CN108226465A - Detect the time-resolved fluoroimmunoassay kit of aflatoxin B1 - Google Patents
Detect the time-resolved fluoroimmunoassay kit of aflatoxin B1 Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a kind of time fluorescence resolved fluorometric immunoassay kits for detecting aflatoxin B1.The time resolved fluoro-immunoassay detection kit of the detection aflatoxin B1 is made of sheep anti-mouse antibody, cleaning solution and the enhancement solution of porous coating plate, buffer solution, aflatoxin B1 standard items, the antibody dried frozen aquatic products of aflatoxin B1, europium label.The detection method of the time fluorescence immunoassay kit of the detection aflatoxin B1 includes the following steps：(1) preparation of 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 has detection high specificity, in batch and differences between batches are small, high sensitivity and easy to operate quick, and the advantages that detection particularly suitable for batch samples.
Description
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 aflatoxin B1
Light immunoassay kits.
Background technology
Aflatoxin（AFT）It is the similar compound of a kind of chemical constitution, is the derivative of dihydrofuran cumarin
Object.Aflatoxin is mainly by aspergillus flavus（aspergillus flavus）Aspergillus parasiticus（a.parasiticus）It generates
Secondary metabolite, occur the probability highest of aflatoxin in damp-heat area food and feed.They be present in soil,
In animals and plants, various nuts, the grain oil products such as peanut, corn, rice, soybean, wheat are particularly easily polluted, 1993 yellow bent
Mould toxin is by the World Health Organization（WHO）Agency for Research on Cancer delimit as 1 class carcinogenic substance, be a kind of extremely strong violent in toxicity of toxicity
Matter.The harmfulness of aflatoxin is there is destruction to people and animal's liver tissue, can cause liver cancer even dead when serious
It is the most common with aflatoxin B1 in the food of natural contamination to die, toxicity and carcinogenicity are also most strong.
B1 is most dangerous carcinogenic substance, and often in corn, peanut, cotton seeds can be often detected in some dry fruits.They
Fluorescence can be generated under ultraviolet light irradiation, according to fluorescence color difference, is classified as B races and two major class of G races and its derivative.
AFT has found that more than 20 plant.AFT is present in soil, animals and plants, various nuts, mainly pollutes oil and foodstuffs, animals and plants food
Deng；As peanut, corn, rice, wheat, beans, nut fruits, meat, milk and milk products, aquatic products etc. have aflatoxin dirt
Dye is a kind of mycotoxin that mycotoxicosis is maximum, extremely protrudes human health risk.
And Timed resolved fluoroimmunoassay（TR-FIA）Due to its high specificity, high sensitivity, easy to operate, honest and clean
Valency, and be valued by the people the advantages that detection particularly suitable for batch samples and increasingly and use.There is presently no for
The patent and document report of the time fluoroimmunoassay of aflatoxin B1 detection.
Invention content
For solution more than technical problem, the purpose of the present invention is to provide aflatoxin B1s in a kind of vegetable and fruit to remain
Detection time resolved fluorometric immunoassay kits.
The second object of the present invention be to provide it is a kind of quickly and easily detect aflatoxin B1 in vegetable and fruit when
Between resolved fluorometric immunoassay kits detection method, it is residual for either quantitatively or qualitatively detecting aflatoxin B1 in crops
Allowance.
What one of the object of the invention was realized in：Detect the time resolved fluoro-immunoassay reagent of aflatoxin B1
Box, key are by the porous antibody for being coated with plate, buffer solution, aflatoxin B1 standard solution, aspergillus flavus resisting toxin B1
Dried frozen aquatic products, the rabbit anti-mouse antibody of europium label, cleaning solution and enhancing liquid are formed.
What the two of the object of the invention were realized in：Detect the time resolved fluoro-immunoassay reagent of aflatoxin B1
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：By haptens aflatoxin B1 and bovine serum albumin(BSA)（BSA）Coupling, obtains immunogene
（Aflatoxin B1-BSA）；
(2) preparation of coating antigen：By haptens aflatoxin B1 and ovoserum albumin（OVA）Coupling, obtains coating antigen
（Aflatoxin B1-OVA）；
(3) preparation of monoclonal antibody：
A. the immunogene of step (1) is used（Aflatoxin B1-BSA）By hybridoma technology, it is anti-to obtain secretion for immune mouse
The hybridoma cell strain of the monoclonal antibody of aflatoxin B1；
B. antibody is largely prepared to induce ascites method in vivo, is purified using Protein G columns, obtain aspergillus flavus resisting toxin
The monoclonal antibody IgG of B1；
C. with 96 hole of coating primordial covering of step (2) coating plate；
(4) pre-treatment and detection of sample：
It takes and is coated with coating antigen（Aflatoxin B1-OVA）Porous coating plate, add in 50 μ L aflatoxin B1 to respectively
From micropore in, add 50 μ L with the diluted aspergillus flavus resisting toxin B1 antibody of buffer solution, 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 buffer solution3+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 the Huang calculated according to standard curve in sample is bent
Mould toxin B1 contents.
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 detects aflatoxin B1 using time-resolved fluorescence immunoassay method.Using time resolution
Mainly there are two aspects for the technology of fluoroimmunoassay：First, prepared by monoclonal antibody specific, exempted from the immunogene of coupling
Epidemic disease mouse by hybridoma technology, obtains the hybridoma cell strain of the monoclonal antibody of secretion aspergillus flavus resisting toxin B1；With internal
It induces ascites method and largely prepares antibody, purified using Protein G columns, obtain the monoclonal antibody of aspergillus flavus resisting toxin B1
IgG.Second, Eu3+The preparation of labelled antibody.
Assay method of the present invention：The basis of measure is labelled immune reaction.It is coated with the porous of aflatoxin B1-OVA
Plate is coated with, adds in test sample to respective micropore, adds aspergillus flavus resisting toxin B1 antibody, oscillating reactions, free Huang
Aspertoxin B1 does not have with the aflatoxin B1-OVA competition aspergillus flavus resisting toxin B1 antibody on microwell plate, cleaning solution washing
The aflatoxin b1 antibody of connection is removed in washing step.Add in Eu3+Rabbit anti-mouse antibody, is marked immune response,
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, its fluorescence intensity cps, fluorescence intensity are measured with time-resolved fluorescence instrument
It is inversely proportional with the concentration in sample, reference standard curve is the amount that can determine aflatoxin B1 in sample.
Detection method does not need to expensive instrument, and sample pre-treatments are simple, energy execute-in-place detection, using wide
General, this method is sensitive, accurate, quick, easy to operate, high specificity, suitable for the quick detection of gross sample.
Description of the drawings
Fig. 1 is the aflatoxin B1 standard items of the present invention and the TR-FIA standard suppression curve figures of antibody.
Specific embodiment
Embodiment
1st, prepared by immunogene and coating antigen
Immunogene of the present invention（Aflatoxin B1-BSA）Synthesis：Accurately weighing aflatoxin B1 324mg 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（320mg BSA are dissolved in 5mL physiology salts
Water）, add n-hydroxysuccinimide（NHS）23mg, N, N- dicyclohexylcarbodiimide（DCC）45.4mg, 4 DEG C were reacted
Night is centrifuged off precipitating, and takes supernatant phosphate buffer（PBS）Dialysis 3 days replaces dialyzate in every 6 hours, by products therefrom
Lyophilized is saved backup in -20 DEG C；
Coating antigen（Aflatoxin B1-OVA）Synthesis：In above-mentioned reaction, after changing BSA into OVA, reaction conjugate is obtained
Aflatoxin B1-OVA is used when the conjugate is detected as TR-FIA as coating antigen.
2nd, 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, with sterile 0.01mol/L pH7.4 PBS lytic immunities original（Aflatoxin B1-BSA）, then with equivalent
Freund's complete adjuvant mixes, and emulsifies completely, 2~3 points of injections of strength dorsal sc point；Booster immunization, with 0.01mol/L pH7.4
PBS lytic immunities 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 immune mouse tail vein blood sampling, to collect serum, mice serum potency is detected 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 intraperitoneal injection aflatoxin B1-BSA antigen 1s
0.2mL/ only, pays attention to observation daily after injection, mouse is in good condition before ensureing fusion.
It is prepared by 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 detaches 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 skin is torn with tweezers, exposes peritonaeum, transducer set
Tweezers and scissors cut off an osculum on the peritonaeum of abdomen center with scissors.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）It in homogenizer, is ground, squeezes out splenocyte, take out homogenizer
Homogenate stick, then add appropriate basal medium（RPMI-1640）, 2min is stood, after upper cell liquid is drawn, is put into abdominal cavity
In macrophage centrifuge tube, aforesaid operations are repeated 1 time.1200r/min centrifuges 10min, removes supernatant.By 108A immune spleen is thin
Born of the same parents and 1~2 × 107A SP2/0 myeloma cell is according to 1:10 or 1:5 ratio is added in centrifuge tube, carries out mixing, Ran Houyu
1500r/min horizontal centrifugal 10min, discard 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 precipitation is allowed to loosen, then centrifuge tube is placed in 37 DEG C of water-baths.In 1min
Slowly 50% PEG 0.8mL are instilled in centrifuge tube, side edged gently stirs sedimentation cell with pipette tip.It is further continued for stirring 30s
Afterwards, 1min is stood, is then slowly added into the 40mL basal mediums for carrying out 37 DEG C of pre-temperatures in advance（RPMI-1640）.Add basic training
Foster based method is：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, and lightly stir culture base when adding culture medium every time, it finally will be remaining
RPMI-1640 culture mediums are slowly added into.1000r/min centrifuges 5min, removes supernatant.Then suspend what is mixed with HAT culture mediums
Cell adds raising splenocyte.Suitable HAT culture mediums are added as needed, are uniformly mixed, then will contain feeder cells
Cell fusion drop is added in 96 porocyte culture plates, and dripping quantity is about 150 μ L/ holes.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 it to other positive holes, carries out 24 holes and expands culture, with indirect ELISA and indirectly
Competitive ELISA is detected the supernatant for expanding culture hole, is positive hole to indirect ELISA and indirect competitive ELISA
Cell carries out liquid nitrogen frozen preservation.By fusion detection, and hybridoma cell strain is obtained 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,
20 cells will be randomly choosed per strain of hybridoma, carry out the counting of cell chromosome item number, then calculate cell chromosome item
Several average value.Mouse boosting cell chromosome number is 40, and the chromosome number average of SP2/0 cells 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 carries out 1:After 10 dilutions, antibody subtype, the antibody of cell strain secretion are measured with sandwich ELISA method
Hypotype is IgG1.Mouse ascites are purified using caprylic acid-ammonium.The monoclonal antibody is differentiated available for preparation time
Fluorescence detection reagent kit.
The purifying of 2.3 monoclonal antibodies
Mouse ascites are purified using caprylic acid-ammonium：Mouse ascites 10mL is taken, adds in isometric barbital buffering
Liquid, suitable silica mixing, shaken at room temperature 30min.After being stored at room temperature 15min, supernatant is taken in clean centrifuge tube, 4 DEG C,
1800r/min centrifuges 20min；Supernatant 18mL is taken, adds in 36mL 0.06mol/L sodium-acetate buffers, with HCl tune pH value extremely
4.5, it is sufficiently stirred down and 297 μ L of octanoic acid is slowly added in 30min；Continue to stir 10min, be then transferred to 4 DEG C of refrigerators and stand
2h, 4 DEG C, 15000r/min centrifugation 30min, supernatant volume after 0.45 μm of membrane filtration is 50mL；Add in 5mL 0.1mol/
The phosphate buffer of L with NaOH tune pH value to 7.6, is slowly added into ammonium sulfate to final concentration of 0.277g/mL under stirring；4 DEG C of ice
After case stands 2h, 4 DEG C, 12000r/min centrifugation 30min abandon supernatant；Precipitation is resuspended with the phosphate buffer of 5mL 0.1mol/L,
Bag filter is packed into, after fully being dialysed with 5000mL 0.01mol/L pH7.2 PBS buffer solution, then it is saturating with 2000 mL distilled water
Analysis finally boils off ionized water with 3000mL tri- and dialyses；Then 4 DEG C, 12000r/min centrifugation 30min abandon precipitation, collect 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 rabbits, the rabbit anti-mouse igg hyper-immune serum of high-titer is prepared, using full
Serum is slightly carried with ammonium sulfate precipitation method, the rabbit anti-mouse igg of high-purity is obtained after G-200 crosses column.
3.1st, reagent preparation box and detection sample
The 5g/L rabbit 1 ~ 2mL of anti-mouse antibody for being dissolved in 50 mmol/L PBS pH7.0 are taken, through the conversion buffered condition of PD-10 columns, are washed
The 50 mmol/L Na that de- liquid is the NaCl containing 0.155mmol/L2CO3-NaHCO3 PH8.5 buffer solutions.Protein peak is collected, through purple
Outer absorption analysis quantifies（1.46A280-0.74A260）, rabbit anti-mouse antibody is diluted to 2g/L with above-mentioned eluent.Take 500 ~ 1000
Rabbit anti-mouse antibody after μ L dilutions adds in the Eu containing 0.2 ~ 0.4mg3+-N2[p- isocyanic acids-benzyl]-diethylenetriamine tetraacethyl
（Eu3+-DTTA）Bottle in, 30 DEG C of magnetic agitations are reacted 20 hours.Reaction solution with 80mmol/L Tris-HCl pH7.8 through being delayed
The Sepharose CL-6B columns of fliud flushing balance（1×40cm）Chromatography, A280Protein peak is collected in monitoring, and dilution packing is spare.
It is prepared by 3.2 coating plate solid phase antigens
By aflatoxin B1-OVA 50mmol/LNa2CO3-NaHCO3 PH9.6 buffer solutions are diluted to the coating buffer of 1mg/L,
96 holes each hole of coating 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, lath sealing -20 DEG C of freezen protectives of postposition.
The preparation of 3.3 reagents
（1）Aflatoxin B1 standard solution is prepared：By aflatoxin B1 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 buffers；
（2）Buffer solution：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 is：14.5mmol/L NaCl, 0.2mL/L Tweeen-80 and 0.2%NaN350mmol/L Tris-
HCl pH7.8；
（4）The preparation of enhancement solution：Led to by 15 μm of ol β-naphthoyltrifluoroacetones, 50 μm of ol tri-n-octylphosphine oxide and 1mL Qulas
X-100 is added in pH3.2 Potassium Hydrogen Phthalate buffer solutions, 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 fluoro-immunoassay kit of aflatoxin B1
Including following material：
(1) 96 hole elisa Plates × 1 piece；
(2) aflatoxin B1 standard items 1mg/mL/ bottles；
(3) aspergillus flavus resisting toxin B1 antibody dried frozen aquatic products, used time distill water dissolution with 0.5 mL；
(4)Eu3+Rabbit anti-mouse antibody dried frozen aquatic products, used time distill water dissolution with 0.5 mL；
(5) enhancement solution：15mL；
(6) 10 × cleaning solutions：30mL；
(7) buffer solution：30 mL.
Points for attention before 3.5 measure：
A. all reagents are returned before use and be 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 of quantity need to be used by taking out, and no microwell plate is put into former Fresco Bag and the drying with providing
Agent reseals together, is stored in 2-8 DEG C.
3.6 specific detecting steps are as follows：
Aflatoxin B1-OVA laths are taken, are added in the aflatoxin B1 to respective micropore of 50 μ L, add 50 μ L with slow
The diluted aspergillus flavus resisting toxin B1 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 solution
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, adds 200 μ L enhancement solutions
Oscillation measures fluorescence intensity cps after five minutes, and the aflatoxin B1 content in sample is calculated from standard curve.
3.7 follow these steps reagent preparation box and detection apple, corn, vegetable sample：
(1) the same embodiment of reagent preparation box；
(2) specific detecting step is as follows：
Aflatoxin B1-OVA laths are taken, are added in the aflatoxin B1 to respective micropore of 50 μ L, add 50 μ L with slow
The diluted aspergillus flavus resisting toxin B1 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 solution
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, adds 200 μ L enhancement solutions
Oscillation measures fluorescence intensity cps after five minutes, and the aflatoxin B1 content in sample is calculated according to standard curve.
Claims (6)
1. a kind of time resolved fluoro-immunoassay kit for detecting aflatoxin B1, it is characterised in that：By porous coating
Plate, buffer solution, aflatoxin B1 standard items, the antibody dried frozen aquatic products of aflatoxin B1, the sheep anti-mouse antibody of europium label, washing
Liquid and enhancement solution are formed.
2. a kind of time resolved fluoro-immunoassay kit for detecting aflatoxin B1 according to claim 1, including
Immunogene, the preparation of coating antigen and monoclonal antibody and sample pre-treatments, it is characterised in that：
(1) aflatoxin B1 and bovine serum albumin(BSA) are coupled, obtain immunogene；
(2) by aflatoxin B1 and ovoserum albumen coupling, coating antigen is obtained；
(3) with the immunogen immune mouse of step (1), by hybridoma technology, the Dan Ke of secretion aspergillus flavus resisting toxin B1 is obtained
The hybridoma cell strain of grand antibody；
(4) antibody is largely prepared to induce ascites method in vivo, is purified using Protein G columns, obtain aspergillus flavus resisting toxin
The monoclonal antibody of B1
(5) with the coating primordial covering solid phase carrier of step (2)；
(6) it after animal tissue being first passed through acidolysis extraction, after MAX column purifications, 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 B1 aflatoxin B1s content.
3. the time fluoroimmunoassay kit of aflatoxin B1 is detected according to claim 1, it is characterised in that：
The solid phase carrier is porous coating plate, and plate is coated with as solid phase carrier using more micropores in 96 holes.
4. the time fluorescence resolved immuno analytic approach kit of aflatoxin B1, feature are detected according to claim 1
It is：The derivative reagent is butylamine.
5. the Timed resolved fluoroimmunoassay kit of aflatoxin B1, feature are detected according to claim 1
It is：The catalyst is itrile group diethyl phosphate.
6. the time fluoroimmunoassay kit of aflatoxin B1 is detected 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 aflatoxin B1-OVA, add in what 50 μ L were handled well
In sample to respective micropore, 50 μ L are added in the diluted aflatoxin b1 antibody of buffer solution, 25 ~ 37 DEG C of oscillations 0.5 ~ 1 are small
When, cleaning solution is washed three times, is subject to the diluted 100 μ L Eu of buffer solution3+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, the Huang in sample is calculated from standard curve
Aspertoxin B1 contents.
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