CN109752520A - Detect the time-resolved fluoroimmunoassay kit and its detection method of thiabendazole - Google Patents

Abstract

The invention discloses a kind of time fluorescence resolved fluorometric immunoassay kits and its detection method for detecting thiabendazole.The time-resolved fluoroimmunoassay detection kit of the detection thiabendazole is made of sheep anti-mouse antibody, cleaning solution and the enhancement solution of porous coating plate, buffer, thiabendazole standard items, the antibody dried frozen aquatic products of thiabendazole, europium label.The detection method of the time fluorescence immunoassay kit of the detection thiabendazole 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 and its detection method of thiabendazole

Technical field

The invention belongs to field of biological detection, and specifically, the time-resolved fluorescence for being related to a kind of detection thiabendazole is exempted from Epidemic disease assay kit and its detection method.

Background technique

Thiabendazole is the antisepsis antistaling agent being commonly used at present, is usually used in the fruits and vegetables before harvesting or after harvesting, through spraying Or the fruits and vegetables after dipping, it can not only extend period of storage, also freshness-retained, guarantor's color.These food additives have certain life Object poisonous effect, residual quantity is exceeded unavoidably to be had an impact human health.There are these fungicide in China specific residual Limitation requirement group is stayed, State General Administration for Quality Supervision's publication bulletin is forbidden adding 33 kinds of products such as light the third vinegar of yl benzoic acid as food Agent is produced and sold and is used, and Department of Qulity Supervision, province, which has stopped accepting its production permit application, to close, wherein including thiabendazole.

Currently, the detection method for the fruits and vegetables residual preservative reported both at home and abroad has related generally to spectrophotometry, micella electricity Dynamic chromatography is tired, high phase liquid chromatography, liquid chromatography tandem mass spectrometry, gas-chromatography tandem mass spectrometry etc., in various detections The foundation of method, sample pre-treatments are indispensable preconditions, and when in particular for trace analysis, sample pre-treatments are shadows The key link of analysis result is rung, Chang Yinqi sample detection error limits detection sensitivity, although extraction and purification methods have Soxhlet The technologies such as extraction, mechanical shaking extraction, liquid-liquid distribution, column chromatography, although these technologies do not need expensive equipment and specific apparatus, But time-consuming and laborious, Yi Yinqi error link in entire analytic process；And the use of a large amount of organic solvents, it easily causes to ring The pollution in border.Therefore, it is necessary to invent a kind of new method of few, the easy to operate detection of process.

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 thiabendazole detection.

Summary of the invention

In order to solve the above technical problems, when detection remaining the purpose of the present invention is to provide thiabendazole in a kind of apple Between resolved fluorometric immunoassay kits.

It is glimmering that the second object of the present invention is to provide a kind of time resolution for quickly and easily detecting thiabendazole in apple The detection method of light immunoassay kits, for either quantitatively or qualitatively detecting thiabendazole residual quantity in apple.

One of the object of the invention is achieved in that the time-resolved fluoroimmunoassay kit of detection thiabendazole, Its key is by porous coating plate, buffer, thiabendazole standard solution, the antibody dried frozen aquatic products of anti-thiabendazole, europium mark Rabbit anti-mouse antibody, cleaning solution and the enhancing liquid of note are formed.

The second object of the present invention is to be achieved: detect the time-resolved fluoroimmunoassay kit of thiabendazole Detection method, preparation and sample pre-treatments and detection, key including immunogene, coating antigen and monoclonal antibody are:

(1) preparation of immunogene: haptens thiabendazole and bovine serum albumin(BSA) (BSA) are coupled, immunogene (thiophene benzene miaow is obtained Azoles-BSA)；

(2) preparation of coating antigen: haptens thiabendazole and ovoserum albumin (OVA) are coupled, coating antigen (thiophene benzene miaow is obtained Azoles-OVA)；

(3) preparation of monoclonal antibody:

A. mouse is immunized with the immunogene (thiabendazole-BSA) of step (1) to obtain secreting anti-thiophene benzene miaow by hybridoma technology The hybridoma cell strain of the monoclonal antibody of azoles；

B. antibody is largely prepared to induce ascites method in vivo, is purified using Protein G column, obtains the list of anti-thiabendazole Clonal antibody IgG；

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

(4) pre-treatment and detection of sample:

It takes the porous coating plate for being coated with coating antigen (thiabendazole-OVA), the thiabendazole of 50 μ L is added to respective micropore In, add 50 μ L with the diluted anti-thiabendazole antibody of buffer, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, is added With the diluted 100 μ L Eu of buffer³⁺Rabbit anti-mouse antibody, 25 DEG C~37 DEG C oscillations 0.5 ~ 1

Hour, cleaning solution is washed 6 times, adds the oscillation of 200 μ L enhancement solutions to measure fluorescence intensity cps after five minutes, according to standard curve meter Calculate the thiabendazole content in sample.

