CN105566494A - Monoclonal antibody and enzyme-linked immunosorbent assay method for detecting aflatoxin M1 - Google Patents
Monoclonal antibody and enzyme-linked immunosorbent assay method for detecting aflatoxin M1 Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody which can specifically identify aflatoxin M1. The monoclonal antibody is secreted by a hybridoma cell strain AFM 1/3D8, the hybridoma cell strain AFM 1/3D8 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:C201559. The invention further discloses an enzyme-linked immunosorbent assay method and kit for detecting the aflatoxin M1. Compared with the prior art, the prepared monoclonal antibody only identifies the aflatoxin M1, and the enzyme-linked immunosorbent assay method and kit have the advantages of being high in detection sensitivity and accuracy, good in precision, simple, convenient, rapid and the like.
Description
Technical field
The invention belongs to wild animal resources and immunological technique field, being specifically related to a kind ofly can identify the monoclonal antibody of Aflatoxins M1 and a kind of enzyme-linked immunoassay method (ELISA) for detecting Aflatoxins M1.
Background technology
The secondary metabolite that aflatoxin is mainly secreted by flavus and Aspergillus parasiticus.It is the similar compound of a class formation, all contains two furan nucleuss and tonka bean camphor.It has kind more than 20, current isolation identification have 12 kinds, common is mainly aflatoxin B1, B2, G1, G2, M1, M2.The main contaminated milk of Aflatoxins M1 and milk preparation thereof, the toxicity of Aflatoxins M1 is extremely strong, and has carinogenicity.Along with the raising of people's living standard, the demand of people to milk preparation is increasing, therefore strengthens the residual monitoring of Aflatoxins M1 in milk very necessary.
The method of current detection Aflatoxins M1 is mainly instrumental method and immunological method.Although instrumental method is sensitive, accurate, resolution is high and can carry out the qualitative and quantitative study of multi-residue determination, need expensive instrument, loaded down with trivial details pre-treatment, skilled professional operation person.If adopt instrumental method to carry out the detection of batch samples, its cost will be very high; And in current national feeler mechanism, major part only has provincial, and municipal level to be just equipped with accurate analytical instrument, therefore needs to set up one from the detection system screening confirmation.Instrumental method is not obviously suitable for a large amount of screenings of sample.Immunochemical analyses method particularly enzyme-linked immunosorbent analytical technique has the advantages such as quick, highly sensitive, simple to operate, strong adaptability, is applicable to the screening of high-throughput sample, residual therefore for rapid detection aflatoxin, and ELISA method has more advantage.And the antibody preparing higher sensitivity is the basic of ELISA method, only prepare the antibody of higher sensitivity, just can prepare and there is emulative ELISA kit.Jiang Tao etc. (2007) have prepared the monoclonal antibody of anti-AFM1 and have established ELISA method, and the linearity range of typical curve is 0.02 ~ 2ng/mL, and detecting of the method is limited to 0.07ng/mL; Zhang Yuanyuan etc. (2008) take AFM1-BSA as immunogen, and prepared anti-AFM1 antibody, detecting of this antibody is limited to 0.08ng/mL, and linearity range is 0.08 ~ 5ng/mL.Therefore the detection sensitivity of existing antibody is not high enough.
Summary of the invention
Object of the present invention is:
(1) a kind of monoclonal antibody of energy specific recognition Aflatoxins M1 is provided;
(2) described monoclonal antibody is provided to prepare the application in Aflatoxins M1 enzyme-linked immunologic detecting kit;
(3) a kind of enzyme linked immunological kit that can detect Aflatoxins M1 is provided;
(4) utilize this monoclonal antibody, set up a kind of ELISA method that can detect Aflatoxins M1;
Above-mentioned purpose is achieved through the following technical solutions:
A monoclonal antibody for energy specific recognition Aflatoxins M1, it is secreted by hybridoma cell strain AFM1/3D8.
Described hybridoma cell strain AFM1/3D8, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201559.
