CN109752553A - Detect the time-resolved fluoroimmunoassay kit of Aflatoxins M1 - Google Patents

Abstract

The invention discloses a kind of time fluorescence resolved fluorometric immunoassay kits for detecting Aflatoxins M1.The time-resolved fluoroimmunoassay detection kit of the detection Aflatoxins M1 is made of sheep anti-mouse antibody, cleaning solution and the enhancement solution of porous coating plate, buffer, Aflatoxins M1 standard items, the antibody dried frozen aquatic products of Aflatoxins M1, europium label.The detection method of the time fluorescence immunoassay kit of the detection Aflatoxins M1 includes the following steps: the preparation of (1) immunogene；(2) preparation of coating antigen；(3) preparation of monoclonal antibody；(4) pre-treatment and detection of sample.Detection time of the present invention is short, average recovery rate is high, sample pre-treatments are simple, energy execute-in-place detection, it is widely used, testing cost is low, while having detection high specificity, in batch and differences between batches are small, high sensitivity and it is easy to operate quickly, and the advantages that detection particularly suitable for batch samples.

Description

Detect the time-resolved fluoroimmunoassay kit of Aflatoxins M1

Technical field

The invention belongs to field of biological detection, specifically, it is glimmering to be related to a kind of time resolution for detecting Aflatoxins M1 Light immunoassay kits.

Background technique

Aflatoxin (Aflatoxins, AFT) is the secondary metabolite of fungi, mainly by aspergillus flavus (A. Flavus), some bacterial strains of aspergillus parasiticus (A. parasiticus) sum aggregate bee aspergillus (A. nonius) generate；Aspergillus flavus Toxin producing C due to bacterial strain difference and difference is very big；All bacterial strains of aspergillus parasiticus can generate aflatoxin.Aspergillus flavus Toxin is a kind of very strong hepatotoxin of toxicity, can cause the acute or chronic damage of liver, it and hepatitis B are considered as liver Most important two big inducements of cancer；In addition to the liver of damage body, aflatoxin is to other Various Tissues organs such as kidney Also it can cause serious harm, more seriously aflatoxin has been shown to have carcinogenic, teratogenesis, causes " the three of cell mutation Cause " effect.Aflatoxin is widely present in the cereal agricultural product such as peanut, corn, wheat, paddy, in macaroni, seasoning It is also frequently found in the food such as product, milk and edible oil, seriously endangers people, animal, avian health.Aflatoxin B1 is recognized To be the strongest natural materials of current carcinogenicity, aflatoxin in 1993 is by the cancer research machine of the World Health Organization (WHO) It is I class carcinogenic substance that structure, which delimited,.Since discovery aflatoxin in 1961, the type of aflatoxin is more and more.Mesh It is preceding it is separated identify 12 kinds, be aflatoxin B1, B2, G1, G2, M1, M2, B2a, G2a, GM1, R0 and poison respectively Alcohol；It is chemically seen in structure, the structure of various aflatoxin is closely similar, all contains a bifuran and cumarin (cumarin), the former be basic toxin moiety, the latter may with it is carcinogenic related.The toxicity of aflatoxin is than cyanide and arsenic It is many times larger.It has found out at present, aflatoxin all shows violent toxicity to people and a variety of animals, and has obvious Carciongenic potency；Wherein aflatoxin B1 toxicity is maximum, and carciongenic potency is most strong, G1, M1 secondly, B2, G2 and M2 compared with It is weak.Aflatoxin B1 is in addition to carcinogenic, moreover it is possible to mutagenesis and lead to deformity.The LD 50 of aflatoxin B1 is 0.249 mg/kg belongs to special extremely toxic substance less than 1 mg/kg, and toxicity is 10 times of potassium cyanide, is 68 times of arsenic. It has been found that aflatoxin B1 is strongest one kind of carcinogenicity in known carcinogenic substances all at present.China provides in fresh milk Aflatoxins M1 content is no more than 0.5 μ g/kg, and the aflatoxin in infant food and milk powder must not be examined Out.

Detection method about aflatoxin has had already appeared many chemical detection methods, such as gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography, spectrophotometry, LC-MS instrument (LC-MS), gas chromatograph-mass spectrometer (GC-MS) etc..There are sample size needed for testing cost height, analysis time length, detection (usual > 500 mL) greatly for chemical detection method And pre-treatment step it is cumbersome the deficiencies of, can not in a big way system, comprehensively, quickly analysis aflatoxin content.

