CN109400682A - Simulate the ring type polypeptide and its detection kit of aflatoxin B1 epitope - Google Patents
Simulate the ring type polypeptide and its detection kit of aflatoxin B1 epitope Download PDFInfo
- Publication number
- CN109400682A CN109400682A CN201811126720.7A CN201811126720A CN109400682A CN 109400682 A CN109400682 A CN 109400682A CN 201811126720 A CN201811126720 A CN 201811126720A CN 109400682 A CN109400682 A CN 109400682A
- Authority
- CN
- China
- Prior art keywords
- afb1
- aflatoxin
- liquid
- ring type
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
Abstract
The invention discloses a kind of ring type polypeptides and its detection kit for simulating aflatoxin B1 epitope, and the ring type polypeptide amino acid sequence is CVPSKPGLC, as shown in SEQ ID No.1.The kit includes AFB1 competition antigen, the ELISA Plate for being embedded with anti-AFB1 monoclonal antibody, the standard solution of aflatoxin B1 and horseradish peroxidase-labeled Streptavidin etc.；Polypeptide sequence of the invention can simulate the epitope of AFB1, can combine with antibody specificity used is screened, high sensitivity, high specificity reduce exposure of the AFB1 to operator, are a kind of safer, more efficient detection methods.The invention has important practical significance to pollution monitoring of the AFB1 in agricultural product is solved.
Description
Technical field
The present invention relates to technical field of immunoassay chemistry, and in particular to a kind of simulation aflatoxin B1 epitope
Ring type polypeptide and its detection kit.
Background technique
Aflatoxin (Aflatoxins, AFTs) is mainly by one kind of aspergillus flavus and aspergillus parasiticus generation with virulent
The secondary metabolite of property.AFTs can be divided mainly into B race, G race and M race.B race AFTs is to issue in 365nm ultraviolet excitation
Blue-fluorescence out, and Aflatoxin B1 (AFB1) and AFB2 can be divided into.Wherein AFB1 is toxicity maximum, by the World Health Organization
Agency for Research on Cancer is defined as I class carcinogenic substance, i.e., has the substance of confirmation carcinogenicity to the mankind.Damp and hot environment improperly harvests
The generation of AFB1 can all be caused with stored manner, mainly pollute rice, peanut, corn, grain and oil product etc..
Detection aflatoxin B1 routine immunoassay method taken it is mostly be competitive analysis.First prepare competitiveness
Antigen: introducing carboxyl by succinimide ester process on AFB1 molecule, generates aflatoxin B1 oxime, then with active ester method and
Carrier protein (bovine serum albumin(BSA) or ovalbumin) coupling, the epitope which is exposed can compete anti-with AFB1
AFB1 antibody is usually coated on ELISA Plate and uses as envelope antigen.But the envelope antigen is in the process for preparing and applying
In, need the professions such as high temperature and pressure to set when operator can be made to be exposed in AFB1 environment, and preparing aflatoxin B1 oxime
It is standby, there is certain potential risk, and the competitiveness antigen preparation cost is high, batch is affected.
Therefore, there is an urgent need to prepare and develop safer more efficient tachysynthesis kit and analysis method.
Summary of the invention
It is anti-that it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of simulation aflatoxin B1s (AFB1)
The ring type polypeptide and its detection kit of former epitope, the kit have high sensitivity, high specific, operating method simply, more
Safe and efficient feature；It can be used for the rapid screening of AFB1 and monitoring in agricultural product.
To achieve the above object, a kind of ring type polypeptide for simulating aflatoxin B1 epitope designed by the present invention,
Be characterized in that: its amino acid sequence is CVPSKPGLC, as shown in SEQ ID No.1.
One of method is prepared in the ring type polypeptide of above-mentioned simulation aflatoxin B1 epitope:
1) by anti-AFB1 monoclonal antibody it is purified after be coated on ELISA Plate, commercialized phage display ring seven is added
(the peptide library is purchased from New England Biolabs company to peptide library Ph.D.-C7C, and polypeptide display is in filobactivirus in the peptide library
On the piii protein of M13), the phage display for then simulating AFB1 epitope with specificity is obtained after the elution of AFB1 competitiveness
Polypeptide translates into amino acid sequence, chemical synthesis polypeptide sequence, such as SEQ ID No.1 after the polypeptide is measured DNA sequence dna
It is shown.