Described monoclonal antibody reacts with Aflatoxins M1 (AFM1) and carboxymethyl azanol half hydrochloride (CMO) to generate Aflatoxins M1 oxime (AFM1O), by AFM1O and carrier protein couplet, the conjugate obtained prepares as immunogen.In the present invention, preferred immunogenic carrier albumen is hemocyanin (KLH).
Described monoclonal antibody is for setting up corresponding ELISA method.
Described monoclonal antibody may be used for preparing the enzyme-linked immunologic detecting kit detecting Aflatoxins M1.
Detect the ELISA method that in milk, Aflatoxins M1 is residual, comprise the preparation of immunogen, coating antigen and antibody, the foundation of indirect competitive ELISA method, its step is as follows:
(1) haptens AFM1O AFM1 and CMO is obtained by reacting in the basic conditions;
(2) use EDC method by AFM1O and carrier protein couplet, obtain immunogen;
(3) coating antigen is obtained by EDC method by after aflatoxin B1 oxime (AFB1O) and carrier protein couplet;
(4) the hybridoma cell strain AFM1/3D8 of preserving number CCTCCNO:C201559 has been prepared by the immunogen that step (2) obtains;
(5) monoclonal antibody is prepared with the hybridoma cell strain AFM1/3D8 that preserving number is CCTCCNO:C201559;
(6) the coating antigen bag obtained by step (3) is by solid phase carrier;
(7) sample pre-treatments: get milk sample 10 ± 0.5mL in 50mL centrifuge tube, the centrifugal 5min of 4000r/min, gets supernatant, is determinand solution;
(8) enzyme linked immunosorbent detection is carried out to determinand solution.
Preferably, in aforesaid method, immunogenic carrier albumen is hemocyanin (KLH), and coating antigen carrier proteins is oralbumin (OVA).
The invention has the beneficial effects as follows:
1) the present invention is when preparing monoclonal antibody, adopts Aflatoxins M1 oxime to be haptens, obtains immunogen, the monoclonal anti physical efficiency specific recognition Aflatoxins M1 adopting this immunogen to prepare by this hapten conjugation albumen;
2) ELISA method set up of the present invention and test kit energy specific detection Aflatoxins M1, and detection sensitivity, accuracy are high, and precision is good, IC
50value reaches 64.75ng/L, linearity range 5 ~ 405ng/L.
Accompanying drawing explanation
Fig. 1 is the indirect competitive ELISA reaction normal graphic representation of monoclonal antibody of the present invention and Aflatoxins M1 standard substance, X-axis is Aflatoxins M1 (AFM1) concentration of standard solution logarithmic value, and Y-axis is that the optical density value of aflatoxin standard solution is divided by " zero " hole optical density value (B/B
0).
Embodiment
Below by embodiment, the invention will be further described, but do not limit the present invention.
The preparation of embodiment 1 immunogen and coating antigen
1.1 immunogenic preparations
Get the AFM1 methanol solution that 2mL concentration is 1mg/mL, add the sodium carbonate of 3mgCMO and 5mg, at room temperature stirring reaction 24h, then dry up with nitrogen, add the hydrochloric acid 2mL of 0.05mol/L, it is fully dissolved, add the extraction into ethyl acetate of 2mL, get ethyl acetate layer, repeat 3 times; The liquid obtained is dried up under a nitrogen, namely obtains haptens, react as follows:
To obtain haptens with DMF to dissolve, and add the NHS of EDC and 5mg of 10mg, activation 12h is A liquid; Being dissolved in the PBS solution of 5mL by the KLH of 2mL, is B liquid.Then A liquid is slowly added in B liquid, under condition of ice bath, react 12h; After question response is complete, loads in dialysis tubing through it, dialyse 3 ~ 5 days in PBS solution, change liquid every day 3 times.Namely obtain complete A antigen FM1-EDC-KLH, react as follows:
The preparation of 1.2 coating antigen AFB1-EDC-OVA
Be dissolved in DMF by haptens AFB1O, add the NHS of EDC and 5mg of 12mg, activation 4h is A liquid; Being dissolved in the PBS solution of 5mL by the OVA of 15mg, is B liquid; Then A liquid is slowly added in B liquid, under condition of ice bath, react 12h; After question response is complete, loads in dialysis tubing through it, dialyse 3 ~ 5 days in PBS solution, change liquid every day 3 times.Namely obtain complete A antigen FB1-EDC-OVA, react as follows:
The preparation of embodiment 2 monoclonal antibody
2.1 animal immune
The Aflatoxins M1 oxime utilizing the national basic veterinary drug at contriver place to remain to prepare benchmarks room and the immune Balb/C mouse of limpet hemocyanin conjugate (AFM1-EDC-KLH) (purchased from Hubei Prov. Academy of Medical Sciences's Experimental Animal Center).Immune programme for children gets containing injecting in Mice Body after the protein solution of AFM1-EDC-KLH conjugate 50 ~ 80 μ g and adjuvant balanced mix, makes it produce specific serum.