And Timed resolved fluoroimmunoassay (TR-FIA) is due to its high specificity, high sensitivity, easy to operate, honest and clean Valence, and be valued by the people the advantages that detection particularly suitable for batch samples and increasingly and use.There is presently no be directed to The patent and document report of the time fluoroimmunoassay of Aflatoxins M1 detection.

Summary of the invention

In order to solve the above technical problems, the purpose of the present invention is to provide the remaining inspections of Aflatoxins M1 in a kind of milk Survey time-resolved fluoroimmunoassay kit.

The second object of the present invention is to provide a kind of time for quickly and easily detecting Aflatoxins M1 in milk point The detection method of fluorescence immunoassay kit is distinguished, for either quantitatively or qualitatively detecting Aflatoxins M1 residual in crops Amount.

One of the object of the invention is achieved in that the time-resolved fluoroimmunoassay reagent of detection Aflatoxins M1 Box, key are by the porous antibody for being coated with plate, buffer, Aflatoxins M1 standard solution, aspergillus flavus resisting toxin M1 Dried frozen aquatic products, the rabbit anti-mouse antibody of europium label, cleaning solution and enhancing liquid are formed.

The second object of the present invention is to be achieved: detect the time-resolved fluoroimmunoassay reagent of Aflatoxins M1 The detection method of box, preparation and sample pre-treatments and detection including immunogene, coating antigen and monoclonal antibody, key exist In:

(1) preparation of immunogene: haptens Aflatoxins M1 and bovine serum albumin(BSA) (BSA) are coupled, immunogene is obtained (Aflatoxins M1-BSA)；

(2) preparation of coating antigen: haptens Aflatoxins M1 and ovoserum albumin (OVA) are coupled, coating antigen is obtained (Aflatoxins M1-OVA)；

(3) preparation of monoclonal antibody:

A. mouse is immunized with the immunogene (Aflatoxins M1-BSA) of step (1), by hybridoma technology, it is anti-obtains secretion The hybridoma cell strain of the monoclonal antibody of Aflatoxins M1；

B. antibody is largely prepared to induce ascites method in vivo, is purified using Protein G column, obtain aspergillus flavus resisting toxin The monoclonal antibody IgG of M1；

C. plate is coated with 96 hole of coating primordial covering of step (2)；

(4) pre-treatment and detection of sample:

The porous coating plate for being coated with coating antigen (Aflatoxins M1-OVA) is taken, the Aflatoxins M1 of 50 μ L is added to respectively From micropore in, add 50 μ L with the diluted aspergillus flavus resisting toxin M1 antibody of buffer, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, wash It washs liquid to wash 3 times, is subject to the diluted 100 μ L Eu of buffer³⁺Rabbit anti-mouse antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, washing Liquid is washed 6 times, and the oscillation of 200 μ L enhancement solutions is added to measure fluorescence intensity cps after five minutes, and it is bent to calculate the Huang in sample according to standard curve Mould toxin M1 content.

Above-mentioned solid phase carrier is porous coating plate, using the porous coating plate in 96 holes as solid phase carrier.

The present invention mainly uses time-resolved fluorescence immunoassay method to detect Aflatoxins M1.Using time resolution Mainly there are two aspects for the technology of fluoroimmunoassay: first, monoclonal antibody specific preparation is exempted from the immunogene of coupling Epidemic disease mouse obtains the hybridoma cell strain of the monoclonal antibody of secretion aspergillus flavus resisting toxin M1 by hybridoma technology；With internal It induces ascites method and largely prepares antibody, purified using Protein G column, obtain the monoclonal antibody of aspergillus flavus resisting toxin M1 IgG.Second, Eu³⁺The preparation of labelled antibody.

Measuring method of the present invention: the basis of measurement is labelled immune reaction.It is coated with the porous of Aflatoxins M1-OVA It is coated with plate, test sample is added into respective micropore, adds aspergillus flavus resisting toxin M1 antibody, oscillating reactions, free Huang Aflatoxins M1-OVA on aspertoxin M1 and microwell plate competes aspergillus flavus resisting toxin M1 antibody, and cleaning solution washing does not have The Aflatoxins M1 antibody of connection is removed in washing step.Eu is added³⁺Immune response is marked in rabbit anti-mouse antibody, It is washed again with cleaning solution, there is no the Eu of connection after reaction³⁺Rabbit anti-mouse antibody is removed in washing step.Enhancement solution is added to vibrate Afterwards, very strong fluorescence is emitted under the excitation of ultraviolet lamp, measures its fluorescence intensity cps, fluorescence intensity with time-resolved fluorescence instrument It is inversely proportional with the concentration in sample, reference standard curve is the amount that can determine Aflatoxins M1 in sample.

