CN104897651B - A kind of chemical luminescence reagent kit of Aflatoxins M1 and its application - Google Patents
A kind of chemical luminescence reagent kit of Aflatoxins M1 and its application Download PDFInfo
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Abstract
(CLEIA) detection kit, including box body, the Chemiluminescent plate being located in box body and the reagent being located in box body is immunized the invention discloses a kind of chemiluminescent enzyme-linked immunosorbent of Aflatoxins M1；Characterized in that, each hole of the Chemiluminescent plate is coated with aspergillus flavus resisting toxin M1 antibody；The reagent includes：Liquid is redissolved in 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, concentration；The characteristics of chemical luminescence ELISA detection kit of the present invention has high sensitivity, simplicity is quick, the degree of accuracy is high, is compared, the operating time is greatly reduced with traditional ELISA method.It can be used as Aflatoxins M1 residue detection in detection milk, milk powder.
Description
Technical field
The present invention relates to it is a kind of detect Aflatoxins M1 chemiluminescence immune detection reagent kit, for detect milk,
Aflatoxins M1 content or residual quantity in milk powder.Belong to field of immunological detection.
Background technology
One kind that Aflatoxins M1 belongs in the similar compound of the class formation of aflatoxin one, in damp-heat area food
With occur the probability highest of aflatoxin in feed.Physicochemical properties quite stable, is not destroyed by pasteurization.Lactation
After feed or food that the intake of class animal is polluted by aflatoxin B1, Aflatoxins M1 is changed into by hydroxylation.
Aflatoxins M1 harm is mainly manifested in carcinogenicity and mutagenicity, has destruction to people and animal's liver tissue, can lead
Cause liver cancer even dead.Therefore, most of government organs all have strict to the aflatoxin amount that human body and animal can be taken in
Regulation is limited, and there are clear and definite limit standard in many countries to the Aflatoxins M1 content in milk and dairy products, yellow
It is 1 class carcinogenic substance that the aspertoxin Agency for Research on Cancer by the World Health Organization (WHO) in 1993, which delimited,.
At present, determine Aflatoxins M1 main method be fluorimetry, high performance liquid chromatography, double fluid exempt to enzyme-linked
Epidemic disease method etc..Instrumental method is highly effective, accurate, sensitive method, but measuring samples need to be cumbersome through a series of pretreatment
Time-consuming, from sample pretreatment to showing that assay needs the long period, testing cost is very high；On the other hand this detection method
There must also be the instrument and equipment of costliness, only specific professional could apply, and the limited sample detected every time, it is uncomfortable
The examination of gross sample is answered, the popularization and application of the detection method is seriously hindered, is less suitable for field test.By comparison,
Chemiluminescence immunoassay has quick, sensitive, conveniently, the features such as testing cost is relatively low, operating error can be reduced to greatest extent
And working strength, it is a kind of screening technique for being especially suitable for Aflatoxins M1 in basic unit's detection milk, milk powder.
The content of the invention
It is an object of the invention to provide a kind of chemiluminescence detection kit of Aflatoxins M1.The kit has inspection
Survey sensitivity it is high, using it is flexible, facilitate the characteristics of, the residual quantity inspection available for Aflatoxins M1 in the samples such as milk, milk powder
Survey.
To achieve the above object, the present invention mainly uses the general principle of the specific immune response of antigen and antibody
Realize.Chemiluminescence immunoassay is the product that chemoluminescence method and immunoassay are combined, therefore has chemistry simultaneously
The high sensitivity of luminescence method and the high specific of immunoassay.In whole course of reaction, Aflatoxins M1 contains in sample
Amount is higher, and luminous intensity is weaker in reaction system；Conversely, Aflatoxins M1 content is fewer in sample, luminous intensity is higher.
The chemiluminescence immune detection reagent kit of the detection Aflatoxins M1 of the present invention contains box body, is located in box body
Chemiluminescent plate and the reagent being located in box body.Specifically contain following component：
Be coated with aspergillus flavus resisting toxin M1 antibody the hole of White-opalescent 96 is detachable or non-removable polystyrene
Learn luminescent screen.