2.2 cytogamy and cloning
With reference to Yang Hanchun " animal immunology ", utilize AFM1-EDC-KLH immunogen immune Balb/C mouse (purchased from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center) prepared by contriver the country one belongs to residue of veterinary drug benchmarks room, immune programme for children is: fundamental immunity is by after immunogen and isopyknic Freund's complete adjuvant emulsification, in the subcutaneous multi-point injection of mouse back, once at interval of 2 weeks booster immunizations, use Freund's incomplete adjuvant emulsification instead later.Finally in first three sky of fusion (be better than most immunity terminate rear rest and reorganization January) abdominal injection, reinforced immunological, antigen amount doubles, and does not add adjuvant.
During fusion, the Balb/C mouse of last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum), soaks 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen, isolates splenocyte, with the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of fresh preparation by 1 ~ 2 × 10
7individual and 10
8individual immune spleen cell, it, between 1:10 ~ 1:15, is all placed in 50mL centrifuge tube by its ratio, and with the RPMI-1640 basal liquid re-suspended cell of 15mL, the centrifugal 5min of 1500r/min, washes cell 1 time.The substratum that temperature is bathed by centrifugal gap, the water of temperature bath, super clean bench put into by the polyoxyethylene glycol (PEG) etc. of temperature bath.Then take out the thieving paper of sterilizing, by the centrifuge tube that myeloma cell and immune spleen cell be housed emptying to the greatest extent, tipping upside down on and thieving paper controls solid carbon dioxide dripping, rapping at the bottom of pipe and cell is loosened.Open timing register, draw the PEG of 0.8mL, the hand-held centrifuge tube that cell mixing is housed, place it in a moment in water-bath, be added drop-wise to slowly on cell mixing by PEG, limit edged stirs gently, adds in 1min, Keep agitation 30s.Then add 10mL basic culture solution, be slowly added on fused cell along tube wall, limit edged shakes gently (can not blow and beat), add in 5min, finally add basic culture solution to 40mL, after adding, cover lid, puts upside down several times repeatedly, and cell is mixed.800r/min5min is centrifugal, abandons supernatant.Draw the HAT substratum 1mL containing feeder cell, slowly add, and stir gently, avoid piping and druming; Then be dropwise added dropwise in the serum bottle containing feeder cell by this cell, mix, be then seeded on Tissue Culture Plate by cell, two, every hole, is placed in incubator and cultivates.Single cell fusion can inoculate 4 ~ 6 piece of 96 orifice plate.Also can plant less as required, the cell count of generally pressing SP2/0 calculates, and every hole inoculum size is about containing 10
4a left and right SP2/0 cell.In 37 DEG C, 5%CO
2cultivate in incubator.
The same day of merging counts 0d, and front 3d tries not kinetocyte plate, keeps incubator homeostasis.3d adds in every hole 1 HAT perfect medium; 5d every hole sucking-off l/2 culture supernatant (100 μ L), then add 1 HT perfect medium; Suck l/2 ~ 3/4 culture supernatant every the same method of 2d later, after 7d, change to HT perfect medium.