It 2) is CVPSKPGLC by the artificial synthesized amino acid sequence that can be obtained of sequence shown in SEQ ID No.1.
The invention discloses a kind of detection aflatoxin B1 kit comprising aforementioned polypeptides, the kit include such as
Lower part:
(1) AFB1 competes antigen, and AFB1 competition antigen is that ring-type described in the claim 1 of biotin labeling is more
Peptide；
(2) it is embedded with the ELISA Plate of anti-AFB1 monoclonal antibody
(3) standard solution of aflatoxin B1；
(4) horseradish peroxidase-labeled Streptavidin, as ELIAS secondary antibody；
(5) washing buffer；
(6) sample diluting liquid；
(7) develop the color A liquid；
(8) develop the color B liquid；
(9) the sulfuric acid liquid of 2M.
Further, the AFB1 competition antigen is coupled by the ring type polypeptide and biotin of detection aflatoxin AFB1
It forms.Still further, the formula of the washing buffer PBST are as follows: sodium chloride 40g, potassium dihydrogen phosphate 8g, disodium hydrogen phosphate
29g, potassium chloride 8g, Tween-20 2.5ml, distilled water 20mL.
The concentration of standard solution of the aflatoxin B1 includes 30,10,2,1,0.2,0.02ng/mL；
The sample diluting liquid: being the phosphate buffer of 10% methanol, the formula of phosphate buffer containing volume fraction
For sodium chloride 8g, potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 2.9g, potassium chloride 0.2g.
The colour developing A liquid: urea peroxide 1g, citric acid 10.3g, Na2HPO4·12H2100 μ L of O35.8g, Tween-20,
Distilled water 1000mL, pH5；
The colour developing B liquid: tetramethyl benzidine (TMB) 300mg (50mL DMSO dissolution), 10.3g citric acid, distilled water
1000mL, pH2.4-2.6.
The invention also discloses it is a kind of using mentioned reagent box detection aflatoxin B1 competitive immunization analysis method,
It is characterized by comprising following steps:
1) after mixing AFB1 competition antigen in equal volume with sample to be tested, the enzyme for being embedded with anti-AFB1 monoclonal antibody is added
In target；
2) after washing ELISA Plate with washing buffer, horseradish peroxidase-labeled Streptavidin (enzyme mark two is added
It is anti-), then ELISA Plate is washed with washing buffer, the mixed liquor that colour developing A liquid and the B liquid that develops the color is added develops the color, and 2M is then added
Sulfuric acid liquid terminate；
3) the OD value to gaging hole is measured in 450nm wavelength with microplate reader, is established using the standard solution of aflatoxin B1
Standard curve, according to the conversion corresponding with the OD value to gaging hole of established standard curve to get the aspergillus flavus poison into sample to be tested
The content of plain B1.
The present invention also provides application of the above-mentioned detection aflatoxin B1 kit in checking rice in AFB1 content.
The principle of the present invention:
The competitive immunization analysis method principle of ring type polypeptide detection AFB1 based on biotin labeling is: on ELISA Plate
Each Kong Jun is coated with same amount of anti-AFB1 antibody, be added isometric biotin labeling ring type polypeptide and AFB1 standard to be measured
The mixture of product or sample, the ring type polypeptide of biotin labeling and AFB1 to be measured vies each other and antibody response, due to each hole
In coated antibody and the ring type polypeptide uniform content of biotin labeling of addition cause, so when AFB1 concentration to be measured is high,
It is just few to be then bonded to the ring type polypeptide of the biotin labeling on solid-phase coating antibody, the ELIAS secondary antibody of addition and is fixed biology
The ring type polypeptide binding capacity of element label is few, substrate developing solution is added after being washed with cleaning solution, chromogenic reaction is shallow, is detected with microplate reader
OD value it is low, show inhibiting rate height；Conversely, the OD value then surveyed is high, and inhibiting rate is low when AFB1 concentration to be measured is low.According to
Known AFB1 standard concentration detects made standard curve, can extrapolate the concentration of AFB1 in sample to be tested.