The Aflatoxins M1 system obtained using pH=7.40.05mol/L PBS solution dilution Aflatoxins M1 sterling
Row standard solution, concentration range has comprised at least 0.05~4.05ng/mL concentration ranges.
Enzyme-labelled antigen concentrate：The artificial antigen being made and horseradish peroxidating are coupled by Aflatoxins M1 and ovalbumin
The coupling of thing enzyme is prepared.
Enzyme-labelled antigen dilution：PH7.6 0.01M sodium phosphate, 0.25M NaCl solution.
Luminous substrate liquid：Luminous substrate liquid is divided into A, B liquid.A liquid be chemical luminous substrate-luminol and luminescence enhancer-
P-cresol solution, B liquid is hydrogen peroxide urea solution.
Liquid is redissolved in concentration：It is specially 2 times of concentrated phosphoric acid salt buffers that liquid is redissolved in concentration, and with distilled water work is diluted to using preceding
Make to use after concentration, for sample pre-treatments.
Concentrated cleaning solution：Thickening and washing solution is specially 20 times of concentration phosphorus containing Tween-20 (Tween-20) buffer solution
Phthalate buffer, is used using preceding be diluted to distilled water after working concentration, for washing chemistry luminescent screen in experimentation.
The preparation of solution of the present invention：
The Aflatoxins M1 standard solution that is related in kit of the present invention, enzyme mark Aflatoxins M1 antigenic solution,
The sensitivity influence that chemiluminescent solution and wash solution and its formula are detected on kit of the present invention is very big；Wherein each solution
Main component and its compound method are：
1st, Aflatoxins M1 standard liquid：In conventional manner by Aflatoxins M1 sterling 0.05mol/L, pH=
7.4 PBS be configured to concentration be respectively 0,0.05,0.15,0.45,1.35,4.05ng/mL Aflatoxins M1 standard it is molten
Liquid.
2nd, enzyme mark Aflatoxins M1 antigenic solution：The artificial antigen prepared is coupled with Aflatoxins M1 and coupling protein
Prepared with horseradish peroxidase, gained enzyme mark Aflatoxins M1 antigen is diluted with enzyme mark Aflatoxins M1
Liquid is diluted to 1:4000 working concentration.
3rd, enzyme-labelled antigen dilution：It is cushioning liquid that 0.01M, NaCl are 0.25M for pH7.6 sodium phosphate.
4th, chemiluminescent solution：A liquid is that luminol content is that 0.01M, p-cresol content are 0.001M pH=8.8
Three (methylol) aminomethane solution, B liquid is per 100mL solution 2.1g containing citric acid, anhydrous Na2HPO42.82g, 0.75%
The carbamide peroxide 0.64mL aqueous solution.
5th, working solution is redissolved in concentration：NaH2PO4·2H2O5.74g、Na2HPO4·12H2O32.6g is dissolved in 1L deionized water
In.
6th, thickening and washing solution：Tween-20 is added to pH=7.4,0.1mol/L phosphate by volume fraction 0.05%
In buffer solution.
7th, it is coated with solution：1.59g sodium carbonate and 2.53g sodium acid carbonates are dissolved in 1L water, adjust pH=9.5.
8th, lock solution is prepared：10g BSA are dissolved in 1L wash solutions, add weight than the NaN for 5 ‰3。
The coating of Chemiluminescent plate of the present invention：
Coating Chemiluminescent plate, which is used, in the present invention is placed in aspergillus flavus resisting toxin M1 antibody in the coating solution of setting, with
The concentration of setting, reacts coating in 37 DEG C of insulating boxs.
The present invention uses pH=9.5 sodium carbonate-bicarbonate cushioning liquid.It is coated with the present invention in microwell plate
Aspergillus flavus resisting toxin M1 antibody can be very good under alkaline environment combine on microwell plate frosting, can be subjected to repeatedly
Board-washing, the antibody used is coated with concentration for 5.0 μ g/mL.
The microwell plate being coated with can be closed with lock solution, the preferred BSA of inert protein in confining liquid, need to add NaN3It is anti-
Only go bad.
The preparation of enzyme mark Aflatoxins M1 antigenic solution：
Enzyme mark Aflatoxins M1 antigen solution concentration is to determine Aflatoxins M1 chemistry hair in the present invention in the present invention
Light detection kit measurement range and the key factor of sensitivity.
