use of the kit described in claim 5 to be measured
The step of sample is detected.
7. application of the kit described in claim 5 in quantitative detection rice aspergillus cellulose content.
8. the monoclonal antibody described in claim 2 to be mutually coupled with solid phase carrier to obtained immune affinity sorbent or with described
Immune affinity sorbent is the immune affinity chromatographic column of filler.
9. the kit containing the immune affinity sorbent described in claim 8 contains being immunized described in claim 8
The kit of affinity chromatographic column.
10. the reagent described in immune affinity sorbent or immune affinity chromatographic column or claim 9 described in claim 8
Application of the box in rice aspergin is isolated and purified.
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| CN102590527A (en) * | 2012-01-16 | 2012-07-18 | 中国农业大学 | Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof |
| CN103760353A (en) * | 2014-02-08 | 2014-04-30 | 中国农业大学 | Method and special ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting ustiloxin A |
| CN104569416A (en) * | 2015-01-22 | 2015-04-29 | 中国农业大学 | Method for detecting ustiloxin B and special enzyme-linked immunosorbent kit for method |
| CN106279359A (en) * | 2016-08-11 | 2017-01-04 | 中国水稻研究所 | A kind of method preparing five kinds of ustilaginoidea virens toxin |
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| CN102590527A (en) * | 2012-01-16 | 2012-07-18 | 中国农业大学 | Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof |
| CN103760353A (en) * | 2014-02-08 | 2014-04-30 | 中国农业大学 | Method and special ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting ustiloxin A |
| CN104569416A (en) * | 2015-01-22 | 2015-04-29 | 中国农业大学 | Method for detecting ustiloxin B and special enzyme-linked immunosorbent kit for method |
| CN106279359A (en) * | 2016-08-11 | 2017-01-04 | 中国水稻研究所 | A kind of method preparing five kinds of ustilaginoidea virens toxin |
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