Polypeptide can not only simulate antigen table of the AFB1 in conjunction with antibody as the element competed with AFB1 in the present invention
The detection signal can also be amplified by the high-affinity of biotin and Streptavidin, establish safe and non-toxic AFB1's by position
Competitive immunization analysis method.Therefore, the ring type polypeptide of biotin labeling is that a kind of very safe and efficient AFB1 competitiveness is anti-
It is former.
In the present invention screening obtain specificity simulation AFB1 epitope ring type polypeptide (polypeptide has 9 amino acid altogether, two
Ring-type is formed after the cysteine oxidation of side, such as Fig. 1 can simulate the antigenic determinant of AFB1, and it is competitive to become substitution AFB1
The reagent of antigen), sequence CVPSKPGLC, the sequence can effectively simulate the combination epitope of AFB1 and antibody, can be with anti-AFB1
Monoclonal antibody combines, by adding quantitative AFB1 in rice sample, with immune point established after simple pre-treatment
The measurement of analysis method, and this method is verified.
Beneficial effects of the present invention:
1. detect agricultural product in AFB1 general immunisation analysis method be indirect immunoassays method, generally require by
It is connected to competitive antigen with carrier protein after AFB1 processing, this process operator can be exposed in the environment of AFB1,
With potentially hazardous property.
The present invention screens to have obtained specific simulation AFB1 epitope, the polypeptide that sequence is CVPSKPGLC, which can
With in conjunction with anti-AFB1 monoclonal antibody, by the biotin labeling polypeptide, success is built instead of traditional competitive antigen
Vertical method is the immunoassay method of safe and non-toxic detection AFB1 a kind of.
2. the ring type polypeptide of biotin labeling is as competitive antigen, using Streptavidin and horseradish peroxidase
Conjugate can significantly improve the sensitivity of detection method as ELIAS secondary antibody, using the ring-type of biotin labeling in the present invention
IC of the polypeptide as competitive antigen institute method for building up50For 0.92ng/mL, can in Accurate Determining rice sample AFB1 content.
3. including that the ring type polypeptide of biotin labeling, anti-AFB1 monoclonal are anti-in claimed kit in the present invention
The reagents such as body, ELIAS secondary antibody can satisfy the testing requirements of agricultural samples, be the inspection of safe, simple, portable AFB1 a kind of
Survey method.
In summary: the present invention relates to the screening of the phage polypeptide of Specific competition AFB1, polypeptide amino acid sequence,
The coupling of polypeptide and biotin, the foundation of immunoassay method,
The present invention uses the ring type polypeptide in the bacteriophage source of biotin labeling for the first time and detects agricultural production as competitive antigen
The content of AFB1 in product.This method replaces making and using for tradition AFB1 envelope antigen, greatly reduces AFB1 pairs
The exposure of operator, therefore, this method are a kind of sensitive measuring methods of highly effective and safe.
Detailed description of the invention
Fig. 1 is the ring type polypeptide schematic diagram from phage display system；
Fig. 2 is to identify positive plaque schematic diagram with Phage ELISA method,
Fig. 3 is the standard curve that the standard solution of aflatoxin B1 in kit is established.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, so as to those skilled in the art understand that.
Following laboratory samples are purchased from market, and part description of commodity buys source:
The specific elutriation and amplification of 1 phage-displayed polypeptides of embodiment
The elutriation of phage display specific polypeptide
5 holes are coated in 96 hole elisa Plates, the 1st hole is 100 μ g/mL antibody, remaining 4 coating 3%BSA, 4 DEG C of packets
It is stayed overnight, next day is added 200 μ L 0.05%PBST board-washing 3 times；1%BSA closes ELISA Plate, 25 DEG C of incubation 2h, and board-washing 3 times；Hole
1 μ L phage display ring seven peptide library is added in 1, is incubated for 1h, the elution of 500ppb AFB1 competitiveness, 25 DEG C of incubations are added after board-washing
Eluent is transferred in 2,3,4,5 hole of hole by 1h, 25 holes μ L/, after being incubated for 1h, is collected supernatant, is then the elution after elutriation
Liquid utilizes the titre of upper panel method measurement eluent pnagus medius.Second wheel elutriation and third round elutriation antibody concentration are successively
Halve (50 μ g/mL, 25 μ g/mL), the concentration of Tween-20 is respectively 0.1% and 0.15% in the PBST cleaning solution used.
