CN109752522A - Detect the time-resolved fluoroimmunoassay kit and its detection method of o-phenyl phenol in fruits and vegetables - Google Patents

Abstract

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

Technical field

The invention belongs to field of biological detection, specifically, are related to a kind of time-resolved fluorescence for detecting o-phenyl phenol Immunoassay kits and its detection method.

Background technique

Food safety and sanitation is related to the life and health of the mankind, causes the extensive concern of international community.With food The reduction for producing natural way, industrializes the increase of specific gravity, and food is by people are intentional or the chance of unintentional pollution is just gradually increasing Add, and with the increasingly prosperity of international trade, the speed of food pollution diffusion is fastly, range is extensively, harm is big and preceding Do not have.Present fruit and vegetable fresh preservation technology, mainly by suitable low temperature and air conditioning, but in fruit vegetables storing period Between their temperature for being able to bear using under degree and gas condition, lose control of the growth of mould；In addition fruit and vegetable fresh preservation needs Higher levels of humidity, the good condition of formal fungus growth, therefore the use of preservative is essential in preserving fruit and vegetable utilizing.Anti-corrosion Agent is not only advantageous to preserving fruit and vegetable utilizing, and it also can control and generates mould, to make people from the infringement of mycotoxin.

The use of preservative is of great significance to food storage.But inevitably people is allowed to carry on a shoulder pole using chemicals in food The heart, preservative can sterilize, while also harmful to human body.People in food for using chemicals, especially with anti-corrosion Agent worries very much, but actually without using again not all right, preservative is at one double-edged sword.For this purpose, relevant expert proposes that several points are built View selects suitable dosage wherein a little wanting highest specified in strict implement GB2760 using limitation and maximum residue limit, Drug effect is improved to the maximum extent, to reduce usage amount, pollution is minimized into level.This just needs to develop corresponding detection side Method fast and accurately detects remaining preservative in fruits and vegetables, provides maximum Bo Ahu to consumer.Preservative exists When practical application, the characteristics of being acted on due to the diversity and preservative of microbe species, people endeavour to reinforce opening for compounded technology Hair and application, using the mixing application of a variety of antistaling agents.Therefore, it is necessary to develop while measuring the analysis of a variety of antistaling agents in fruits and vegetables Method improves efficiency in terms of analysis time and material.

O-phenyl phenol is widely used in countries in the world, prevents fruit from rotting in growth, storage, transportational process. But remaining preservative has certain toxicity, main liver kidney, nervous system and the marrow for encroaching on human body to human body in fruit.Have Document report o-phenyl phenol has potential cause bladder cancer effect to experimental animal.

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 o-phenyl phenol detection.

Summary of the invention

In order to solve the above technical problems, the purpose of the present invention is to provide the remaining detections of o-phenyl phenol in a kind of apple Time-resolved fluoroimmunoassay kit.

The second object of the present invention is to provide a kind of time resolution for quickly and easily detecting o-phenyl phenol in apple The detection method of fluorescence immunoassay kit, for either quantitatively or qualitatively detecting o-phenyl phenol residual quantity in apple.

One of the object of the invention is achieved in that the time-resolved fluoroimmunoassay reagent of detection o-phenyl phenol Box, key are by the antibody freeze-drying of porous coating plate, buffer, o-phenyl phenol standard solution, anti-o-phenyl phenol Product, 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 kit of o-phenyl phenol Detection method, preparation and sample pre-treatments and detection including immunogene, coating antigen and monoclonal antibody are crucial to exist In:

(1) preparation of immunogene: haptens o-phenyl phenol and bovine serum albumin(BSA) (BSA) are coupled, immunogene (adjacent benzene is obtained Base phenol-BSA)；

(2) preparation of coating antigen: haptens o-phenyl phenol and ovoserum albumin (OVA) are coupled, coating antigen (adjacent benzene is obtained Base phenol-OVA)；

(3) preparation of monoclonal antibody:

A. mouse is immunized with the immunogene (o-phenyl phenol-BSA) of step (1), by hybridoma technology, obtains secreting anti-adjacent benzene The hybridoma cell strain of the monoclonal antibody of base phenol；

B. antibody is largely prepared to induce ascites method in vivo, is purified using Protein G column, obtains anti-o-phenyl phenol Monoclonal 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 (o-phenyl phenol-OVA), the o-phenyl phenol of 50 μ L is added to respective In micropore, add 50 μ L with the diluted anti-o-phenyl phenol antibody of buffer, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, it is subject to 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 o-phenyl phenol 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 o-phenyl phenol.It is glimmering using time resolution Mainly there are two aspects for the technology of light immunoassay: first, monoclonal antibody specific preparation, with the immunogen immune of coupling Mouse obtains the hybridoma cell strain for secreting the monoclonal antibody of anti-o-phenyl phenol by hybridoma technology；To induce in vivo Ascites method largely prepares antibody, is purified using Protein G column, and the monoclonal antibody IgG of anti-o-phenyl phenol is obtained.The Two, 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 packet of o-phenyl phenol-OVA By plate, test sample is added into respective micropore, adds anti-o-phenyl phenol antibody, oscillating reactions, free adjacent phenyl O-phenyl phenol-OVA on phenol and microwell plate competes anti-o-phenyl phenol antibody, cleaning solution washing, the adjacent benzene not connected Base phenol antibody is removed in washing step.Eu is added³⁺Rabbit anti-mouse antibody is marked immune response, then is washed with cleaning solution It washs, there is no the Eu of connection after reaction³⁺Rabbit anti-mouse antibody is removed in washing step.After adding enhancement solution to vibrate, in ultraviolet lamp The very strong fluorescence of the lower transmitting of excitation, measures its fluorescence intensity cps with time-resolved fluorescence instrument, the concentration in fluorescence intensity and sample It is inversely proportional, reference standard curve is the amount that can determine o-phenyl phenol 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 (o-phenyl phenol-BSA) of the present invention: it accurately weighs o-phenyl phenol 324mg and is dissolved in 2mL N, N- In dimethylformamide, γ-aminobutyric acid solution is added dropwise under stirring, is stirred to react 3 hours, it is left to adjust reaction solution pH 10 It is right.Sediment is removed in centrifugation.Above-mentioned reaction is added dropwise in BSA solution (320mg BSA is dissolved in 5mL physiological saline), then N-hydroxysuccinimide (NHS) 23mg, N, N- dicyclohexylcarbodiimide (DCC) 45.4mg is added, 4 DEG C of reactions are stayed overnight, from The heart removes precipitating, takes supernatant phosphate buffer (PBS) to dialyse 3 days, every 6 hours replacement dialyzates, by products therefrom low pressure Freeze-drying, saves backup in -20 DEG C.