Detection method does not need expensive instrument, and sample pre-treatments are simple, energy execute-in-place detects, using wide General, this method is sensitive, accurate, quick, easy to operate, high specificity, the quick detection suitable for gross sample.

Specific embodiment

Embodiment

1, prepared by immunogene and coating antigen

The synthesis of immunogene (Aflatoxins M1-BSA) of the present invention: it accurately weighs Aflatoxins M1 324mg and is dissolved in 2mL In n,N-Dimethylformamide, γ-aminobutyric acid solution is added dropwise under stirring, is stirred to react 3 hours, adjusts reaction solution pH 10 or so.Sediment is removed in centrifugation.Above-mentioned reaction is added dropwise in BSA solution to (320mg BSA is dissolved in 5mL physiology salt Water), n-hydroxysuccinimide (NHS) 23mg, N, N- dicyclohexylcarbodiimide (DCC) 45.4mg is added, 4 DEG C were reacted Night is centrifuged off precipitating, takes supernatant phosphate buffer (PBS) to dialyse 3 days, every 6 hours replacement dialyzates, by products therefrom Lyophilized is saved backup in -20 DEG C；

The synthesis of coating antigen (Aflatoxins M1-OVA): in above-mentioned reaction, after changing BSA into OVA, reaction conjugate is obtained Aflatoxins M1-OVA is used when the conjugate is detected as TR-FIA as coating antigen.

2, prepared by monoclonal antibody

2.1 animal immune

6 week old female Balb/c mouse are immunized respectively with immunogene prepared by step 1, the immunizing dose of every mouse is 100 μ g/ 0.2mL.First immunisation, it is former (Aflatoxins M1-BSA) with sterile 0.01mol/L pH7.4 PBS lytic immunity, then with equivalent Freund's complete adjuvant mixing, emulsifies completely, 2~3 points of injections of strength dorsal sc point；Booster immunization, with 0.01mol/L pH7.4 PBS lytic immunity original is mixed with equivalent Freund's complete adjuvant, fully emulsified, mouse peritoneal injection.Every minor tick 14~21 days, the 3 times it is immune after start within 7~10 days to take a blood sample to immune mouse tail vein, collect serum, detect mice serum potency with ELISA.End It is secondary it is immune after be spaced 4 weeks or more, 3~4 days before cell fusion, 00 μ g/ of Aflatoxins M1-BSA antigen 1 is injected intraperitoneally 0.2mL/ only, pays attention to observation daily after injection, guarantee that mouse is in good condition before merging.