The amplification of wash-out bacteriophage
E. coli ER2738 bacterium solution is accessed in 20mL LB liquid medium, to OD600nmIt is 0.6~1.0
When, it is added the bacteriophage of above-mentioned elution, after 37 DEG C of shake culture 4.5h, bacterium solution is transferred in 50mL centrifuge tube, 4 DEG C
8000rpm is centrifuged 10min.The 20%PEG/NaCl precipitating phage of 1/6 volume is added to supernatant, 4 DEG C stand overnight；It is secondary
Day, 4 DEG C of 10000rpm are centrifuged 15min, are dissolved and are precipitated with 1mL PBS solution, after 4 DEG C of 10000rpm centrifugation 5min, supernatant is taken,
And the 20%PEG/2.5M NaCl that 1/6 volume is added is precipitated again, is incubated for 15~60min, 4 DEG C of 10000rpm centrifugations on ice
After 10min, precipitating is dissolved with 200 μ L sterile PBS buffers, is the phage solution after expanding after mixing.After amplification
Phage peptide library surveys titre, carries out next round elutriation.
The identification and sequencing of 2 specific polypeptide of embodiment
From picking monoclonal on the plate of above-mentioned third round measurement wash-out bacteriophage, surveyed using the method for Phage ELISA
Determine the ability of phage-displayed polypeptides clone's simulation AFB1 epitope, binding antibody, concrete operations are as follows: by the plaque of picking
It is seeded in 2mL LB culture medium (E.coli ER2738 early period of logarithm containing 1:100), after 37 DEG C of shake culture 6h, 10000rpm
It is centrifuged 10min, collects supernatant.Each clone is measured with 3 holes, and hole 1 and the coating of hole 2 screen 100 μ L of antibody (10 μ g/ used
ML), hole 3 is coated with 1%BSA and does blank control, and after being washed with PBST, 3% skim milk closing 2h (300 hole μ L/), hole 1 is added
The mixed liquor of 50 μ L500ppb AFB1 and 50 μ L plaque culture supernatants is added, 50 μ L, 10% methanol is added in hole 2 and hole 3
The mixed liquor of PBS and 50 μ L plaque culture supernatants, 37 DEG C of incubation 30min, PBST board-washing 3 times.It is anti-that 100 μ L are added in each hole
After M13 phage antibody-HRP, 37 DEG C of incubation 30min, board-washing 3 times, 100 μ L substrate developing solutions are added in every hole, and room temperature is protected from light aobvious
After color 5-15min, 50 μ L 2M H are added2SO4Terminate reaction.Each hole light absorption value under 450nm, 2 absorbance of hole are measured with microplate reader
It is positive plaque that value, which is higher than hole 1 and the clone in hole 3,.It is sequenced after extracting DNA after positive bacteriophage is expanded, is drawn using general
Object -96g III (5-CCCTCATAGTTAGCGTAACG-3).
Competitive binding ability through Phage ELISA measurement plaque, as shown in Fig. 2, wherein clone 3-C-43 is competitive
Inhibitory effect is best, therefore will be sequenced after the clonal expansion, which is GTGCCTAGTAAGCCGGGGCTG, translation
Obtaining amino acid sequence is CVPSKPGLC, as detects the ring type polypeptide of aflatoxin AFB1.
The synthesis of the ring type polypeptide of 3 biotin labeling of embodiment
Biotin is reacted with-the NH2 in ring type polypeptide, is total in the ring type polypeptide sequence there are two free-NH2,
One in N-terminal, another is connected on the R base of lysine, therefore by ring type polypeptide and biotin with the ratio of 1:2.Weigh 2mg
Ring type polypeptide, be added 50 μ L DMF and 2mL PBS buffer solution dissolve polypeptide；
4.6mg biotin is weighed, 50 μ LDMF are added and are dissolved, the 22 dissolved biotins of μ L is taken to be added dropwise to dissolution
In polypeptide afterwards, dialyse after ice bath 2h for 24 hours to get the ring type polypeptide for arriving biotin labeling.