The synthesis of coating antigen (o-phenyl phenol-OVA): in above-mentioned reaction, after changing BSA into OVA, reaction coupling is obtained Object o-phenyl phenol-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 (o-phenyl phenol-BSA) with sterile 0.01mol/L pH7.4 PBS lytic immunity, then not with equivalent The mixing of family name's Freund's complete adjuvant, 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 3rd time Start within 7~10 days to take a blood sample to immune mouse tail vein after immune, collect serum, detects mice serum potency with ELISA.Last is exempted from It is spaced after epidemic disease 4 weeks or more, 3~4 days before cell fusion, 00 μ of intraperitoneal injection o-phenyl phenol-BSA antigen 1 g/0.2mL/, Observation is paid attention to after injection daily, 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 o-phenyl phenol-OVA 50mmol/LNa₂CO₃-NaHCO₃ PH9.6 buffer is diluted to the coating buffer of 1mg/L, and 96 Hole 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 above-mentioned Buffer blind, 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) o-phenyl phenol standard solution is prepared: by o-phenyl phenol 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.

4.4 the reagent that kit provides

Based on the reagent of above-mentioned preparation, the present invention is used to detect the time-resolved fluoroimmunoassay kit packet of o-phenyl phenol Include following material:

(1) 96 hole elisa Plates × 1 piece；

(2) o-phenyl phenol standard items 1mg/mL/ bottles；

(3) anti-o-phenyl phenol 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:

O-phenyl phenol-OVA lath is taken, the o-phenyl phenol of 50 μ L is added into respective micropore, adds 50 μ L with buffer Diluted anti-o-phenyl phenol antibody, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, and it is diluted to be subject to buffer 100 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 Fluorescence intensity cps is measured after five minutes, calculates the o-phenyl phenol 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.

O-phenyl phenol-OVA lath is taken, the o-phenyl phenol of 50 μ L is added into respective micropore, adds 50 μ L with slow The diluted anti-o-phenyl phenol antibody of fliud flushing, 25 DEG C~37 DEG C vibrate 0.5 ~ 1 hour, and cleaning solution is washed 3 times, are subject to buffer dilution 100 μ L 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 shake Measurement fluorescence intensity cps after five minutes is swung, the o-phenyl phenol content in sample is calculated according to standard curve.

Claims (6)

1. a kind of time-resolved fluoroimmunoassay kit for detecting o-phenyl phenol, it is characterised in that: by porous coating plate, Buffer, o-phenyl phenol standard items, the antibody dried frozen aquatic products of o-phenyl phenol, the sheep anti-mouse antibody of europium label, cleaning solution and increasing Strong liquid is formed.

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

O-phenyl phenol and bovine serum albumin(BSA) are coupled, immunogene is obtained；

By o-phenyl phenol and ovoserum albumen coupling, coating antigen is obtained；

The monoclonal antibody for secreting anti-o-phenyl phenol is obtained by hybridoma technology with the immunogen immune mouse of step (1) Hybridoma cell strain；

Antibody is largely prepared to induce ascites method in vivo, is purified using Protein G column, obtains the list of anti-o-phenyl phenol Clonal antibody

With the coating primordial covering solid phase carrier of step (2)；

After animal tissue is first passed through acidolysis extraction, after MAX column purification, it is eventually adding at derivative reagent and catalyst Reason, obtains product to be measured；

The object to be checked of step (6) is measured into fluorescence intensity cps, the o-phenyl phenol that reference standard curve calculates in sample contains Amount.

3. the detection method of the time fluoroimmunoassay kit of o-phenyl phenol is detected according to claim 1, Be characterized 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 detection side of the time fluorescence resolved immuno analytic approach kit of o-phenyl phenol according to claim 1 Method, it is characterised in that: the derivative reagent is butylamine.

5. detecting the detection side of the Timed resolved fluoroimmunoassay kit of o-phenyl phenol according to claim 1 Method, it is characterised in that: the catalyst is itrile group diethyl phosphate.

6. the detection method of the time fluoroimmunoassay kit of o-phenyl phenol is detected according to claim 1, Be characterized in that: the step (6) and (7) specially take the micropore coating plate for being coated with o-phenyl phenol-OVA, are added at 50 μ L 50 μ L are added into respective micropore with the diluted o-phenyl phenol antibody of buffer, 25 ~ 37 DEG C of oscillations 0.5 in the sample managed ~ 1 hour, cleaning solution was washed three times, was subject to the diluted 100 μ L Eu of buffer³⁺Sheep anti-mouse antibody, 25 ~ 37 DEG C of oscillations 0.5 ~ 1 are small When, cleaning solution is washed six times, is added the oscillation of 200 μ L enhancement solutions to measure fluorescence intensity cps after five minutes, is calculated in sample from standard curve O-phenyl phenol content.