The preparation of 2.2 monoclonal antibodies

The splenocyte of separating immune mouse, and carry out homogenate and prepare immune spleen cell.1 immune Balb/c mouse is taken, from eye Socket of the eye bloodletting separates serum as negative serum, puts to death.Mouse impregnates 5min with 75% alcohol, carries out overall disinfection.By mouse four limbs It is fixed, mouse lower abdomen skin then is clamped with tweezers, an osculum is cut off, then tear skin with tweezers, exposes peritonaeum, transducer set Tweezers and scissors cut off an osculum with scissors on the peritonaeum of abdomen center.Transducer set tweezers and scissors cut off peritonaeum with scissors, Expose spleen, then transducer set instrument clamps spleen with tweezers, broken spleen outer membrane with scissors, be then placed in sterilize in advance it is even It starches in device.Add appropriate basal medium (RPMI-1640) in homogenizer, ground, squeeze out splenocyte, takes out homogenizer Homogenate stick, then add appropriate basal medium (RPMI-1640), stand 2min, after upper cell liquid is drawn, be put into abdominal cavity In macrophage centrifuge tube, repeat aforesaid operations 1 time.1200r/min is centrifuged 10min, removes supernatant.By 10⁸A immune spleen is thin Born of the same parents and 1~2 × 10⁷A SP2/0 myeloma cell according to 1:10 or 1:5 ratio be added centrifuge tube in, mixed, then in 1500r/min horizontal centrifugal 10min, discards supernatant.Centrifuge tube is tipped upside down on the blotting paper of sterilizing, liquid in pipe is blotted. Tube bottom is gently tapped with finger or desktop, the cell of precipitating is allowed to loosen, then centrifuge tube is placed in 37 DEG C of water-baths.In 1min Slowly 50% PEG 0.8mL is instilled in centrifuge tube, side edged gently stirs sedimentation cell with pipette tip.It is further continued for stirring 30s Afterwards, 1min is stood, the 40mL basal medium (RPMI-1640) for carrying out 37 DEG C of pre-temperatures in advance is then slowly added into.Add basic training Support based method are as follows: 1mL is instilled in 1min dropwise, instills 2mL in 2min dropwise, instills 3mL in 3min dropwise, the 4mL is instilled in 4min dropwise, need to be slowly added to when adding culture medium every time, and lightly stir culture base, it finally will be remaining RPMI-1640 culture medium is slowly added into.1000r/min is centrifuged 5min, removes supernatant.Then with the suspension mixing of HAT culture medium Cell adds raising splenocyte.Suitable HAT culture medium is added as needed, is uniformly mixed, then will contain feeder cells Cell fusion drop is added in 96 porocyte culture plates, and dripping quantity is about 150 holes μ L/.Culture plate is placed in 37 DEG C, 5% CO₂ In saturated humidity incubator, cultivated.Positive cell clone is screened with the indirect ELISA of foundation.Selection strong positive collection is born Long hole, is cloned with limiting dilution assay.And to other positive holes, carries out 24 holes and expand culture, with indirect ELISA and indirectly Competitive ELISA detects the supernatant for expanding culture hole, is positive hole to indirect ELISA and indirect competitive ELISA Cell carries out liquid nitrogen frozen preservation.It is detected by fusion, and obtains hybridoma cell strain after carrying out 3 subclones.Hybridoma is thin Born of the same parents' strain by repeatedly passing on, freezing, recovering, stablize by hybridoma secretory antibody.The counting of hybridoma chromosome is carried out, Every strain of hybridoma is randomly choosed into 20 cells, carries out the counting of cell chromosome item number, then calculates cell chromosome item Several average value.Mouse boosting cell chromosome number is 40, and the chromosome number average of SP2/0 cell is 62 ~ 68, and this The 20 strain of hybridoma chromosome numbers obtained are tested all between 92 ~ 103, average out to 96.8.This hybridoma Chromosome number is higher than the chromosome number of two parental cells, and explanation is the hybrid product of two kinds of cells.Take cell strain cell point The culture supernatant secreted after carrying out 1:10 dilution, measures antibody subtype, the antibody of cell strain secretion with sandwich ELISA method Hypotype is IgG1.Mouse ascites are purified using caprylic acid-ammonium.The monoclonal antibody can be used for preparation time resolution Fluorescence detection reagent kit.

The purifying of 2.3 monoclonal antibodies

Mouse ascites are purified using caprylic acid-ammonium: taking mouse ascites 10mL, isometric barbital buffering is added Liquid, suitable silica mixing, shaken at room temperature 30min.After being stored at room temperature 15min, take supernatant in clean centrifuge tube, 4 DEG C, 1800r/min is centrifuged 20min；Supernatant 18mL is taken, 36mL 0.06mol/L sodium-acetate buffer is added, extremely with HCl tune pH value 4.5, it is sufficiently stirred down and is slowly added to 297 μ L of octanoic acid in 30min；Continue to stir 10min, is then transferred to 4 DEG C of refrigerators and stands 2h, 4 DEG C, 15000r/min is centrifuged 30min, and supernatant volume after 0.45 μm of membrane filtration is 50mL；5mL 0.1mol/ is added The phosphate buffer of L is slowly added into ammonium sulfate to final concentration of 0.277g/mL with NaOH tune pH value to 7.6 under stirring；4 DEG C of ice After case stands 2h, 4 DEG C, 12000r/min is centrifuged 30min, abandons supernatant；Precipitating is resuspended with the phosphate buffer of 5mL 0.1mol/L, It is packed into bag filter, after sufficiently being dialysed with 5000mL 0.01mol/L pH7.2 PBS buffer solution, then it is saturating with 2000 mL distilled water Analysis finally steams deionized water dialysis with 3000mL tri-；Then 4 DEG C, 12000r/min is centrifuged 30min, abandons precipitating, collects supernatant Liquid surveys protein concentration.SDS-PAGE electrophoresis is done, identifies the purity of monoclonal antibody.