The kit of ring type polypeptide immunoassay method detection AFB1 of the embodiment 4 based on biotin labeling, kit packet
Containing following part:
(1) ring type polypeptide of above-mentioned biotin labeling；
(2) 96 hole elisa Plates of anti-AFB1 monoclonal antibody are coated with；
(3) standard solution of aflatoxin B1；
(4) horseradish peroxidase-labeled Streptavidin (ELIAS secondary antibody)；
(5) formula of thickening and washing buffer (PBST) are as follows: sodium chloride 40g, potassium dihydrogen phosphate 8g, disodium hydrogen phosphate
14.5g, potassium chloride 8g, Tween-20 2.5ml, distilled water 20ml；
(6) sample diluting liquid: the phosphate buffer containing 10% methanol, the formula of phosphate buffer be sodium chloride 8g,
Potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 2.9g, potassium chloride 0.2g；
(7) develop the color A liquid: urea peroxide 1g, 10.3g citric acid, 35.8g Na2HPO4·12H2100 μ l of O, Tween-20,
Distilled water 1000ml, pH5；Develop the color B liquid: tetramethyl benzidine (TMB) 300mg (50ml DMSO dissolution), 10.3g citric acid steam
Distilled water 1000mL, pH2.4-2.6.
(8) the sulfuric acid liquid of 2M.
After this kit is completed, the ring type polypeptide of antibody coating plate and biotin labeling can at least be protected at -20 DEG C
It deposits 1 year, other reagents are stored at room temperature.
The immunoassay method of ring type polypeptide of the embodiment 5 based on biotin labeling measures AFB1
Use above-mentioned anti-AFB1 monoclonal antibody as coated antibody, first by the ring type polypeptide of biotin labeling and AFB1 sample
Product are added after mixing in equal volume and are coated in the enzyme mark hole of anti-AFB1 monoclonal antibody, compete the antibody jointly, add enzyme mark
Secondary antibody and substrate pass through the content of AFB1 in the degree judgement sample of substrate colour developing.AFB1 is measured with competitive immunoassay method,
Its step are as follows:
(1) it balances: the ELISA Plate of processed good anti-AFB1 monoclonal antibody being taken out from the package, is put in equilibrium at room temperature
15min；
(2) board-washing: the PBST that 200 μ L are added in every hole (contains the PBS (pH 7.4) of 0.05% (v/v) Tween20, places
1min, then cleaning solution is got rid of, it is repeated 3 times, is patted dry PBST is remained in plate on blotting paper；
(3) competitive reaction: now the ring type polypeptide of biotin labeling and isometric AFB1 standard or sample to be tested are mentioned
Liquid is taken to mix, standard solution and sample diluting liquid are with the PBST for containing 10% methanol；100 μ L are added to the enzyme mark hole closed
In, 1h, board-washing are reacted at room temperature；
(4) add ELIAS secondary antibody: ELIAS secondary antibody (conjugate of Streptavidin and horseradish peroxidase) is pressed with PBST
1:1,000 dilution, every hole is added 100 μ L, reacts 1h, board-washing at room temperature；
(5) develop the color: A liquid is prepared: urea peroxide 1g, 10.3g citric acid, 35.8g Na2HPO4·12H2O, Tween-20
100 μ L, distilled water 1000mL, pH5；B liquid is prepared: tetramethyl benzidine (TMB) 700mg (40ml DMSO dissolution), 10.3g lemon
Lemon acid, distilled water 1000mL, pH 2.4-2.6.A liquid and B liquid are mixed by 1:1, then 100 μ L are added in every hole, under room temperature
It is protected from light colour developing 10-15min；
(6) terminate and measure: every hole is added the sulfuric acid liquid that 50 μ L concentration are 2M and terminates reaction, immediately with microplate reader in
450nm wavelength measures the OD value in each hole.
The anti-AFB1 monoclonal antibody for using screening used as envelope antigen, establish by the ring type polypeptide based on biotin labeling
Immunoassay method detection AFB1 standard suppression curve.The series standard concentration setting range of AFB1 is 0~30ng/mL.
With quadruplex parameters fit standard curve (see Fig. 3), the sensitivity concentration table of AFB1 required when inhibiting highest OD value half
Show, i.e. concentration IC in inhibition50Value, the IC of the standard curve50Value is 0.92ng/mL.The range of linearity IC of curve20-IC80Value is
0.23-3.36ng/mL。
6 specificity experiments of embodiment
Select aflatoxin B 2, aflatoxin G 1, aflatoxin G 2, zearalenone, ochratoxin A and
Citrinin measures the IC of each substance as determinand50Value, then the cross reaction with following equation calculating antibody to these substances
Property；Cross reacting rate is smaller, then specific more strong, on the contrary then poor specificity of the polypeptide to AFB1.