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 thiabendazole.Using time-resolved fluorescence Mainly there are two aspects for the technology of immunoassay: first, monoclonal antibody specific preparation is small with the immunogen immune of coupling Mouse obtains the hybridoma cell strain for secreting the monoclonal antibody of anti-thiabendazole by hybridoma technology；To induce ascites in vivo Method largely prepares antibody, is purified using Protein G column, and the monoclonal antibody IgG of anti-thiabendazole is obtained.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 coating of thiabendazole-OVA Plate is added test sample into respective micropore, adds anti-thiabendazole antibody, oscillating reactions, free thiabendazole with Thiabendazole-OVA on microwell plate competes anti-thiabendazole antibody, cleaning solution washing, and the thiabendazole antibody not connected exists It is removed in washing step.Eu is added³⁺Rabbit anti-mouse antibody is marked immune response, then is washed with cleaning solution, does not have after reaction There is the Eu of connection³⁺Rabbit anti-mouse antibody is removed in washing step.After adding enhancement solution to vibrate, emit under the excitation of ultraviolet lamp Very strong fluorescence measures its fluorescence intensity cps with time-resolved fluorescence instrument, and fluorescence intensity is inversely proportional with the concentration in sample, right Sighting target directrix curve is the amount that can determine thiabendazole 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 (thiabendazole-BSA) of the present invention: it accurately weighs thiabendazole 324mg and is dissolved in 2mL N, N- diformazan In base formamide, γ-aminobutyric acid solution is added dropwise under stirring, is stirred to react 3 hours, adjusts reaction solution pH 10 or so.From The heart removes sediment.(320mg BSA is dissolved in 5mL physiological saline) is added dropwise in BSA solution in above-mentioned reaction, adds N- HOSu NHS (NHS) 23mg, N, N- dicyclohexylcarbodiimide (DCC) 45.4mg, 4 DEG C of reactions overnight, are centrifuged off Precipitating takes supernatant phosphate buffer (PBS) to dialyse 3 days, every 6 hours replacement dialyzates, by products therefrom lyophilized, It is saved backup in -20 DEG C.

The synthesis of coating antigen (thiabendazole-OVA): in above-mentioned reaction, after changing BSA into OVA, reaction conjugate is obtained Thiabendazole-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 (thiabendazole-BSA) with sterile 0.01mol/L pH7.4 PBS lytic immunity, then with equivalent Freund Freund's complete adjuvant mixing, emulsifies completely, 2~3 points of injections of strength dorsal sc point；Booster immunization, it is molten with 0.01mol/L pH7.4 PBS Solution immunogene is mixed with equivalent Freund's complete adjuvant, fully emulsified, mouse peritoneal injection.Every minor tick 14~21 days, exempts from for the 3rd time Start within 7~10 days after epidemic disease to take a blood sample to immune mouse tail vein, collect serum, detects mice serum potency with ELISA.Final immunization After be spaced 4 weeks or more, 3~4 days before cell fusion, 00 μ g/0.2mL/ of intraperitoneal injection thiabendazole-BSA antigen 1 only, injection Pay attention to observation daily afterwards, guarantees 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, sample pretreating method: taking 1g homogeneous sample in 50 mL centrifuge tubes, and 5 mL water are added, acutely vibrate 1 min；5 mL acetonitriles are added, acutely vibrate 3 min, 4000g or more is centrifuged 5 min；Take 1ml supernatant in 10ml teat glass In, it is dried up under 50~60 DEG C of water-bath nitrogen streams；1ml normal hexane vortex instrument whirling motion 30s is added, adds 1mL sample diluting liquid, Whirling motion 30s, 4000 r/min are centrifuged 5 min；Upper organic phase is removed, lower liquid is transferred in another clean centrifuge tube, For detecting.

4, 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.

4.2 coating plate solid phase antigen preparations

By thiabendazole-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, the above-mentioned of 150 μ L OVA containing 3g/L is added to delay Fliud flushing closing, 4 DEG C stand overnight.Confining liquid is discarded, vacuum is drained, and lath seals -20 DEG C of freezen protectives of postposition.