The enzyme mark Aflatoxins M1 antigenic solution being related in the present invention can be dilute with enzyme mark Aflatoxins M1 dilution
It is interpreted into 1:4000 working concentration.
The kit prepared according to above-mentioned enzyme mark Aflatoxins M1 antigen solution concentration can reach linear model well
Enclose (normal line scope can reach 0.05~4.05ng/mL) and good sensitivity (IC50For 0.065ng/mL).
The preparation of chemiluminescent solution：
The chemical luminescence for liquid A liquid be luminol content be 0.01M, three that p-cresol content is 0.001M pH=8.8
(methylol) aminomethane solution, B liquid is 100mL solution 2.1g containing citric acid, anhydrous Na2HPO42.82g, 0.75% peroxide
Change the hydrogen urea 0.64mL aqueous solution.The luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
The principle of the present invention is that the high sensitivity for the high degree of specificity and enzymatic for reacting antibody-antigene has been combined
Come, production concentration is detected using the chemiluminescence reaction of substrate for enzymatic activity.
The chemiluminescence detection kit of the present invention has sensitivity height, easy fast and accurately feature, with traditional ratio
Color ELISA method compares, and sensitivity can improve an order of magnitude.It is expected to the Aflatoxins M1 residual inspection in milk, milk powder
Played a significant role in survey.
Brief description of the drawings
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
React a：Aflatoxins M1 5mg, plus the acetonitrile 10ml dissolvings dried are taken, Boron tribromide solution containing 3mg is added dropwise
1ml, is stirred at room temperature 4h, stops reaction, and detection, raw material total overall reaction is completed.Stop reaction, be evaporated, add water and appropriate acetic acid second
Ester, is extracted, and is stood, and layering divides and goes aqueous phase, anhydrous sodium sulfate drying is evaporated, and obtains product 1.
React b：Product 1 plus DMF dissolvings, plus sodium carbonate 20mg, stirring, plus bromo-butyric acid 10mg, 60 DEG C of reaction 8h, are detected,
Raw material total overall reaction is completed, and is added water, ethyl acetate is extracted, washing, drying is evaporated, and is crossed short column of silica gel, is obtained haptens product.
Above-mentioned product is taken through nuclear magnetic resonance hydrogen spectruming determining, as shown in Fig. 2 chemical shift δ=11ppm's resonates for carboxyl hydrogen
Absworption peak, chemical shift δ=4.0ppm, 2.0ppm, 2.3ppm is the hydrogen resonance absorbing peak of methylene on spacerarm, it was demonstrated that interval
Arm successful connection, haptens structure is correct, illustrates hapten synthesis success.
(2) synthesis of immunogene (Aflatoxins M1-BSA)
18mg haptens is taken, is dissolved in 1mL DMF, takes 30mg EDC and NHS 0.2ml water fully to dissolve after addition
(1) in, 24h is stirred at room temperature, you can obtain reaction solution (1).BSA50mg is weighed, is allowed to be substantially dissolved in 3.8mLPB, will
Reaction solution (1) is slowly dropped in protein solution dropwise, and stirring 24h. 0.01mol/lPBS at room temperature, 4 DEG C of dialysis 3d
3 dialyzates are changed daily, to remove unreacted small-molecule substance.Packing, is saved backup in -20 DEG C.
Haptens 18mg and OVA50mg are weighed, envelope antigen is prepared by above-mentioned steps reaction, for enzyme mark.
(3) preparation of Aflatoxins M1 monoclonal antibody
A, animal immune：With the above-mentioned immunogene (Aflatoxins M1-BSA) prepared by 100 μ g/ only, with physiology salt
Water dissolving immunogene is mixed in equal volume with Freund's complete adjuvant, and immune 6~8 week old Balb/c female mices are subcutaneously injected in nape part, just
It is secondary it is immune after the 7th, 14,28 days mixed in equal volume with immunogene and incomplete Freund's adjuvant, each supplementary immunization once, merges preceding 3
It with the μ g/ of immune complex 100 only, being not added with Freund's adjuvant, supplementary immunization is once again.