Cross reaction (CR%)=IC50(AFB1)/IC50(for trying object) × 100%
Measuring the results are shown in Table 1, and using the method for case study on implementation 3, the ring type polypeptide of the biotin labeling can not be known
Other determinand (cross reacting rate < 1%), can only identify AFB1, therefore this method high specificity, it can be ensured that AFB1 in sample
The reliability of assay result.
Cross reaction of the table 1 based on the immunoassay method of polypeptide from different mycotoxins
7 sample detection of embodiment
Rice sample is bought from supermarket, through being free of AFB1 in HPLC-FLD identification sample.5.0g rice is weighed in 50mL
In beaker, 1.5mLAFB1 standard items (being dissolved with methanol) is added into sample, makes that AFB1's in rice sample is final concentration of
10ng/g, 20ng/g and 40ng/g.Sample pre-treatments: 15mL methanol being added into 5g rice sample, mixes, ultrasonic extraction
5min, 5000rpm are centrifuged 10min, and supernatant detects the content of AFB1 with 4 the method for embodiment after diluting 10 times with PBS.Add
The testing result that add-back is received is shown in Table 2, and the average recovery rate of intraassay is 84.1~96.7%, the coefficient of variation 2.1~6.3%；
The average recovery rate analyzed between batch is 80.5~89.1%, the coefficient of variation 5.4~9.2%.The accuracy and precision of this method
Meet actually detected requirement, can be used for rapid screening of the AFB1 in rice sample.
Measurement result is recycled in the addition of AFB1 in 2 rice sample of table
Other unspecified parts are the prior art.Although above-described embodiment is made that the present invention and retouches in detail
State, but it is only a part of the embodiment of the present invention, rather than whole embodiments, people can also according to the present embodiment without
Other embodiments are obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.
Sequence table
<110>Hua Zhong Agriculture University
<120>ring type polypeptide and its detection kit of aflatoxin B1 epitope are simulated
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213>artificial synthesized sequence (Synthetic sequence)
<400> 1
Cys Val Pro Ser Lys Pro Gly Leu Cys
1 5
Claims (6)
1. a kind of ring type polypeptide for simulating aflatoxin B1 epitope, it is characterised in that: its amino acid sequence is
CVPSKPGLC, as shown in SEQ ID No.1.
2. a kind of detection aflatoxin B1 kit comprising polypeptide described in claim 1, it is characterised in that: the kit
Include following part:
(1) AFB1 competes antigen, and AFB1 competition antigen is ring type polypeptide described in the claim 1 of biotin labeling；
(2) it is embedded with the ELISA Plate of anti-AFB1 monoclonal antibody；
(3) standard solution of aflatoxin B1；
(4) horseradish peroxidase-labeled Streptavidin, as ELIAS secondary antibody；
(5) washing buffer；
(6) sample diluting liquid；
(7) develop the color A liquid；
(8) develop the color B liquid；
(9) the sulfuric acid liquid of 2M.
3. detecting aflatoxin B1 kit according to claim 2, it is characterised in that: AFB1 competition antigen be by
The ring type polypeptide of detection aflatoxin AFB1 is coupled with biotin.
4. detecting aflatoxin B1 kit according to claim 2, it is characterised in that: the washing buffer PBST's
Formula are as follows: sodium chloride 40g, potassium dihydrogen phosphate 8g, disodium hydrogen phosphate 29g, potassium chloride 8g, Tween-20 2.5ml, distilled water
20mL；
The concentration of standard solution of the aflatoxin B1 includes 30,10,2,1,0.2,0.02ng/mL；
The sample diluting liquid: being the phosphate buffer of 10% methanol containing volume fraction, and the formula of phosphate buffer is chlorine
Change sodium 8g, potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 2.9g, potassium chloride 0.2g；
The colour developing A liquid: urea peroxide 1g, citric acid 10.3g, Na2HPO4·12H2100 μ L of O 35.8g, Tween-20 steams
Distilled water 1000mL, pH 5；
The colour developing B liquid: tetramethyl benzidine 300mg, 10.3g citric acid, distilled water 1000mL, pH 2.4-2.6.