Cell colony to be fused grows to about 1/4 of culture hole, screens with the indirect ELISA method set up.Compared with zero medicine hole, medicine hole OD value repressedly can be judged to the positive.According to inhibiting rate and cell colony upgrowth situation, select the cell hole only having 1-2 single colony of 2 ~ 6 strong positives, adopt limited dilution method to carry out cloning.
Through 3 ~ 4 time clonings, finishing screen selects the monoclonal antibody hybridoma cell strain of secretion aspergillus flavus resisting toxin M1, applicant is by its called after AFM1/3D8, and China typical culture collection center (CCTCC) preservation being positioned at Wuhan City, Hubei Province Wuhan University is sent on April 24th, 2015, deposit number is CCTCCNO:C201559.Chromosome counting has been carried out to this clone, result shows, and the chromosome number of SP2/0 is 62 ~ 68, and splenocyte karyomit(e) is 40, and the chromosome number mean value of hybridoma is 99.1, the cell of SP2/0 really of fused cell and the hybrid product of splenocyte are described.By this cell strain through abdominal injection Balb/C mouse, manufacture order clonal antibody.Adopt and identify the hypotype of the monoclonal antibody that the present invention obtains and light chain purchased from mouse monoclonal antibody (Monoclonalantibody, Mab) the Rapid ELISA isotyping kit of ThermoSxientific company, result is mouse IgG
1hypotype.
The foundation of embodiment 3AFM1 racing ELISA detecting method
The preparation (reagent that the present embodiment uses all adopts following methods preparation except another indicating) of 3.1 reagent
Phosphate buffered saline buffer: NaCl8.0g, KH
2pO
40.2g, Na
2hPO
412H
2o2.9g, KCl0.2g, add distilled water to 1000mL, regulates pH to 7.4;
Coating buffer: get Na
2cO
31.59g, NaHCO
32.93g, adds tri-distilled water to 1000mL, adjust ph to 9.6;
Washings: NaCl8.0g, KH
2pO
40.2g, Na
2hPO
412H
2o2.9g, KCl0.2g, Tween200.5mL, add distilled water to 1000mL, regulates pH to 7.4;
Confining liquid: skim-milk 1.5g is dissolved in 100mL phosphate buffered saline buffer;
Substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;
Substrate solution B:Na
2hPO
414.6g, citric acid 9.3g, 0.75% Urea Peroxide 6.4mL, adds distilled water to 1000mL;
Substrate cocktail: by A liquid and B liquid by volume 1:1 mix and get final product, now with the current;
Stop buffer: 2mol/L sulphuric acid soln.
Tentatively determining of 3.2 coating antigen concentration and antibody working concentration
Select the AFB1-EDC-OVA of above-mentioned synthesis as coating antigen, 8.0mg/L, 4.0mg/L, 2.0mg/L, 1.0mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L, 0.0625mg/L8 concentration is diluted to coating buffer, at 96 hole enzyme plates, from first to the 8th leu adds, and 4 DEG C are spent the night; Wash 3 times, pat dry, add confining liquid 200 μ L, 37 DEG C of closed 60min; Wash 3 times, pat dry, the first row to the 8th row of enzyme plate add successively 100 μ L phosphate buffered saline buffers dilution extension rate be 4000,8000,16000,32000,64000,128000,256000,512000 monoclonal antibody, hatch 30min for 37 DEG C, wash 3 times, pat dry; The sheep anti-mouse igg antibody that each hole adds the HRP mark of 1:5000 times of phosphate buffered saline buffer dilution (is called for short two to resist, the anti-sheep anti-mouse igg antibody being HRP mark of following indication two, purchased from Wuhan Fei Yuan Science and Technology Ltd.) 100 μ L, hatch 30min for 37 DEG C, wash 5 times, pat dry; Each hole adds 100 μ L Substrate cocktail, and lucifuge colour developing 15min, adds 50 μ L stop buffers, measure optical density value (OD value), the results are shown in Table 1 by automatic microplate reader at 450nm wavelength place.