The preparation of 2.4 rabbit anti-mouse igg antibody

With Balb/C mouse IgG immune health new zealand white rabbit, the rabbit anti-mouse igg hyper-immune serum of high-titer is prepared, using full Serum is slightly mentioned with ammonium sulfate precipitation method, the rabbit anti-mouse igg of high-purity is obtained after G-200 crosses column.

3.1, reagent preparation box and test sample

The 5g/L rabbit 1 ~ 2mL of anti-mouse antibody for being dissolved in 50 mmol/L PBS pH7.0 is taken to wash through the conversion buffered condition of PD-10 column The 50 mmol/L Na that de- liquid is the NaCl containing 0.155mmol/L₂CO₃-NaHCO₃ PH8.5 buffer.Protein peak is collected, through purple Outer absorption analysis is quantitative (1.46A280-0.74A260), dilutes rabbit anti-mouse antibody to 2g/L with above-mentioned eluent.Take 500 ~ 1000 The Eu for containing 0.2 ~ 0.4mg is added in rabbit anti-mouse antibody after μ L dilution³⁺-N₂[p- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu³⁺- DTTA) bottle in, 30 DEG C of magnetic agitations are reacted 20 hours.Reaction solution is through slow with 80mmol/L Tris-HCl pH7.8 Sepharose CL-6B column (1 × 40cm) chromatography of fliud flushing balance, A₂₈₀Protein peak is collected in monitoring, and dilution packing is spare.

3.2 coating plate solid phase antigen preparations

By Aflatoxins M1-OVA 50mmol/LNa₂CO₃-NaHCO₃ PH9.6 buffer is diluted to the coating buffer of 1mg/L, 96 holes coating each hole of plate adds 100 μ L, and 4 DEG C stand overnight.Coating buffer is discarded, is flushed three times, adds that 150 μ L OVA's containing 3g/L is upper Buffer blind is stated, 4 DEG C stand overnight.Confining liquid is discarded, vacuum is drained, and lath seals -20 DEG C of freezen protectives of postposition.

3.3 the preparation of reagent

(1) Aflatoxins M1 standard solution is prepared: by Aflatoxins M1 standard items, dilution becomes 0ng/mL, 0.01ng/ ML, 0.025ng/mL, 0.1ng/mL, 0.25ng/mL, 1ng/mL, 2.5ng/mL, 10ng/mL, 25ng/mL, 100ng/mL series Concentration, dilution are 0.1mol/L pH7.5 phosphate buffer；

(2) buffer: 8mmol/L NaCl, 0.2% OVA, 50 μm of ol/L diethylene triamine pentacetic acid (DTPA)s (DTPA), 0.1mL/L Tweeen-80 and 0.1%NaN₃50mmol/L Tris-HCl pH7.8；

(3) cleaning solution are as follows: 14.5mmol/L NaCl, 0.2mL/L Tweeen-80 and 0.2%NaN₃50mmol/L Tris- HCl pH7.8；

(4) preparation of enhancement solution: led to by 15 μm of ol β-naphthoyltrifluoroacetones, 50 μm of ol tri-n-octylphosphine oxide and 1mL Qula X-100 is added in pH3.2 Potassium Hydrogen Phthalate buffer, then is settled to 1L and is formulated.

The reagent that 3.4 kits provide

Based on the reagent of above-mentioned preparation, the present invention is used to detect the time-resolved fluoroimmunoassay kit of Aflatoxins M1 Including following material:

(1) 96 hole elisa Plates × 1 piece；

(2) Aflatoxins M1 standard items 1mg/mL/ bottles；

(3) aspergillus flavus resisting toxin M1 antibody dried frozen aquatic products, used time are dissolved with 0.5 mL distilled water；

(4)Eu³⁺Rabbit anti-mouse antibody dried frozen aquatic products, used time are dissolved with 0.5 mL distilled water；

(5) enhancement solution: 15mL；

(6) 10 × cleaning solutions: 30mL；

(7) buffer: 30 mL.

Points for attention before 3.5 measurements:

A. all reagents are returned before use and is warmed to room temperature (18-30 DEG C)；

B. all reagents are put back to 2-8 DEG C immediately after use；

C. suggest using Multi-channel liquid transfer device if sample size is big；

D. during all constant-temperature incubations, light is avoided to irradiate, with cap covers micropore；

E. the microwell plate and frame that need to use quantity are taken out, by unused microwell plate put into former Fresco Bag and with the drying that provides Agent reseals together, is stored in 2-8 DEG C.