5. a kind of competitive immunization analysis method using the detection aflatoxin B1 of kit described in claim 2, feature
It is: the following steps are included:
1) after mixing AFB1 competition antigen in equal volume with sample to be tested, the ELISA Plate for being embedded with anti-AFB1 monoclonal antibody is added
In；
2) after washing ELISA Plate with washing buffer, horseradish peroxidase-labeled Streptavidin is added, then use washing buffer
Liquid washs ELISA Plate, and the mixed liquor that colour developing A liquid and the B liquid that develops the color is added develops the color, and the sulfuric acid liquid that 2M is then added terminates；
3) the OD value to gaging hole is measured in 450nm wavelength with microplate reader, establishes standard using the standard solution of aflatoxin B1
Curve, according to the conversion corresponding with the OD value to gaging hole of established standard curve to get the aflatoxin B1 into sample to be tested
Content.
6. application of the detection aflatoxin B1 kit in checking rice in AFB1 content described in a kind of claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811126720.7A CN109400682B (en) | 2018-09-26 | 2018-09-26 | cyclic polypeptide simulating aflatoxin B1 epitope and detection kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811126720.7A CN109400682B (en) | 2018-09-26 | 2018-09-26 | cyclic polypeptide simulating aflatoxin B1 epitope and detection kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109400682A true CN109400682A (en) | 2019-03-01 |
| CN109400682B CN109400682B (en) | 2019-12-10 |
Family
ID=65466391
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811126720.7A Active CN109400682B (en) | 2018-09-26 | 2018-09-26 | cyclic polypeptide simulating aflatoxin B1 epitope and detection kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109400682B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110615831A (en) * | 2019-09-27 | 2019-12-27 | 天津科技大学 | Cyclic peptide and application thereof |
| CN111999502A (en) * | 2020-08-24 | 2020-11-27 | 湖南农业大学 | Aflatoxin B1 detection kit and method for regulating multimode signal output based on PBNPs in-situ growth |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018458A (en) * | 2012-12-06 | 2013-04-03 | 北京普赞生物技术有限公司 | Ultra-sensitive aflatoxin B1 enzyme-linked immunosorbent assay kit |
| CN104262489A (en) * | 2014-09-30 | 2015-01-07 | 陈仁奋 | Cyclic chimeric citrullinated peptide antigen and application thereof |
| CN104311637A (en) * | 2013-03-06 | 2015-01-28 | 南昌大学 | Antigen mimic epitope capable of mimicking fumonisins B1 and application of antigen mimic epitope |
| CN104569380A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit |
| CN105277708A (en) * | 2015-03-20 | 2016-01-27 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili |
| CN108444992A (en) * | 2018-02-09 | 2018-08-24 | 河南工业大学 | A kind of quantitative aflatoxin detection kit and its detection method |
-
2018
- 2018-09-26 CN CN201811126720.7A patent/CN109400682B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018458A (en) * | 2012-12-06 | 2013-04-03 | 北京普赞生物技术有限公司 | Ultra-sensitive aflatoxin B1 enzyme-linked immunosorbent assay kit |
| CN104311637A (en) * | 2013-03-06 | 2015-01-28 | 南昌大学 | Antigen mimic epitope capable of mimicking fumonisins B1 and application of antigen mimic epitope |
| CN104262489A (en) * | 2014-09-30 | 2015-01-07 | 陈仁奋 | Cyclic chimeric citrullinated peptide antigen and application thereof |
| CN104569380A (en) * | 2015-01-23 | 2015-04-29 | 天津伯克生物科技有限公司 | Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit |
| CN105277708A (en) * | 2015-03-20 | 2016-01-27 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili |
| CN108444992A (en) * | 2018-02-09 | 2018-08-24 | 河南工业大学 | A kind of quantitative aflatoxin detection kit and its detection method |
Non-Patent Citations (6)
| Title |
|---|
| HINA MUKHTAR ET AL.: "Cyclic peptide: a safe and effective alternative to synthetic aflatoxin B1-competitive antigens", 《ANALYTICAL AND BIOANALYTICAL CHEMISTRY》 * |