B, cell fusion：Carry out according to a conventional method, take the splenocyte and the Mouse Bone in exponential phase of immune mouse
Myeloma cells (SP2/0) are mixed, and the fusion agent (PEG4000) that preheating was then slowly added in 45 seconds is merged, and is trained with HAT
Support base to suspend uniformly, add appropriate feeder cells, be incubated at 96 well culture plates, in 37 DEG C, 5%CO2Cultivated in incubator,
Liquid is partly changed after 5 days with HT culture mediums, carries out changing liquid full when 9 days.
C, the screening of hybridoma：After cell fusion, when cell grows to the 1/4 of culture hole area, sieved using substep
Method is selected to screen hybridoma.Primary election uses indirect ELISA method, with envelope antigen (in advance with square formation method conventional titration its most
Good coating concentration and positive serum dilution factor) coating Chemiluminescent plate, measured hole culture supernatant is added, is incubated, is added after cleaning
Sheep anti-mouse igg-HRP and IgM-HRP, OPD carry out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISAs method sieve filtered out
Choosing, first mixes cell conditioned medium with 100 μ g/mL Aflatoxins M1 in equal volume, and 37 DEG C of water-baths act on 30min, are then added to
In the Chemiluminescent plate being coated with.Compared simultaneously with PBS substitution Aflatoxins M1s, remaining step is ibid.If through aspergillus flavus
OD after toxin M1 blockings450Nm values drop to less than the 50% of control wells, then are judged to the positive, are all the positive through 2~3 detections
Hole, carry out subcloning with limiting dilution assay immediately.
It is prepared by D, monoclonal antibody：2~3 subclones are built into the hybridoma after strain and expand culture, supernatant is collected
Potency is determined with indirect ELISA, is frozen；And take 8~10 week old Balb/c mouse peritoneal injection atoleines 0.5mL/ only, 7~
Hybridoma 1~2 × 10 is injected intraperitoneally after 10 days6/ only, mouse ascites are extracted after 7~10 days, centrifuging and taking supernatant determines effect
Valency, and freeze standby.
Embodiment 2：The preparation of enzyme-labelled antigen
A, weighs 2mg HRP and is dissolved in 0.5mL distilled waters；Add the 0.06mol/L NaIO that 0.5mL is newly prepared4It is molten
Liquid, 4 DEG C of lucifuges act on 30min；
B, adds 160m mol/L ethylene glycol 0.5mL, room temperature effect 30min；
C, adds Aflatoxins M1-OVA2mg, is fitted into after mixing in treated bag filter, puts 1000mL 0.05m
Dialysed in mol/L sodium carbonate buffers, 4 DEG C overnight；
D, dialyzate is drawn in 10mL centrifuge tube, plus the 5g/L NaBH that 0.25mL newly matches somebody with somebody4Liquid, mixes rearmounted 4 DEG C of 2h；
Isometric saturated ammonium sulfate solution is added, 4 DEG C act on 30min, and 3000rpm centrifuges 25min at 4 DEG C, abandons supernatant；
E, precipitation is dissolved in 1.5mL0.02mol/L pH7.4PBS, in suction bag filter, in 0.02mol/L pH
7.4PBS is dialysed, and 4 DEG C (are changed PBS3 times midway) overnight；
F, liquid in dialyzate is drawn in microcentrifugal tube, and 10000rpm centrifuges 30min at 4 DEG C, and supernatant is suctioned out,
Plus equivalent glycerine, mix, -20 DEG C save backup.
Embodiment 3：The foundation of CLEIA detection methods
(1) coated antibody and preferred (the square formation method) of enzyme-labelled antigen concentration
80.0,40.0,20.0,10.0,5.0,2.5,1.25,0.625 μ g/mL series is pressed with every kind of coated antibody in longitudinal direction
Dilution factor is coated with Chemiluminescent plate, and 100 μ L/ holes are placed in after 37 DEG C of insulating box 2h, patted dry；With 150 μ L/ holes lock solution closings,
37 DEG C of insulating boxs are placed 2 hours, and board-washing once, is patted dry；Add a series of enzyme mark Aflatoxins M1 antigen of 50 dilutions in μ L/ holes
(1:1000 to 1:512000), (20~25 DEG C) incubation 15min of room temperature, board-washing five times is patted dry for the last time；It is separately added into 50 μ
Chemiluminescence A, B liquid in L/ holes, determines luminous intensity values.There is obvious graded with the concentration of enzyme-labelled antigen with luminous intensity values
Coated antibody concentration and enzyme-labelled antigen dilution factor be optium concentration carry out specific assay.
(2) measure of antibody sensitivity
According to above-mentioned to coated antibody and the optimization experiment of enzyme-labelled antigen concentration, applicant selects and determines that enzyme-labelled antigen is dense
Spend for 1:4000, coated antibody concentration is the measure for the sensitivity that 5.0 μ g/mL carry out antibody：
A, coating：Solution is coated with 0.05M pH=9.6 carbonate, and aspergillus flavus resisting toxin M1 antibody is made into 5.0 μ g/
Add 100 μ L, 37 DEG C of insulating box 2h in mL solution, each XPS Chemiluminescent plate reacting hole.Solution in hole is discarded, is clapped
It is dry.
B, closing：With the above-mentioned coated Chemiluminescent plate of lock solution closing, 150 μ L/ holes, 37 DEG C of insulating box 2h are then
Board-washing once, is patted dry.
C, sample-adding：Plus the μ L/ holes of Aflatoxins M1 standard solution 50 of various concentrations, add the dilution of 50 μ L/ holes
Enzyme mark Aflatoxins M1 antigen (1:4000) in the above-mentioned reacting hole closed, room temperature (20~25 DEG C) lucifuge is incubated
15min, then board-washing five times, are patted dry for the last time.
D, lights：Added in each reacting hole after the μ L/ holes of chemiluminescent solution 100 of Extemporaneous, reaction 3min with change
Learn the detection of luminescence immunoassay instrument.
E, testing result is calculated with inhibiting rate：
Relative luminous intensity (%)=RLU/RLU0, RLU is the luminous intensity values that standard items or sample solution are determined,
RLU0It is the luminous intensity values of blank (concentration is 0 standard liquid).
The concentration of medicine is the sensitivity of the antibody when calculating 50% inhibiting rate.
Embodiment 4：Detect the chemical luminescence reagent kit of Aflatoxins M1
(1) composition of the chemical luminescence reagent kit of detection Aflatoxins M1
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, concentrates enzyme mark Aflatoxins M1-OVA solution：Manually antigen (Aflatoxins M1-OVA) and horseradish peroxide
The coupling of compound enzyme is prepared, with diluted to working concentration when using.
D, enzyme mark Aflatoxins M1-OVA dilutions：Sodium phosphate, NaCl cushioning liquid.
E, luminescent solution：A liquid is luminol, p-cresol solution, B liquid hydrogen peroxide urea solutions, A liquid, B liquid etc. when using
Volume is mixed, now with the current.
Liquid is redissolved in F, 2 times of concentrations, and working concentration is diluted to distilled water when using.
G, 20 times of thickening and washing solution：When using working concentration is diluted to distilled water.
(2) preparation of Chemiluminescent plate
Aspergillus flavus resisting toxin M1 monoclonal antibodies are diluted to 5.0 μ g/mL with coating buffer, 100 μ L, 37 DEG C of perseverances are added per hole
Incubator places 2h, and coating buffer of inclining is patted dry, and the μ L of confining liquid 150 are then added per hole, and 37 DEG C of insulating boxs are placed 2h, inclined in hole
Liquid, cleaning solution washed once, and pat dry, and be preserved with masking foil vacuum sealing.
Embodiment 5：Detect the application of the chemical luminescence reagent kit of Aflatoxins M1
(1) preparation of reagent
A, wash solution：By the concentrated cleaning solution deionized water provided in kit by 1:Used after 19 times of dilutions.
B, redissolves working solution：The concentrated phosphoric acid salt buffer provided in kit is spent into ionized water by 1:After 1 times of dilution
Use.
C, chemiluminescent solution：Using preceding by A liquid and B liquid by volume 1:1 mixes.
D, enzyme-labelled antigen working solution：Enzyme-labelled antigen dilution and enzyme-labelled antigen concentrate are pressed 10:1 volume ratio is mixed and mixed
It is even.