3.6 specific detecting steps are as follows:

Aflatoxins M1-OVA lath is taken, the Aflatoxins M1 of 50 μ L is added into respective micropore, adds 50 μ L with slow The diluted aspergillus flavus resisting toxin M1 antibody of fliud flushing, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, and it is dilute to be subject to buffer The 100 μ L Eu released³⁺Rabbit anti-mouse antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 6 times, and 200 μ L enhancement solutions are added Oscillation measures fluorescence intensity cps after five minutes, calculates the Aflatoxins M1 content in sample from standard curve.

3.7 follow these steps reagent preparation box and detection milk sample:

(1) the same embodiment of reagent preparation box；

(2) specific detecting step is as follows:

Aflatoxins M1-OVA lath is taken, the Aflatoxins M1 of 50 μ L is added into respective micropore, adds 50 μ L with slow The diluted aspergillus flavus resisting toxin M1 antibody of fliud flushing, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, and it is dilute to be subject to buffer The 100 μ L Eu released³⁺Rabbit anti-mouse antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 6 times, and 200 μ L enhancement solutions are added Oscillation measures fluorescence intensity cps after five minutes, calculates the Aflatoxins M1 content in sample according to standard curve.

Claims (6)

1. a kind of time-resolved fluoroimmunoassay kit for detecting Aflatoxins M1, it is characterised in that: by porous coating Plate, buffer, Aflatoxins M1 standard items, the antibody dried frozen aquatic products of Aflatoxins M1, the sheep anti-mouse antibody of europium label, washing Liquid and enhancement solution are formed.

2. a kind of time-resolved fluoroimmunoassay kit for detecting Aflatoxins M1 according to claim 1, including The preparation and sample pre-treatments of immunogene, coating antigen and monoclonal antibody, it is characterised in that:

(1) Aflatoxins M1 and bovine serum albumin(BSA) are coupled, obtain immunogene；

(2) by Aflatoxins M1 and ovoserum albumen coupling, coating antigen is obtained；

(3) the immunogen immune mouse for using step (1) obtains the Dan Ke of secretion aspergillus flavus resisting toxin M1 by hybridoma technology The hybridoma cell strain of grand antibody；

(4) antibody is largely prepared to induce ascites method in vivo, is purified using Protein G column, obtain aspergillus flavus resisting toxin The monoclonal antibody of M1

(5) the coating primordial covering solid phase carrier of step (2) is used；

(6) it after animal tissue being first passed through acidolysis extraction, after MAX column purification, is eventually adding derivative reagent and catalyst carries out Processing, obtains product to be measured；

(7) object to be checked of step (6) is measured into fluorescence intensity cps, reference standard curve calculates the aspergillus flavus poison in sample Plain M1 Aflatoxins M1 content.

3. detecting the time fluoroimmunoassay kit of Aflatoxins M1 according to claim 1, it is characterised in that: The solid phase carrier is porous coating plate, using more micropores coating plate in 96 holes as solid phase carrier.

4. detecting the time fluorescence resolved immuno analytic approach kit of Aflatoxins M1, feature according to claim 1 Be: the derivative reagent is butylamine.

5. detecting the Timed resolved fluoroimmunoassay kit of Aflatoxins M1, feature according to claim 1 Be: the catalyst is itrile group diethyl phosphate.

6. detecting the time fluoroimmunoassay kit of Aflatoxins M1 according to claim 1, it is characterised in that: The step (6) and (7) are specially to take the micropore coating plate for being coated with Aflatoxins M1-OVA, are added what 50 μ L were handled well Into respective micropore 50 μ L are added with the diluted Aflatoxins M1 antibody of buffer, 25 ~ 37 DEG C of oscillations 0.5 ~ 1 are small in sample When, cleaning solution is washed three times, and the diluted 100 μ L Eu of buffer is subject to³⁺Sheep anti-mouse antibody, 25 ~ 37 DEG C vibrate 0.5 ~ 1 hour, wash It washs liquid to wash six times, the oscillation of 200 μ L enhancement solutions is added to measure fluorescence intensity cps after five minutes, calculate the Huang in sample from standard curve Aspertoxin M1 content.