The preparation of 4.3 reagents

(1) thiabendazole standard solution is prepared: by thiabendazole 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 are dense Degree, 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: bent by 15 μm of ol β-naphthoyltrifluoroacetones, 50 μm of ol tri-n-octylphosphine oxide and 1mL It draws logical X-100 to be added in pH3.2 Potassium Hydrogen Phthalate buffer, then is settled to 1L and is formulated.

The reagent that 4.4 kits provide

Based on the reagent of above-mentioned preparation, the time-resolved fluoroimmunoassay kit that the present invention is used to detect thiabendazole includes Following material:

(1) 96 hole elisa Plates × 1 piece；

(2) thiabendazole standard items 1mg/mL/ bottles；

(3) anti-thiabendazole 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 4.5 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. take out the microwell plate and frame that need to use quantity, by unused microwell plate put into former Fresco Bag and with provide Desiccant reseals together, is stored in 2-8 DEG C.

4.6 specific detecting steps are as follows:

Thiabendazole-OVA lath is taken, the thiabendazole of 50 μ L is added into respective micropore, 50 μ L is added to dilute with buffer Anti- thiabendazole antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, cleaning solution is washed 3 times, and the diluted 100 μ L of buffer is subject to Eu³⁺Rabbit anti-mouse antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 6 times, add the oscillation of 200 μ L enhancement solutions after five minutes Fluorescence intensity cps is measured, calculates the thiabendazole content in sample from standard curve.

4.7 follow these steps reagent preparation box and detection apple sample:

(1) reagent preparation box with embodiment 4.

(2) specific detecting step is as follows:

Apple pre-treating method: taking 1g homogeneous sample in 50 mL centrifuge tubes, and 5 mL water are added, acutely vibrate 1 min；It is added 5 ML acetonitrile, acutely vibrates 3 min, and 4000g or more is centrifuged 5 min；Take 1ml supernatant in 10ml teat glass, 50~60 DEG C It is dried up under water-bath nitrogen stream；1ml normal hexane vortex instrument whirling motion 30s is added, adds 1mL sample diluting liquid, whirling motion 30s, 4000 r/min are centrifuged 5 min；Upper organic phase is removed, lower liquid is transferred in another clean centrifuge tube, for detecting.

Thiabendazole-OVA lath is taken, the thiabendazole of 50 μ L is added into respective micropore, adds 50 μ L with buffer Diluted anti-thiabendazole antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, and buffer diluted 100 is subject to µL Eu³⁺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 is added to vibrate 5 points Fluorescence intensity cps is measured after clock, and the thiabendazole content in sample is calculated according to standard curve.

Claims (6)

1. a kind of time-resolved fluoroimmunoassay kit for detecting thiabendazole, it is characterised in that: by porous coating plate, delay Fliud flushing, thiabendazole standard items, the antibody dried frozen aquatic products of thiabendazole, the sheep anti-mouse antibody of europium label, cleaning solution and enhancement solution institute Composition.

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

Thiabendazole and bovine serum albumin(BSA) are coupled, immunogene is obtained；

By thiabendazole and ovoserum albumen coupling, coating antigen is obtained；

(3) the immunogen immune mouse for using step (1), by hybridoma technology, the monoclonal for obtaining secreting anti-thiabendazole is anti- The hybridoma cell strain of body；

(4) antibody is largely prepared to induce ascites method in vivo, is purified using Protein G column, obtains anti-thiabendazole Monoclonal antibody

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 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 thiabendazole in sample Content.

3. the detection method of the time fluoroimmunoassay kit of thiabendazole is detected according to claim 1, it is special Sign is: the solid phase carrier is porous coating plate, using more micropores coating plate in 96 holes as solid phase carrier.

4. the detection method of the time fluorescence resolved immuno analytic approach kit of thiabendazole is detected according to claim 1, It is characterized by: the derivative reagent is butylamine.

5. the detection method of the Timed resolved fluoroimmunoassay kit of thiabendazole is detected according to claim 1, It is characterized by: the catalyst is itrile group diethyl phosphate.

6. the detection method of the time fluoroimmunoassay kit of thiabendazole is detected according to claim 1, it is special Sign is: the step (6) and (7) specially take the micropore coating plate for being coated with thiabendazole-OVA, and 50 μ L are added and handle well Sample into respective micropore, 50 μ L are added with the diluted thiabendazole antibody of buffer, 25 ~ 37 DEG C of oscillations 0.5 ~ 1 are small 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 thiophene in sample from standard curve Benzene imidazole content.