CN105136779A - Chemiluminescence immune kit for detection of aflatoxin M1 and application thereof - Google Patents
Chemiluminescence immune kit for detection of aflatoxin M1 and application thereof Download PDFInfo
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- CN105136779A CN105136779A CN201510283031.7A CN201510283031A CN105136779A CN 105136779 A CN105136779 A CN 105136779A CN 201510283031 A CN201510283031 A CN 201510283031A CN 105136779 A CN105136779 A CN 105136779A
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Abstract
The invention discloses a chemiluminescence immune kit for detection of aflatoxin M1, and is characterized in that each hole of a chemiluminescence plate is coated with an anti aflatoxin M1 antibody; the reagent includes a series of aflatoxin M1 standard solutions, the chemiluminescence plate, an enzyme-labelled antigen concentrated liquid, an enzyme-labelled antigen diluent, a luminous substrate liquid, a concentrated washing liquid, and a concentration reconstitution liquid. The established chemiluminescence immunoassay has the advantages of high flux, high sensitivity, strong specificity, low detection cost, accuracy, rapidness and the like, and can meet the requirements of rapid screening testing of on-site large-scale detection.
Description
Technical field
The present invention relates to a kind of chemiluminescence immunoassay kit detecting Aflatoxins M1, for detecting the content of Aflatoxins M1, belonging to field of immunological detection.
Background technology
Aflatoxins M1 (AflatoxinM1, abbreviation AFM
1) belong to one in the similar compound of aflatoxin one class formation, this toxoid is the metabolic product produced by common Aspergillus flavus (AsperillusFlavus) and aspergillus parasiticus bacterium (AsperillusParasiticus), occurs that the probability of aflatoxin is the highest in the food and feed of damp-heat area.
Aflatoxin is a kind of strong carcinogenic substance, and be the mushrooms such as Aspergillus height poison for thing.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.Aflatoxin delimited as I class carcinogenic substance by the Agency for Research on Cancer of the World Health Organization (WHO) (WHO) for 1993.
AFM
1it is the metabolin of aflatoxin B1, its basic structure is the coumarin of two furan nucleuss, be dissolved in multiple organic solvent, as chloroform, acetonitrile, first alcohol and water, but be insoluble in the non-polar solvents such as normal hexane, sherwood oil, ether, physicochemical property quite stable, is not destroyed by pasteurization.Wherein aflatoxin B1 is topmost a kind of toxin, after mammal takes in the feed or food polluted by aflatoxin B1, under hepatomicrosome MO system catalysis in vivo, by CYP1A regulating action, and AFB
1end furan nucleus C-10 is generated AFM by hydroxylation
1, a part is discharged from urine and milk, and another part is present in the edible part of animal; AFM
1also directly can be produced by some Aspergillus flavus and aspergillus parasiticus.Aflatoxins M1 is mainly present in soil, animals and plants, the cereal that goes mouldy, various nut, food, flavouring, edible oil, milk and dairy produce etc. go mouldy in goods, directly or indirectly enter human foods chain, the mutagenic poisonous substance of carcinogenic teratogenesis, serious threat human health and life security.Cow's milk and goods thereof are one of food being vulnerable to aflatoxin contamination.For ensureing its safety and sanitation, each state has all formulated strict limit standard.U.S. FDA specifies that the limitation level of Aflatoxins M1 in cow's milk is 0.5 μ g/kg, and in China's regulation cow's milk, Aflatoxins M1 content must not more than 0.5 μ g/kg.
At present, the detection method of aflatoxin mainly contains: immunoaffinity chromatography purification high performance liquid chromatography, multiple detection method such as immunoaffinity chromatography purification fluorimetry, thin-layer chromatography etc.Immunoaffinity chromatography purification high performance liquid chromatography, sample pretreatment process is comparatively complicated, consuming time, need purification process, detect the laboratory large-scale experiment instrument and equipment that needs are expensive, be equipped with professional testing staff and carry out test operation, be difficult to meet on-the-spot extensive detection.Immunoaffinity chromatography purification fluorimetry, sample pretreatment process is comparatively complicated, consuming time, need purification process, and detection needs fluorophotometer, is difficult to meet Site Detection needs.Thin-layered chromatography sample pre-treatments is loaded down with trivial details, and experimentation is complicated, consuming time, poor accuracy, and sensitivity is low, specificity is not strong, and larger to experiment operator harm.The present invention set up Chemiluminescence immunoassay has high flux, highly sensitive, high specificity, testing cost are low, the accurate advantage such as quick, the requirement of the on-the-spot extensive rapid screening inspection detected can be met.
Summary of the invention
The object of the invention is to, not enough for prior art, a kind of chemiluminescence immunoassay kit detecting dairy products is provided, there is high flux, highly sensitive, high specificity, testing cost are low, the accurate advantage such as quick, the requirement of the on-the-spot extensive rapid screening inspection detected can be met.
The present invention mainly utilizes the ultimate principle of the specific immune response of antigen and antibody to realize.Chemiluminescence immune analysis method is the product that chemoluminescence method and immunoassay combine, therefore it 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.
A kind of chemiluminescence immunoassay kit detecting Aflatoxins M1 of the present invention, comprises 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.
Described Aflatoxins M1 standard solution 6 bottles, concentration is respectively 0ug/L, 0.05ug/L, 0.1ug/L, 0.2ug/L, 0.4ug/L, 0.6ug/L.
Enzyme-labelled antigen concentrate: be that the artificial antigen of being made up of Aflatoxins M1 and bovine serum albumin coupling and horseradish peroxidase prepare.
Enzyme-labelled antigen dilution: 0.01-0.02M phosphate buffer, the bovine serum albumin of pH7.0-8.0,0.5-1%.
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 enzyme mark Aflatoxins M1 antigenic solution, chemiluminescent solution and the wash solution that relate in kit of the present invention and formula thereof detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method as follows:
1, 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 diluent.
2, enzyme-labelled antigen dilution: 0.01-0.02M phosphate buffer, the bovine serum albumin of pH7.0-8.0,0.5-1%.
3, luminous substrate liquid: three (methylol) aminomethane solution that A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH8.8, 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%.
4, the concentrated working fluid that redissolves: pH value is 7.5-7.8, and containing 2-4% casein, the phosphate buffer of 0.1-0.2mol/L, described number percent is w/v.
5, thickening and washing solution: pH value is 7.2-7.5, containing 0.8-1.2% Tween-20,0.3-0.6 ‰ sodium azide, the phosphate buffer of 0.1-0.2mol/L, described number percent is mass volume ratio.
6, bag is buffered liquid: pH value is the phosphate buffer of 9.2-9.6,0.1-0.2mol/L, and described number percent is w/v.
7, lock solution preparation: containing 5-8% skimmed milk power, the phosphate buffer of pH value 7.2-7.6,0.1-0.2mol/L, described number percent is mass volume ratio.
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 pH9.2-9.6 phosphate buffer.In the present invention, in microwell plate, the aspergillus flavus resisting toxin M1 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 can from 5mg/ml-20mg/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 0.05-0.60ug/L).
The preparation of chemiluminescent solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly luminol-hydrogen peroxide system.
Three (methylol) aminomethane solution that described luminous substrate liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH8.8, 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.
Chemical luminescence ELISA detection kit of the present invention has highly sensitive, easy feature fast and accurately, is expected to play a significant role to Aflatoxins M1 residue detection in milk.
Accompanying drawing explanation
Fig. 1 is that (horizontal ordinate is Aflatoxins M1 canonical plotting: AMF1 drug concentration (ug/L), ordinate is: RLU/RLU
0).
Embodiment
The synthesis of embodiment 1 Aflatoxins M1 hapten-carrier protein conjugate and qualification
(1) Aflatoxins M1 hapten synthesis
Get the mixing in 2ml dimethyl sulfoxide (DMSO) (DMSO) of 0.10g Aflatoxins M1, slowly 0.1ml1 is dripped at 60 DEG C, 3-propane diamine and 0.1ml pyridine are in 2mlDMSO, after dropwising, continue reaction 15 hours, rotary evaporation removes solvent and unreacted propane diamine, quantitatively obtains the propane diamine list condensation product of Aflatoxins M1.
(2) immunogenic preparation
A. get the water-soluble solution of haptens 5mg 0.8ml, after dissolving completely, dropwise add 0.2ml0.02mol/L glutaraldehyde, stirring at room temperature reaction 24h, obtains being dissolved in I;
B. get bovine serum albumin(BSA) (BSA) 30mg, be dissolved in 4ml water, obtain solution II;
C. solution I slowly joined in solution II, stirring at room temperature, reaction is spent the night, and obtains solution III;
D. in solution III, 30mgNaBH is added
4reduction;
E. dialyse three days with 0.02mol/LPBS, change dislysate every day three times, obtain Aflatoxins M1 immunogene.
(3) preparation of coating antigen
Get the water-soluble solution of oralbumin (OVA) 50mg 3ml and obtain solution IV; Get carbodiimides (EDC) and the water-soluble solution of N-hydroxysuccinimide (NHS) each 50mg 1.5ml completely after add in solution IV, stirring at room temperature reaction 30min obtains solution V; Get haptens 6.8mg 0.5ml dimethyl formamide (DMF) dissolving and obtain solution VI; Slowly joined by solution V in solution VI, stirring at room temperature reaction 24h, obtains Aflatoxins M1 coating antigen.
(4) qualification of Aflatoxins M1 hapten-carrier protein conjugate
The PBS of carrier protein, Aflatoxins M1 haptens, Aflatoxins M1 hapten-carrier protein conjugate pH7.4 is made into the solution of 0.5mg/mL, return to zero with 0.01mol/LpH7.4PBS, with ultraviolet spectrophotometer in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of carrier protein, Aflatoxins M1 haptens, Aflatoxins M1 hapten-carrier protein conjugate.There is different absorption curves in three, shows Aflatoxins M1 haptens and carrier protein couplet success.
(5) preparation of Aflatoxins M1 monoclonal antibody
A. animal immune
Immunogene above-mentioned steps obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, screening obtains the Aflatoxins M1 monoclonal antibody hybridoma cell strain of stably excreting Aflatoxins M1 monoclonal antibody.
C. cell cryopreservation and recovery
Hybridoma cryopreserving liquid is made 5 × 10
6the cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in cell culture medium, cultivates under 37 DEG C of conditions, and by sad-saturated ammonium sulfate method, the nutrient solution obtained is carried out purifying, obtain monoclonal antibody, mensuration is tired, and frozen-20 DEG C save backup.
Embodiment 2: the preparation of enzyme-labelled antigen
A. taking 2mgHRP is dissolved in 0.5mL distilled water; Add the 0.06mol/LNaIO that 0.5mL newly prepares
4solution, 4 DEG C of lucifuge effect 30min;
B. the ethylene glycol 0.5mL of 160mmol/L is added, room temperature effect 30min;
C. add Aflatoxins M1 hapten-carrier protein conjugate 2mg, mixing loads in the bag filter processed afterwards, and put in the 0.05mmol/L sodium phosphate 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/LNaBH 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. precipitation is dissolved in 1.5mL0.02mol/LpH7.0-7.50.2-0.4%NaN
3in 0.2-0.3MOL/L borate buffer solution, suck in bag filter, at 0.02mol/LpH=7.0-7.50.2-0.4%NaN
30.2-0.3mol/L borate buffer solution is dialysed, 4 DEG C spend the night (borate buffer solution 3 times are changed in midway);
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 antibody and envelope antigen concentration
Longitudinally press the dilution series bag of 100.0,50.0,25.0,12.5,6.3,3.2,1.6,0.8 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 envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with luminous intensity values with the concentration of envelope antigen.
(2) mensuration of antibody sensitivity
According to the above-mentioned optimization experiment to coated antibody and enzyme-labelled antigen concentration, select and determine that enzyme-labelled antigen concentration is 1:4000, coated antibody concentration is the mensuration that 5.0 μ g/mL carry out the sensitivity of antibody:
A. quilt is wrapped: the solution by solution, aspergillus flavus resisting toxin M1 antibody being made into 5.0 μ g/mL with the carbonate bag of 0.05MpH=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. close: 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 chemiluminescence enzyme linked immunoassay reagent kit detecting Aflatoxins M1
(1) composition of the chemiluminescence enzyme linked immunoassay 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: 0,0.05,0.10,0.20,0.40,0.60ug/L.
C, concentrated enzyme mark Aflatoxins M1 hapten-carrier protein conjugate solution: prepare by artificial antigen and horseradish peroxidase, during use by diluted to working concentration.
D, enzyme mark Aflatoxins M1 hapten-carrier protein conjugate 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 chemiluminescence enzyme linked immunoassay 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 Sample pretreatment method:
Get 400 μ l fresh milk samples; Add 800 μ l acetonitriles, whirling motion 1min, get 100 μ l supernatants and add 500 μ l redissolution working fluids, mixing; Get 50 μ l for analyzing, Sample Dilution multiple: 20.
B. milk powder Sample pretreatment method:
Take 1.0 ± 0.05g milk powder sample in 10ml centrifuge tube, add 2.5ml deionized water, whirling motion 1min; Pipette 400 μ l in 2ml centrifuge tube, add 400 μ l acetonitriles, then add 400 μ l methyl alcohol, whirling motion 1min, more than 3000g, room temperature (20-25 DEG C) centrifugal 3-5min; Get 100 μ l supernatants and add 500 μ l redissolution working fluids, mixing; Get 50 μ l for analyzing, Sample Dilution multiple: 20.
(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. wash: carefully open cover plate film, liquid in hole is dried, with wash operating solution 250 μ L/ hole, fully washing 7 times, every minor tick 10s, pats dry with thieving paper;
C. luminescent solution is added: every hole adds the luminous substrate 100 μ L of new preparation, shakes about about 30 seconds, places 3min by room temperature after cover plate membrane cover plate.
D. detect: 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 preci-sion and accuracy is tested
Accuracy refers to the matching degree between measured value and true value, and kit accuracy is commonly used the recovery and represented.Precision is also known as repeatability, and the conventional coefficient of variation represents.
According to the sample extraction method of embodiment 5, respectively with the Aflatoxins M1 of 0.10ug/kg, 0.20ug/kg two concentration milk, milk powder sample are carried out interpolation and reclaimed, often kind of each concentration of sample each 4 parallel, measure with three batches of kits, calculate average recovery rate and the precision of sample.Experimental result sees the following form.
The kit accuracy of table 1 Aflatoxins M1 and precision measure ng/g (ml)
as seen from the table, the average recovery rate scope that in milk, powdered milk sample, Aflatoxins M1 two concentration are all added between 86.5-91.8%, in batch, batch between all the coefficient of variation be less than 15%.
Claims (8)
1. detect a chemiluminescence immunoassay kit for Aflatoxins M1, comprise Aflatoxins M1 standard solution, Chemiluminescent plate, enzyme-labelled antigen concentrate, enzyme-labelled antigen dilution, luminous substrate liquid, concentrated cleaning solution, the concentrated liquid that redissolves.
2. the chemiluminescence immunoassay kit detecting Aflatoxins M1 as claimed in claim 1, is characterized in that: each hole of described Chemiluminescent plate is coated with aspergillus flavus resisting toxin M1 monoclonal antibody.
3. the chemiluminescence immunoassay kit detecting Aflatoxins M1 as claimed in claim 1 or 2, is characterized in that: described aspergillus flavus resisting toxin M1 monoclonal antibody bag is 5.0mg/mL by concentration.
4. the chemiluminescence immunoassay kit detecting Aflatoxins M1 as claimed in claim 1, is characterized in that: the working concentration of described enzyme-labelled antigen is 1:4000.
5. the as claimed in claim 1 chemiluminescence immunoassay kit detecting Aflatoxins M1, is characterized in that: described Aflatoxins M1 standard solution concentration is respectively: 0,0.05,0.10,0.20,0.40,0.60ug/L.
6. the chemiluminescence 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
2o5.74g, Na
2hPO
412H
2the aqueous solution of O32.6g.
7. the chemiluminescence detection kit of Aflatoxins M1 according to claim 1, it is characterized in that: described thickening and washing solution is pH value is 7.2-7.5, containing 0.8-1.2% Tween-20,0.3-0.6 ‰ sodium azide, the phosphate buffer of 0.1-0.2mol/L, described number percent is mass volume ratio.
8. the chemiluminescence detection kit of Aflatoxins M1 according to claim 1, it is characterized in that: described luminous substrate liquid, be made up of A liquid and B liquid, A liquid is luminol content be 0.01M, three (methylol) aminomethane solution that p-cresol content is 0.001MpH8.8; 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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| CN105806921A (en) * | 2016-03-16 | 2016-07-27 | 济南大学 | Preparation method and application of aflatoxin photoelectrochemical sensor without external light source |
| CN105806921B (en) * | 2016-03-16 | 2018-07-31 | 济南大学 | A kind of preparation method and application of the aflatoxin optical electro-chemistry sensor of no peripheral hardware light source |
| 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 |
| CN115469106B (en) * | 2022-09-16 | 2024-05-17 | 天津科技大学 | Freeze-dried cell membrane fragment reconstitution solution, reconstitution method and application |
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