Result shows, tentatively determine that the bag of coating antigen AFB1-EDC-OVA is 1.0mg/L or 0.5mg/L by concentration, antibody working concentration is 1:32000 or 1:16000.
Tentatively determining of table 1 coating antigen concentration and antibody working concentration
The determination of 3.3 best coating antigen concentration and antibody working concentration
Wrap by concentration with the coating antigen AFB1-EDC-OVA tentatively determined, 1.0mg/L, 0.5mg/L2 concentration coated elisa plate is set.Aflatoxins M1 phosphate buffered saline buffer is diluted to 405,135,45,15,5, a 0ng/L6 concentration, the monoclonal antibody of being diluted by 1:32000 and 1:16000 phosphate buffered saline buffer is respectively (because when making indirect competitive ELISA, the injection volume of monoclonal antibody reduces half, and correspondingly monoclonal antibody extent of dilution reduces half) and above-mentioned Aflatoxins M1 solution respectively add 50 μ L and carry out indirect competitive ELISA.Using the log concentration value of Aflatoxins M1 as X-coordinate, with the ratio (B/B of the OD value of Aflatoxins M1 standardized solution with " zero " hole OD value
0) draw suppression curve as ordinate zou, Aflatoxins M1 concentration (IC when select linear is better, generation 50% suppresses
50) junior as bag by concentration.With best coating antigen concentration coated elisa plate, Aflatoxins M1 is diluted to 405,135,45,15,5, a 0ng/L6 concentration, antibody is arranged extent of dilution with centre concentration 1:16000 equal difference, the Aflatoxins M1 standardized solution of monoclonal antibody and series concentration respectively adds 50 μ L and carries out indirect competitive ELISA, draw and suppress curve, select linear is better, IC
50junior is as optimum antibody working concentration for value.The results are shown in Table 2,3.
The best coating antigen concentration optimization of table 2
| Coating antigen concentration (mg/L) | Antibody dilution multiple (1:X) | 0 hole OD value | IC 50Value (μ g/L) |
| 1.0 | 32000 | 2.166 | 0.11 |
| 0.5 | 16000 | 2.035 | 0.08 |
Table 3 optimum antibody extent of dilution is optimized
| Antibody dilution multiple | 0 hole OD | IC 50(μg/ |
| 18000 | 2.308 | 0.10 |
| 17000 | 2.127 | 0.089 |
| 16000 | 2.047 | 0.081 |
| 15000 | 1.996 | 0.07 |
| 14000 | 1.745 | 0.086 |
Result shows, wrapping by concentration is 0.5mg/L, when antibody dilution is 1:15000, and its IC
50minimum.
The foundation of 3.4 typical curves
Aflatoxins M1 phosphate buffered saline is become 405,135,45,15,5, a 0ng/L6 concentration, each concentration repeats 5 holes, measures, replication 5 times according to indirect competitive ELISA method.With the logarithmic value of Aflatoxins M1 strength of solution for X-coordinate, B/B0 is ordinate zou drawing standard curve, obtains IC
50.The IC of this test kit
50value is 64.75ng/L.
3.5 cross reaction tests
Five kinds of aflatoxin phosphate buffered saline are become proper concn, measures the IC of each medicine by the ELISA method set up
50value, the multiple hole of each medicine 3, with monoclonal antibody to the cross reacting rate of Aflatoxins M1 for 100%, utilize formula 1 to calculate the cross reacting rate of monoclonal antibody to each medicine, the results are shown in Table 4.
Table 4 the present invention is to the cross reacting rate of various aflatoxin
Result shows, monoclonal antibody, to aflatoxin B1, B2, G1, G2 nonrecognition, only identifies M1.
The assembling of embodiment 4ELISA test kit
4.1 ELISA kit of the present invention are made up of following part:
1) solid phase carrier (enzyme plate) of coating antigen AFB1-EDC-OVA is coated with;
2) 6 bottles, Aflatoxins M1 standardized solution, concentration is respectively 405ng/L, 135ng/L, 45ng/L, 15ng/L, 5ng/L, 0ng/L;
3) preserving number is the monoclonal antibody of the hybridoma AFM1/3D8 secretion of CCTCCNO:C201559;
4) the sheep anti-mouse igg antibody working fluid that marks of horseradish peroxidase (HRP);
5) concentrated phosphoric acid salt buffer: NaCl80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o29.0g, KCl2.0g, add distilled water to 1000mL;
6) concentrated cleaning solution: NaCl80.0g, KH
2pO
42.0g, Na
2hPO
412H
2o29.0g, KCl2.0g, Tween205mL, add distilled water to 1000mL;
7) substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;
8) substrate solution B:Na
2hPO
414.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, adds distilled water to 1000mL, regulates pH to 5.0 ~ 5.4;
9) stop buffer: 2mol/L sulphuric acid soln.
The preparation of 4.2 enzyme plates
With coating buffer, AFB1-EDC-OVA is diluted to 0.5mg/L, every hole adds 100 μ L, and 4 DEG C are spent the night, and incline coating buffer, every hole adds 250 μ L washingss and washs 3 times, pat dry, then every hole adds confining liquid 250 μ L, hatches 120min for 37 DEG C, incline liquid in hole, washings washs 3 times, pats dry, and preserves with masking foil vacuum-sealing.
The mensuration program of embodiment 5 enzyme linked immunological kit
The preparation of 5.1 reagent
1) sample diluting liquid: use after the concentrated phosphoric acid salt buffer tri-distilled water provided in test kit is diluted 10 times.
2) washings: use after the washings tri-distilled water provided in test kit is diluted 10 times.
3) Substrate cocktail: according to each institute expense, by the substrate solution A of preparation and substrate solution B by volume 1:1 mixing, now with the current.
5.2 sample pre-treatments: get milk sample 10 ± 0.5mL in 50mL centrifuge tube, the centrifugal 5min of 4000r/min, gets supernatant.
5.3 determination step
1) application of sample: add Aflatoxins M1 series concentration standardized solution or sample solution 50 μ L in enzyme plate micropore, then add monoclonal antibody working fluid 50 μ L, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;
2) wash: pour out the liquid in hole, add washings 250 μ L in every hole and wash 3 times and pat dry;
3) the sheep anti-mouse igg antibody working fluid that horseradish peroxidase (HRP) marks is added: in every hole, add the sheep anti-mouse igg antibody working fluid 100 μ L that horseradish peroxidase (HRP) marks, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;
4) wash: pour out the liquid in hole, in every hole, add washings 250 μ L, wash 3 times and pat dry;
5) substrate is added: add Substrate cocktail 100 μ L in every hole, be placed in wet box, 37 DEG C of constant-temperature incubation 15min;
6) stop buffer is added: in every hole, add stop buffer 50 μ L;
7) measure: the optical density value (OD value) measuring every hole by microplate reader at 450nm place.
5.4 results judge
Typical curve:
With measured standard substance OD value divided by " zero " hole OD value (B/B
0) be ordinate zou, the logarithmic value of the concentration of Aflatoxins M1 is that X-coordinate makes typical curve, and line linearity of going forward side by side returns, and provides regression equation.
In milk, the concentration of Aflatoxins M1 calculates:
The inhibiting rate (the OD value of the sample obtained is divided by " zero " hole OD value) of calculation sample, substitutes into Standard N g/L).
The sensitivity of embodiment 6 ELISA method of the present invention, precision, accuracy test
The sensitivity test of 6.1 test kits of the present invention
With the IC of typical curve
50be worth the sensitivity index as ELISA method of the present invention.The dilution of Aflatoxins M1 standard substance is become 405,135,45,15,5, a 0ng/L6 concentration, each concentration 5 answers hole, according to indirect competitive ELISA method replication 5 times, and asks the IC of mensuration
50value.The regression equation of typical curve is equation: y=(A-D)/[1+ (x/C) ^B]+D, A=0.89811, B=8.04176, C=1.84093, D=0.04628, R
2=0.99917, wherein X=lg (C (AFM1)), Y=B/B0; IC
50=64.75ng/L, linearity range 5 ~ 405ng/L.
The precision test of 6.2 test kits of the present invention
Aflatoxins M1 standard substance are diluted to 405,135,45,15,5, a 0ng/L6 concentration, every concentration 5 repetition, according to indirect competitive ELISA method replication 5 times, the regression equation calculation of application standard curve goes out the measured value of each concentration Aflatoxins M1 standardized solution, the variation coefficient in computing board and between plate, the results are shown in Table 5.
The variation coefficient in the plate of table 5 typical curve and between plate
The accuracy of 6.3 test kits of the present invention, replica test
Added to by Aflatoxins M1 in Feed Sample, its TIANZHU XINGNAO Capsul, between 60% ~ 120%, criticizes interior and interassay coefficient of variation≤15%; Concrete measurement result is in table 6.
Formula 2:
TIANZHU XINGNAO Capsul in table 6 milk
Claims (8)
1. a monoclonal antibody for energy specific recognition Aflatoxins M1, is characterized in that: it is that the hybridoma cell strain AFM1/3D8 being CCTCCNO:C201559 by preserving number secretes.
2. the hybridoma cell strain AFM1/3D8 described in claim 1, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201559.
3. monoclonal antibody according to claim 1, is characterized in that, it prepares as immunogen after the Aflatoxins M1 oxime obtained after Aflatoxins M1 and carboxymethyl azanol half hydrochloride react and carrier protein couplet.
4. the application of the monoclonal antibody described in claim 1 or 3 in the enzyme-linked immunologic detecting kit preparing Aflatoxins M1.
5. comprise the test kit of monoclonal antibody described in claim 1 or 3.
6. test kit according to claim 5, this test kit is the enzyme linked immunological kit detecting Aflatoxins M1.
7. the application of test kit according to claim 5 in Aflatoxins M1 non-diagnostic object detects.
8. detect the enzyme-linked immunoassay method that in milk, Aflatoxins M1 is residual, its step is as follows:
1) aflatoxin B1 oxime and carrier protein couplet are obtained coating antigen;
2) monoclonal antibody is prepared with the hybridoma cell strain AFM1/3D8 that preserving number is CCTCCNO:C201559;
3) by step 1) coating antigen bag by solid phase carrier;
4) sample pre-treatments: get milk sample 10 ± 0.5mL in 50mL centrifuge tube, the centrifugal 5min of 4000r/min, gets supernatant, is determinand solution;
5) to step 4) determinand solution carry out enzyme linked immunosorbent detection.
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Cited By (3)
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| CN107188961A (en) * | 2017-07-17 | 2017-09-22 | 齐齐哈尔大学 | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof |
| CN108254560A (en) * | 2018-04-19 | 2018-07-06 | 国家食品安全风险评估中心 | Aflatoxins M1 high-throughput enzyme linked immunoassay reagent kit and its detection method in milk |
| CN111505293A (en) * | 2020-04-03 | 2020-08-07 | 北京望尔生物技术有限公司 | Test strip for detecting variegated aspergillin and application thereof |
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| CN103513035A (en) * | 2012-06-28 | 2014-01-15 | 北京勤邦生物技术有限公司 | Test strip and method for detecting aflatoxin M1 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107188961A (en) * | 2017-07-17 | 2017-09-22 | 齐齐哈尔大学 | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof |
| CN108254560A (en) * | 2018-04-19 | 2018-07-06 | 国家食品安全风险评估中心 | Aflatoxins M1 high-throughput enzyme linked immunoassay reagent kit and its detection method in milk |
| CN111505293A (en) * | 2020-04-03 | 2020-08-07 | 北京望尔生物技术有限公司 | Test strip for detecting variegated aspergillin and application thereof |
| CN111505293B (en) * | 2020-04-03 | 2022-11-18 | 北京望尔生物技术有限公司 | Test strip for detecting variegated aspergillin and application thereof |
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