(2) sample pre-treatments
A, milk
Fresh milk sample can be detected directly, and a period of time is stood after the milk sample of freezen protective thaws,
Detected again after removing upper-layer fat.
B, milk powder
1g ± 0.05g milk powder samples are weighed, 8ml is added and redissolves working solution, whirling motion is uniform.
(3) detecting step
A, sample-adding：Plus the μ L of standard items/sample 50 are into corresponding micropore, the μ L/ holes of enzyme-labelled antigen working solution 50 are added, gently
Light vibration is mixed, and 15min is reacted with the rearmounted 25 DEG C of light protected environments of cover plate membrane cover plate.
B, washing：Cover plate film carefully is opened, liquid in hole is dried, with the μ L/ holes of wash operating solution 250,5 are fully washed
It is secondary, per minor tick 10s, patted dry with blotting paper；
C, plus luminescent solution：Each 100 μ L of luminous substrate A, B liquid newly prepared are added per hole, are shaken about 30 seconds or so, with lid
Room temperature places 3min after plate membrane cover plate.
D, detection：It is directly placed into survey measurements in microwell plate luminescence analyzer.
(4) result judges
The standard items and the average value of sample luminous intensity values divided by the luminous intensity of first standard (0 standard) obtained
Value is multiplied by with 100, using inhibiting rate as ordinate, and the logarithm of Aflatoxins M1 concentration makees standard curve for abscissa, each
The concentration of sample can be read from standard curve.
Relative luminous intensity (%)=RLU/RLU0, RLU is the luminous intensity values that standard items or sample solution are determined,
RLU0It is the luminous intensity values of blank (concentration is 0 standard liquid).
Embodiment 6：Kit specific test
Using Aflatoxins M1 as standard, if the cross reacting rate of Aflatoxins M1 is 100%, intersect for antibody
Reaction Journal of Sex Research medicine be and Aflatoxins M1 structure or intimate medicine：Aflatoxin B1, aspergillus flavus
Toxin B2, aflatoxin G 1, aflatoxin G 2.By kit step operation, but the competitor added is respectively different
Aflatoxins M1 analog, makes suppression curve, and each inhibition concentration (IC of competitor 50% is calculated according to linear equation50).Hand over
It is IC of the antibody to Aflatoxins M1 to pitch reactivity (%CR)50With IC of the antibody to Aflatoxins M1 competitor50The ratio between
Percentage, calculated as the following formula：
As a result it is listed in table 1：
The Aflatoxins M1 kit specific test of table 1
| Competitor | Cross reacting rate (%) |
| Aflatoxins M1 | 100 |
| Aflatoxin B1 | 163 |
| Aflatoxin B 2 | 20.7 |
| Aflatoxin G 1 | 99.7 |
| Aflatoxin G 2 | 3 |
Embodiment 7：Kit preci-sion and accuracy is tested
Recovery test is added with Aflatoxins M1 addition milk, the milk powder sample of various concentrations respectively, calculated not
The rate of recovery is obtained in different samples with medicine, so that it is determined that the degree of accuracy of kit, each sample adds 3 concentration, Mei Genong
Degree 5 samples of addition, extract 3 batch kits and are tested.
The quantitative calculating of the rate of recovery is carried out according to the linear equation of the standard curve of formulation, 2 are as a result see the table below.
The Aflatoxins M1 kit accuracy test of table 2
In terms of said determination result, the rate of recovery of milk sample is between 92.0%~107.0%；The recovery of milk powder sample
Rate is between 102.5%~107.5%.Overall variation within batch coefficient is between 6.5%~9.3%, and interassay coefficient of variation exists
Between 9.0%~9.7%.Show that this kit has good preci-sion and accuracy.
Claims (8)
1. a kind of chemical luminescence ELISA detection kit of Aflatoxins M1, including box body, are located at the chemistry in box body
Luminescent screen and the reagent being located in box body；It is characterized in that：Each hole of the Chemiluminescent plate is coated with aspergillus flavus resisting toxin M1
Antibody；The reagent includes：Enzyme mark Aflatoxins M1 antigen concentrate, enzyme mark Aflatoxins M1 antigenic dilution, Huang Qu
Liquid is redissolved in mould toxin M1 serial standards solution, chemical luminous substrate A, B liquid, concentrated cleaning solution, concentration；
Aflatoxins M1 antigen in the enzyme mark Aflatoxins M1 antigen is by Aflatoxins M1 haptens and egg white
Albumen coupling is obtained, and the aspergillus flavus resisting toxin M1 antibody is made up of Aflatoxins M1 haptens with bovine serum albumin coupling
Conjugate prepared as immunogen immune Balb/c mouse, the synthetic method of the Aflatoxins M1 haptens is：
Aflatoxins M1 5mg, plus the acetonitrile 10ml dissolvings dried are taken, the 1ml of Boron tribromide solution containing 3mg is added dropwise, 4h is stirred at room temperature,
Stop reaction, detection, raw material total overall reaction is completed, and is stopped reaction, is evaporated, adds water and appropriate ethyl acetate, is extracted, and is stood, point
Layer, divides and goes aqueous phase, anhydrous sodium sulfate drying is evaporated, and obtains product 1；Product 1 plus DMF dissolvings, plus sodium carbonate 20mg, stirring, plus
Bromo-butyric acid 10mg, 60 DEG C of reaction 8h, detection, raw material total overall reaction is completed, and is added water, ethyl acetate is extracted, washing, drying is evaporated,
Short column of silica gel is crossed, haptens product is obtained.
2. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
Chemiluminescent plate is stated for the opaque hole Chemiluminescent plate of polystyrene 96 of milky.
3. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
It is 5.0 μ g/mL to state antibody coating concentration.
4. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
The working concentration for stating enzyme mark Aflatoxins M1 antigen is 1:4000.
5. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
Stating Aflatoxins M1 serial standards solution concentration is respectively：0、0.05、0.15、0.45、1.35、4.05ng/mL.
6. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
It is specially concentrated phosphoric acid salt buffer to state concentration redissolution liquid, is every liter and contains NaH2PO4·2H2O 5.74g、Na2HPO4·12H2O
32.6g the aqueous solution.
7. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
It is pH=7.4,0.1mol/L phosphate buffer containing the Tween-20 of volume fraction 0.05% to state thickening and washing solution.
8. the chemical luminescence ELISA detection kit of Aflatoxins M1 according to claim 1, it is characterised in that：Institute
It is the trihydroxy methyl that luminol content is 0.01M, p-cresol content is 0.001M pH=8.8 to state chemical luminous substrate A liquid
Aminomethane solution；B liquid is per 100mL solution 2.1g containing citric acid, anhydrous Na2HPO4 2.82g, 0.75% carbamide peroxide
The 0.64mL aqueous solution, the percentage is mass percent.
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| CN106771142A (en) * | 2016-11-30 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of Ofloxacin detection method and 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101393208A (en) * | 2008-10-07 | 2009-03-25 | 江苏省原子医学研究所 | Light induced chemiluminescent immunoassay kit of aflatoxin B1 and detecting method thereof |
| CN102072958A (en) * | 2010-11-09 | 2011-05-25 | 北京科美东雅生物技术有限公司 | Chemiluminescence immunoassay kit used for detecting aflatoxin B1 and preparation method and use method thereof |
| CN103091494A (en) * | 2013-01-14 | 2013-05-08 | 华南农业大学 | Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method |
-
2014
- 2014-12-27 CN CN201410832184.8A patent/CN104897651B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101393208A (en) * | 2008-10-07 | 2009-03-25 | 江苏省原子医学研究所 | Light induced chemiluminescent immunoassay kit of aflatoxin B1 and detecting method thereof |
| CN102072958A (en) * | 2010-11-09 | 2011-05-25 | 北京科美东雅生物技术有限公司 | Chemiluminescence immunoassay kit used for detecting aflatoxin B1 and preparation method and use method thereof |
| CN103091494A (en) * | 2013-01-14 | 2013-05-08 | 华南农业大学 | Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method |
Non-Patent Citations (1)
| Title |
|---|
| 黄曲霉毒素M_1化学发光酶免疫分析方法的研究;王岩;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20140815(第08期);论文全文 * |
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