| J.MARC FREMY ET AL.: "Policy on Characterization of Antibodies Used in Immunochemical Methods of Analysis for Mycotoxins and Phycotoxins", 《FREMY&USLEBER: JOURNAL OF AOAC INTERNATIONAL》 * |
| STEPHEN DALY ET AL.: "Production and Characterization of Murine Single Chain Fv Antibodies to Aflatoxin B 1 Derived From a Pre-immunized Antibody Phage Display Library System", 《FOOD AND AGRICULTURAL IMMUNOLOGY》 * |
| 庞倩等: "基于噬菌体展示技术抗黄曲霉毒素B1单链抗体的筛选及其蛋白结构分析", 《中国生物工程杂志》 * |
| 邓省亮等: "采用噬菌体展示技术筛选黄曲霉毒素B1模拟抗原表位", 《卫生研究》 * |
| 马兰等: "抗黄曲霉毒素B1单链抗体基因克隆及其结构分析", 《南方农业学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110615831A (en) * | 2019-09-27 | 2019-12-27 | 天津科技大学 | Cyclic peptide and application thereof |
| CN111999502A (en) * | 2020-08-24 | 2020-11-27 | 湖南农业大学 | Aflatoxin B1 detection kit and method for regulating multimode signal output based on PBNPs in-situ growth |
| CN111999502B (en) * | 2020-08-24 | 2023-08-04 | 湖南农业大学 | Aflatoxin B1 detection kit and method based on PBNPs in-situ growth regulation multimode signal output |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109400682B (en) | 2019-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103018458B (en) | Ultra-sensitive aflatoxin B1 enzyme-linked immunosorbent assay kit | |
| CN106290889B (en) | The detection method of aflatoxin B1 | |
| He et al. | Phage-borne peptidomimetics as immunochemical reagent in dot-immunoassay for mycotoxin zearalenone | |
| CN108614106A (en) | A kind of time-resolved fluoroimmunoassay chromatography card detecting vomitoxin and its acetyl derivatives | |
| CN109709338A (en) | Detect ox or the enzyme linked immunological kit of ovine skeletal muscle Troponin I and the preparation method and application thereof | |
| CN103792356A (en) | Preparation and detection method for ELISA kit detecting Fumonisins | |
| CN104076154A (en) | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof | |
| CN112213497B (en) | polypeptide-ELISA kit for detecting novel coronavirus S protein unique antibody | |
| CN109400682A (en) | Simulate the ring type polypeptide and its detection kit of aflatoxin B1 epitope | |
| CN103487575A (en) | Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof | |
| Kawatsu et al. | A highly rapid and simple competitive enzyme-linked immunosorbent assay for monitoring paralytic shellfish poisoning toxins in shellfish | |
| CN103091494A (en) | Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method | |
| CN102072958A (en) | Chemiluminescence immunoassay kit used for detecting aflatoxin B1 and preparation method and use method thereof | |
| Pei et al. | Colorimetric ELISA with an acid–base indicator for sensitive detection of ochratoxin A in corn samples | |
| CN105319368A (en) | Enzyme linked immunosorbent assay kit used for detecting zearalenone, and detection method thereof | |
| CN101968483A (en) | One-step semi-double-antigen sandwich immunological detection method | |
| CN103513036B (en) | The CLEIA detection kit of salmonella and detection method | |
| CN109406784A (en) | A kind of detection microcapsule phycotoxin MC-LR time-resolved fluoroimmunoassay quantifies POCT test strips | |
| CN101377494B (en) | Chemiluminescence immune analytic reagent kit for detecting tuberculosis antibody | |
| CN110045130A (en) | A kind of immunoassay detection kit of polypeptide relevant to IgA nephrosis | |
| CN104897651A (en) | Chemiluminescent kit for aflatoxin M1 and application thereof | |
| CN110045123A (en) | A kind of carbohydrate antigen 242 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN108508217A (en) | A kind of indirect ELISA method kit and its application for detecting people's lower respiratory tract bocavirus | |
| CN108051600A (en) | A kind of chemiluminescence detection kit of leptin and preparation method thereof | |
| CN107478827A (en) | A kind of chemiluminescence detection kit of fumonisin and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |