CN104897651A - Chemiluminescent kit for aflatoxin M1 and application thereof - Google Patents
Chemiluminescent kit for aflatoxin M1 and application thereof Download PDFInfo
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Abstract
The invention discloses a chemiluminescent enzyme immunoassay (CLEIA) detection kit for aflatoxin M1. The kit comprises a kit body, a chemiluminescent plate arranged in the kit body and a reagent arranged in the kit body. The kit is characterized in that each hole in the chemiluminescent plate is coated with an anti-aflatoxin M1 antibody and the reagent comprises an enzyme-labeled aflatoxin M1 antigen concentrated solution, an enzyme-labeled aflatoxin M1 antigen diluent solution, aflatoxin M1 series standard solutions, a chemiluminescent substrate liquid A, a chemiluminescent substrate liquid B, a concentrated washing solution and a concentrated complex solution. The chemiluminescent enzyme immunoassay detection kit has the characteristics of high sensitivity, simplicity, rapidness and high accuracy. Compared with traditional ELISA methods, the kit provided by the invention enables operation time to be greatly reduced. The chemiluminescent enzyme immunoassay detection kit can be used for detecting aflatoxin M1 residue in milk and milk powder.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit detecting Aflatoxins M1, for detecting Aflatoxins M1 content in milk, milk powder or residual quantity.Belong to field of immunological detection.
Background technology
Aflatoxins M1 belongs to the one in the similar compound of aflatoxin one class formation, occurs that the probability of aflatoxin is the highest in the food and feed of damp-heat area.Physicochemical property quite stable, is not destroyed by pasteurization.Mammal changes into Aflatoxins M1 by hydroxylation after taking in the feed or food polluted by aflatoxin B1.Aflatoxins M1 harm is mainly manifested in carcinogenicity and mutagenicity, has destruction, liver cancer can be caused even dead to people and animal's liver tissue.Therefore, all there are strict regulation limitations in most of government organs to the aflatoxin amount that human body and animal can be taken in, there has been clear and definite limit standard in a lot of country to the Aflatoxins M1 content in milk and dairy products, and it is 1 class carcinogenic substance that aflatoxin delimited by the Agency for Research on Cancer of the World Health Organization (WHO) (WHO) for 1993.
At present, the main method measuring Aflatoxins M1 is fluorimetry, high performance liquid chromatography, double-flow enzyme-linked immunosorbent method etc.Instrumental method is very effective, accurate, responsive method, but measuring samples need through a series of pre-service, and loaded down with trivial details time-consuming, from sample pretreatment to showing that assay needs the long period, testing cost is very high; This detection method also must have expensive instrument and equipment on the other hand, specific professional is only had to apply, and each limited sample detected, be not suitable with the examination of gross sample, seriously hinder applying of this detection method, be more not suitable for field test.By comparison, Chemiluminescence immunoassay has the features such as quick, sensitive, convenient, testing cost is lower, can reduce operate miss and working strength to greatest extent, is a kind of screening technique being applicable to very much Aflatoxins M1 in basic unit's detection milk, milk powder.
Summary of the invention
The object of this invention is to provide a kind of chemiluminescence detection kit of Aflatoxins M1.This kit has that detection sensitivity is high, applying flexible, easily feature, can be used for the residues detection of Aflatoxins M1 in the sample such as milk, milk powder.
For achieving the above object, the present invention mainly utilizes the ultimate principle of the specific immune response of antigen and antibody to realize.Chemiluminescence immunoassay is the product that chemoluminescence method and immunoassay combine, and therefore has the high sensitivity of chemoluminescence method and the high specific of immunoassay simultaneously.In whole course of reaction, in sample, Aflatoxins M1 content is higher, and in reaction system, luminous intensity is more weak; Otherwise in sample, Aflatoxins M1 content is fewer, and luminous intensity is higher.
The chemiluminescence immune detection reagent kit of detection Aflatoxins M1 of the present invention contains box body, the reagent being located at the Chemiluminescent plate in box body and being located in box body.Concrete containing following composition:
Be coated with the detachable or non-removable polystyrene Chemiluminescent plate in White-opalescent 96 hole of aspergillus flavus resisting toxin M1 antibody.
The Aflatoxins M1 serial standards solution that the PBS solution dilution Aflatoxins M1 sterling applying pH=7.40.05mol/L obtains, concentration range at least contains the concentration ranges of 0.05 ~ 4.05ng/mL.
Enzyme-labelled antigen concentrate: the artificial antigen of being made up of Aflatoxins M1 and ovalbumin coupling and horseradish peroxidase prepare.
The sodium phosphate of the 0.01M of enzyme-labelled antigen dilution: pH7.6, the NaCl solution of 0.25M.
Luminous substrate liquid: luminous substrate liquid is divided into A, B liquid.A liquid is chemical luminous substrate-luminol and luminescence enhancer-p-cresol solution, and B liquid is hydrogen peroxide urea solution.
The concentrated liquid that redissolves: the concentrated liquid that redissolves is specially 2 times of concentrated phosphoric acid salt buffers, uses, for sample pre-treatments after using front distilled water to be diluted to working concentration.
Concentrated cleaning solution: thickening and washing solution is specially 20 times of concentrated phosphoric acid salt buffers containing Tween-20 (Tween-20) damping fluid, uses after using front distilled water to be diluted to working concentration, for washing chemistry luminous plaque in experimentation.
The preparation of solution of the present invention:
The sensitivity impact that the Aflatoxins M1 standard solution related in kit of the present invention, enzyme mark Aflatoxins M1 antigenic solution, chemiluminescent solution and wash solution and formula thereof detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method thereof are:
1, Aflatoxins M1 standard solution: in conventional manner the PBS of Aflatoxins M1 sterling 0.05mol/L, pH=7.4 is mixed with concentration be respectively 0,0.05,0.15,0.45,1.35, the Aflatoxins M1 standard solution of 4.05ng/mL.
2, enzyme mark Aflatoxins M1 antigenic solution: the artificial antigen of preparing with Aflatoxins M1 and coupling protein coupling and horseradish peroxidase prepare, becomes the working concentration of 1:4000 by gained enzyme mark Aflatoxins M1 antigen enzyme mark Aflatoxins M1 diluted.
3, enzyme-labelled antigen dilution: the buffer solution of to be 0.01M, NaCl the be 0.25M of the sodium phosphate for pH7.6.
4, chemiluminescent solution: three (methylol) aminomethane solution that A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8, B liquid is that every 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the aqueous solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.
5, concentrated working fluid: the NaH that redissolves
2pO
42H
2o5.74g, Na
2hPO
412H
2o32.6g is dissolved in the deionized water of 1L.
6, thickening and washing solution: by volume Tween-20 is added into pH=7.4 by mark 0.05%, in 0.1mol/L phosphate buffer.
7, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH=9.5.
8, lock solution preparation: 10g BSA is dissolved in 1L wash solution, then adds the NaN that weight ratio is 5 ‰
3.
The bag quilt of Chemiluminescent plate of the present invention:
Wrap in the present invention, by Chemiluminescent plate employing, aspergillus flavus resisting toxin M1 antibody is placed in the bag of setting by solution, with the concentration set, in 37 DEG C of constant temperature ovens, react bag quilt.
What the present invention adopted is the sodium carbonate-bicarbonate buffer solution of pH=9.5.In the present invention, in microwell plate, the aspergillus flavus resisting toxin M1 antibody of institute's bag quilt can well be combined on microwell plate frosting under alkaline environment, can stand repeatedly to wash plate, and the antibody bag of employing is 5.0 μ g/mL by concentration.
Bag can be closed by lock solution by good microwell plate, and in confining liquid, the preferred BSA of inert protein, need add NaN
3prevent from going bad.
The preparation of enzyme mark Aflatoxins M1 antigenic solution:
In the present invention, enzyme mark Aflatoxins M1 antigen solution concentration is the key factor determining Aflatoxins M1 chemiluminescence detection kit measurement range and sensitivity in the present invention.
The enzyme mark Aflatoxins M1 antigenic solution related in the present invention can become the working concentration of 1:4000 by enzyme mark Aflatoxins M1 diluted.
The kit prepared according to above-mentioned enzyme mark Aflatoxins M1 antigen solution concentration can reach the good range of linearity (standard lines scope can reach 0.05 ~ 4.05ng/mL) and good sensitivity (IC
50for 0.065ng/mL).
The preparation of chemiluminescent solution:
Three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the aqueous solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Principle of the present invention is combined with enzymatic high sensitivity by the high degree of specificity that antibody-antigene reacts, and utilizes the chemiluminescence reaction of substrate for enzymatic activity to detect production concentration.
Chemiluminescence detection kit of the present invention has highly sensitive, easy feature fast and accurately, compares with traditional colorimetric ELISA method, and sensitivity can improve an order of magnitude.Be expected to play a significant role in the Aflatoxins M1 residue detection in milk, milk powder.
Accompanying drawing explanation
Fig. 1 is Aflatoxins M1 hapten synthesis reaction equation.
Fig. 2 is Aflatoxins M1 haptens hydrogen nuclear magnetic resonance spectrogram.
Fig. 3 is chemical luminescence reagent kit working curve of the present invention.
Embodiment
Embodiment 1: the preparation of haptens, antigen and monoclonal antibody
(1) Aflatoxins M1 hapten synthesis
Reaction a: get Aflatoxins M1 5mg, adds dry acetonitrile 10ml and dissolves, and drip containing 3mg Boron tribromide solution 1ml, stirring at room temperature 4h, stop reaction, detect, raw material total overall reaction completes.Stop reaction, evaporate to dryness, adds water and appropriate ethyl acetate, extraction, leaves standstill, layering, and divide phase of anhydrating, anhydrous sodium sulfate drying, evaporate to dryness, obtains product 1.
Reaction b: product 1 adds DMF and dissolves, and adds sodium carbonate 20mg, stirs, adds bromo-butyric acid 10mg, 60 DEG C of reaction 8h, detect, raw material total overall reaction completes, and adds water, extraction into ethyl acetate, washing, and dry evaporate to dryness is crossed short column of silica gel, obtained haptens product.
Get above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as shown in Figure 2, chemical shift δ=11ppm's is carboxyl hydrogen resonance absorbing peak, chemical shift δ=4.0ppm, 2.0ppm, 2.3ppm are the hydrogen resonance absorbing peak of methylene on spacerarm, prove spacerarm successful connection, haptens structure is correct, and hapten synthesis success is described.
(2) synthesis of immunogene (Aflatoxins M1-BSA)
Get 18mg haptens, be dissolved in 1mL DMF, get 30mg EDC and NHS 0.2ml water fully dissolve after in adding in (1), stirred at ambient temperature 24h, can obtain reactant liquor (1).Take BSA50mg, make it fully to be dissolved in 3.8mLPB, reactant liquor (1) is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h. 0.01mol/lPBS, 4 DEG C of dialysis 3d change 3 dislysates, to remove unreacted small-molecule substance every day.Packing, saves backup in-20 DEG C.
Take haptens 18mg and OVA50mg, prepare envelope antigen, for enzyme mark by above-mentioned steps reaction.
(3) preparation of Aflatoxins M1 monoclonal antibody
A, animal immune: by the above-mentioned immunogene (Aflatoxins M1-BSA) prepared by 100 μ g/, mix with physiological saline solution immunogene and Freund's complete adjuvant equal-volume, the female mouse of neck dorsal sc injection immunity Balb/c in 6 ~ 8 week age, within after initial immunity the 7th, 14,28 day, mix with immunogene and incomplete Freund's adjuvant equal-volume, each supplementary immunization once, with immune complex 100 μ g/ only merges first 3 days, and supplementary immunization is once more not add Freund's adjuvant.
B, Fusion of Cells: carry out according to a conventional method, the splenocyte getting immune mouse mixes with the murine myeloma cell being in exponential phase (SP2/0), then the fusion agent (PEG4000) slowly adding preheating in 45 seconds merges, suspend evenly with HAT nutrient culture media, then add appropriate feeder cells, be incubated at 96 well culture plates, in 37 DEG C, 5%CO
2cultivate in incubator, within 5 days, partly change liquid with HT nutrient culture media afterwards, when 9 days, entirely change liquid.
C, the screening of hybridoma: after Fusion of Cells, when cell grows to 1/4 of culture hole area, adopts a point step screening method screening hybridoma.Primary election adopts indirect ELISA method, with envelope antigen (wrapping by concentration and positive serum dilutability by its best of square formation method conventional titration in advance) bag by Chemiluminescent plate, add measured hole culture supernatant, hatch, sheep anti-mouse igg-HRP is added and IgM-HRP, OPD carry out chromogenic reaction after cleaning.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first mixes the Aflatoxins M1 equal-volume of cell conditioned medium with 100 μ g/mL, 37 DEG C of water-bath effect 30min, then joins bag by good Chemiluminescent plate.Replace Aflatoxins M1 with PBS to compare, all the other steps are the same simultaneously.If the OD after Aflatoxins M1 blocks
450nm value drops to less than 50% of control wells, be then judged to the positive, and detecting through 2 ~ 3 times is all positive hole, carries out subcloning immediately with limiting dilution assay.
D, monoclonal antibody preparation: 2 ~ 3 subclones are built the hybridoma after strain and expands cultivation, collects supernatant indirect ELISA mensuration and tires, frozen; And get 8 ~ 10 week age Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, lumbar injection hybridoma 1 ~ 2 × 10 after 7 ~ 10 days
6/ only, extract mouse ascites after 7 ~ 10 days, centrifuging and taking supernatant, mensuration is tired, and frozen for subsequent use.
Embodiment 2: the preparation of enzyme-labelled antigen
A, takes 2mg HRP and is dissolved in 0.5mL distilled water; Add the 0.06mol/L NaIO that 0.5mL newly prepares
4solution, 4 DEG C of lucifuge effect 30min;
B, adds the ethylene glycol 0.5mL of 160m mol/L, room temperature effect 30min;
C, adds Aflatoxins M1-OVA2mg, and mixing loads in the bag filter processed afterwards, and put in the 0.05m mol/L sodium carbonate buffer of 1000mL and dialyse, 4 DEG C are spent the night;
D, dislysate is drawn in the centrifuge tube of 10mL, adds the 5g/L NaBH that 0.25mL newly joins
4liquid, mixes rearmounted 4 DEG C of 2h; Add isopyknic saturated ammonium sulfate solution, 4 DEG C of effect 30min, at 4 DEG C, the centrifugal 25min of 3000rpm, abandons supernatant;
E, is dissolved in 1.5mL0.02mol/L pH7.4PBS by precipitation, suck in bag filter, dialyse at 0.02mol/L pH 7.4PBS, 4 DEG C spend the night (midway is changed PBS3 time);
F, is drawn in microcentrifugal tube by liquid in dislysate, and the centrifugal 30min of 10000rpm at 4 DEG C, by supernatant sucking-off, adds equivalent glycerine, and mixing ,-20 DEG C save backup.
The foundation of embodiment 3:CLEIA detection method
(1) preferred (the square formation method) of coated antibody and enzyme-labelled antigen concentration
Longitudinally press the dilution series bag of 80.0,40.0,20.0,10.0,5.0,2.5,1.25,0.625 μ g/mL by Chemiluminescent plate with often kind of coated antibody, 100 μ L/ holes, after being placed in 37 DEG C of constant temperature oven 2h, pat dry; Close with 150 μ L/ hole lock solution, 37 DEG C of constant temperature ovens place 2 hours, wash plate once, pat dry; Add enzyme mark Aflatoxins M1 antigen (1:1000 to 1:512000) of the 50 a series of dilutions in μ L/ hole, room temperature (20 ~ 25 DEG C) hatches 15min, washes plate five times, pats dry for the last time; Add chemiluminescence A, B liquid in 50 μ L/ holes respectively, measure luminous intensity values.The coated antibody concentration of obvious graded and enzyme-labelled antigen dilutability is had to carry out specific assay for optium concentration with luminous intensity values with the concentration of enzyme-labelled antigen.
(2) mensuration of antibody sensitivity
According to the above-mentioned optimization experiment to coated antibody and enzyme-labelled antigen concentration, applicant selects and determines that enzyme-labelled antigen concentration is 1:4000, and coated antibody concentration is the mensuration that 5.0 μ g/mL carry out the sensitivity of antibody:
A, bag quilt: the solution by solution, aspergillus flavus resisting toxin M1 antibody being made into 5.0 μ g/mL with the carbonate bag of 0.05M pH=9.6, adds 100 μ L in each polystyrene board Chemiluminescent plate reacting hole, 37 DEG C of constant temperature oven 2h.Discard solution in hole, pat dry.
B, closes: close the above-mentioned Chemiluminescent plate having wrapped quilt by lock solution, 150 μ L/ holes, then 37 DEG C of constant temperature oven 2h wash plate once, pat dry.
C, application of sample: the Aflatoxins M1 standard solution 50 μ L/ hole adding variable concentrations, add enzyme mark Aflatoxins M1 antigen (1:4000) of 50 μ L/ hole dilutions again in the above-mentioned reacting hole closed, room temperature (20 ~ 25 DEG C) lucifuge hatches 15min, then wash plate five times, pat dry for the last time.
D, luminous: the chemiluminescent solution 100 μ L/ hole adding Extemporaneous in each reacting hole, detect with chemical illumination immunity analysis instrument after reaction 3min.
E, testing result calculates with inhibiting rate:
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution measure, RLU
0it is the luminous intensity values of blank (concentration is the standard solution of 0).
The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
Embodiment 4: the chemical luminescence reagent kit detecting Aflatoxins M1
(1) composition of the chemical luminescence reagent kit of Aflatoxins M1 is detected
A, is coated with the solid phase carrier (Chemiluminescent plate) of Aflatoxins M1 monoclonal antibody;
B, Aflatoxins M1 standard solution: 0,0.05,0.15,0.45,1.35,4.05ng/mL.
C, concentrated enzyme mark Aflatoxins M1-OVA solution: prepare by artificial antigen (Aflatoxins M1-OVA) and horseradish peroxidase, during use by diluted to working concentration.
D, enzyme mark Aflatoxins M1-OVA dilution: sodium phosphate, NaCl buffer solution.
E, luminescent solution: A liquid is luminol, p-cresol solution, B liquid hydrogen peroxide urea solution, during use, A liquid, the mixing of B liquid equal-volume, now with the current.
F, 2 times of concentrated liquid that redissolve, are diluted to working concentration with distilled water during use.
G, 20 times of thickening and washing solution: be diluted to working concentration with distilled water during use.
(2) preparation of Chemiluminescent plate
With coating buffer, aspergillus flavus resisting toxin M1 monoclonal antibody is diluted to 5.0 μ g/mL, every hole adds 100 μ L, 2h placed by 37 DEG C of constant temperature ovens, and incline coating buffer, pats dry, then every hole adds confining liquid 150 μ L, 37 DEG C of constant temperature ovens place 2h, liquid in hole of inclining, and cleansing solution washing once, pat dry, preserve with masking foil vacuum seal.
Embodiment 5: the application detecting the chemical luminescence reagent kit of Aflatoxins M1
(1) preparation of reagent
A, wash solution: the concentrated cleaning solution deionized water provided in kit is doubly diluted rear use by 1:19.
B, redissolution working fluid: the concentrated phosphoric acid salt buffer provided in kit is spent ionized water and doubly dilutes rear use by 1:1.
C, chemiluminescent solution: before using by A liquid and B liquid by volume 1:1 mix.
D, enzyme-labelled antigen working fluid: enzyme-labelled antigen dilution and enzyme-labelled antigen concentrate are mixed by 10:1 volume ratio and mixes.
(2) sample pre-treatments
A, milk
Fresh milk sample can directly detect, after the milk sample of freezen protective thaws, leave standstill a period of time, detects after removing upper-layer fat again.
B, milk powder
Take 1g ± 0.05g milk powder sample, add 8ml redissolution working fluid, whirling motion is even.
(3) detecting step
A, application of sample: add standard items/sample 50 μ L in the micropore of correspondence, then add enzyme-labelled antigen working fluid 50 μ L/ hole, mixing of vibrating gently, reacts 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
B, washing: carefully open cover plate film, dries liquid in hole, with wash operating solution 250 μ L/ hole, fully washing 5 times, every minor tick 10s, pats dry with thieving paper;
C, adds luminescent solution: every hole adds luminous substrate A, each 100 μ L of B liquid of new preparation, shakes about about 30 seconds, places 3min by room temperature after cover plate membrane cover plate.
D, detects: directly put into microwell plate luminescence analyzer survey measurements.
(4) result judges
The mean value of the standard items obtained and sample luminous intensity values is multiplied by 100 again divided by the luminous intensity values of first standard (0 standard), take inhibiting rate as ordinate, the logarithm of Aflatoxins M1 concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution measure, RLU
0it is the luminous intensity values of blank (concentration is the standard solution of 0).
Embodiment 6: kit specific test
Using Aflatoxins M1 as standard, if the cross reacting rate of Aflatoxins M1 is 100%, the medicine for antibody cross reaction Journal of Sex Research is and Aflatoxins M1 structure or intimate medicine: aflatoxin B1, AFB 2, aflatoxin G 1, AFG 2.By the operation of kit step, but the competitor added is respectively different Aflatoxins M1 analogs, makes and suppresses curve, calculate each competitor 50% inhibition concentration (IC according to linear equation
50).Cross reacting rate (%CR) is the IC of antibody to Aflatoxins M1
50with the IC of antibody to Aflatoxins M1 competitor
50the percentage of ratio, calculate by following formula:
The results are shown in table 1:
Table 1 Aflatoxins M1 kit specific test
| Competitor | Cross reacting rate (%) |
| Aflatoxins M1 | 100 |
| Aflatoxin B1 | 163 |
| AFB 2 | 20.7 |
| Aflatoxin G 1 | 99.7 |
| AFG 2 | 3 |
Embodiment 7: kit preci-sion and accuracy is tested
Interpolation recovery test is carried out respectively with the Aflatoxins M1 of variable concentrations interpolation milk, milk powder sample, calculate different pharmaceutical and obtain the recovery in different sample, thus determine the accuracy of kit, each sample adds 3 concentration, each concentration adds 5 samples, extracts 3 batches of kits and tests.
Carry out the quantitative calculating of the recovery according to the linear equation of the typical curve formulated, the results are shown in following table 2.
Table 2 Aflatoxins M1 kit accuracy test
From said determination result, the recovery of milk sample is between 92.0% ~ 107.0%; The recovery of milk powder sample is between 102.5% ~ 107.5%.Overall variation within batch coefficient is between 6.5% ~ 9.3%, and interassay coefficient of variation is between 9.0% ~ 9.7%.Show that this kit has good preci-sion and accuracy.
Claims (10)
1. a chemical luminescence ELISA detection kit for Aflatoxins M1, comprises box body, the reagent being located at the Chemiluminescent plate in box body and being located in box body; It is characterized in that, each hole of described Chemiluminescent plate is coated with aspergillus flavus resisting toxin M1 antibody; Described reagent comprises: enzyme mark Aflatoxins M1 antigen concentrate, enzyme mark Aflatoxins M1 antigenic dilution, Aflatoxins M1 serial standards solution, chemical luminous substrate A, B liquid, concentrated cleaning solution, the concentrated liquid that redissolves.
2. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: described Chemiluminescent plate is milky opaque polystyrene 96 hole Chemiluminescent plate.
3. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterized in that: described enzyme-labelled antigen is obtained by Aflatoxins M1 haptens and carrier protein couplet, and Aflatoxins M1 haptens is obtained through series reaction by Aflatoxins M1 and bromo-butyric acid.
4. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: described antibody bag is 5.0 μ g/mL by concentration.
5. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: the working concentration of described enzyme mark Aflatoxins M1 antigen is 1:4000.
6. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: described Aflatoxins M1 antibody is that the conjugate be made up of Aflatoxins M1 and bovine serum albumin coupling prepares as immunogen immune Balb/c mouse.
7. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: described Aflatoxins M1 serial standards solution concentration is respectively: 0,0.05,0.15,0.45,1.35,4.05ng/mL.
8. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: described concentrated redissolution liquid is specially concentrated phosphoric acid salt buffer, be often liter containing NaH
2pO
42H
2o 5.74g, Na
2hPO
412H
2the aqueous solution of O32.6g.
9. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: described thickening and washing solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
10. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, is characterized in that: three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8; B liquid is that every 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the aqueous solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%, described number percent is mass percent.
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Cited By (3)
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| CN107677810A (en) * | 2017-09-11 | 2018-02-09 | 扬州市伊绿鲜生态农业科技有限公司 | A kind of aflatoxin chemoluminescence method quantitative determination reagent kit |
| CN107688016A (en) * | 2017-08-23 | 2018-02-13 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of Aflatoxins M1 and preparation method thereof |
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