CN105524174A - Monoclonal antibody for detecting thiamphenicol and florfenicol, ELISA (enzyme-linked immunosorbent assay) method and kit - Google Patents

Abstract

The invention discloses a monoclonal antibody for detecting thiamphenicol and florfenicol. The monoclonal antibody is secreted by a hybridoma cell strain TAP/5F4, the hybridoma cell strain TAP/5F4 is preserved in the CCTCC (China Center for Type Culture Collection), and the collection number is CCTCC NO: C201549. The invention further discloses an ELISA (enzyme-linked immunosorbent assay) method for detecting thiamphenicol and florfenicol and a kit. Compared with the prior art, the prepared monoclonal antibody has higher sensitivity, high detection accuracy and good precision and has the advantages of simplicity, convenience, quickness, easiness in operation and the like.

Description

For detecting the monoclonal antibody of thiamphenicol and florfenicol and enzyme-linked immunoassay method and test kit

Technical field

The invention belongs to wild animal resources and immunological technique field, be specifically related to a kind of monoclonal antibody that can identify thiamphenicol and florfenicol, and for the enzyme-linked immunoassay method (ELISA) that detects thiamphenicol and florfenicol and test kit.

Background technology

Florfenicol and thiamphenicol belong to chloromycetin compound, and paraxin has had more than 50 year as prevention and therapy drug use in edibility animal, but because it has serious harm to the mankind, paraxin is classified as forbidden drugs by many countries.Therefore, just need and to have similar anti-microbial activity to paraxin and the compound of potential harm is not had again to replace paraxin to human body.Florfenicol and thiamphenicol are mainly used in treatment ox, poultry and porcine respiratory and intestinal tract infections.Although do not have chloramphenicol toxicity strong, but have been reported the reversibility anaemia that the use showing thiamphenicol can cause animal, florfenicol has certain embryotoxicity, and many countries such as China, the U.S., Japan and European Union and organizing all define their maximum residue limits in animal tissues.The method of current instrumental analysis can reach the testing requirement to such medicine, and accuracy and precision are all very high, but expensive equipment, sample pre-treatments are loaded down with trivial details, also need the operator of specialty.Immunochemical analyses method particularly enzyme-linked immunosorbent analytical technique has the advantages such as quick, highly sensitive, simple to operate, strong adaptability, be applicable to the screening of high-throughput sample, therefore the remaining in animal tissues for rapid detection thiamphenicol and florfenicol, ELISA method has more advantage.

Enzyme-linked immunosorbent assay is a kind of simple, highly sensitive, detection method that specificity is good, is therefore widely used.The report that simultaneously can detect the ELISA detection method of thiamphenicol and florfenicol is not at present a lot, the detection of most of or single medicine.Wherein Luo (2011) establishes the method for detecting residue of florfenicol and thiamphenicol in pig feed, take florfenicol as haptens, adopts mixed anhydride method to synthesize complete antigen, has prepared polyclonal antibody.The method is to florfenicol IC ₅₀be 1.02 μ g/L, to thiamphenicol IC ₅₀be 2.55 μ g/L, the method can identify florfenicol and thiamphenicol simultaneously, but comparatively florfenicol sensitivity is low for thiamphenicol, and cross reacting rate is 40%, and the method prepare for polyclonal antibody.Gold and silver treasure waits (2012) to take florfenicol amine as haptens, adopts carbodlimide method synthetic immunogen, with thiamphenicol succinyl oxide for haptens, adopts active ester method synthesis coating antigen to detect, to the IC of thiamphenicol ₅₀be 15 μ g/L, be respectively 10.8% and 10.4% to the cross reacting rate of florfenicol amine and florfenicol, therefore this antibody can only detect thiamphenicol, and sensitivity is lower.

Summary of the invention

Object of the present invention is:

(1) monoclonal antibody of a kind of energy specific recognition thiamphenicol and florfenicol is provided;

(2) described monoclonal antibody is provided to prepare the application in thiamphenicol and florfenicol enzyme-linked immunologic detecting kit;

(3) a kind of enzyme-linked immunologic detecting kit containing described monoclonal antibody is provided;

(4) application of described enzyme-linked immunologic detecting kit in thiamphenicol and florfenicol drug residue non-diagnostic object detect is provided;

(5) utilize this monoclonal antibody, set up the ELISA method that a kind of non-diagnostic object detects thiamphenicol and florfenicol drug residue.

Above-mentioned purpose is achieved through the following technical solutions:

A kind of monoclonal antibody, can identify thiamphenicol and florfenicol, it is secreted by hybridoma cell strain TAP/5F4.

Described hybridoma cell strain TAP/5F4, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201549.

Described monoclonal antibody, it is that the conjugate obtained using florfenicol amine (FFA) and succinyl oxide (HS) reacted florfenicol amine hemisuccinic acid ester (FFA-HS) and carrier protein couplet prepares as immunogen.The preferred immunogenic carrier albumen of the present invention is hemocyanin (KLH).

Described monoclonal antibody can be used for preparing the enzyme linked immunological kit detecting thiamphenicol and florfenicol.

Comprise a test kit for described monoclonal antibody, this test kit is the enzyme linked immunological kit detecting thiamphenicol and florfenicol drug residue.

Described test kit can be used for the detection of thiamphenicol and florfenicol drug residue non-diagnostic object.

A non-diagnostic object enzyme-linked immune detection method for thiamphenicol and florfenicol drug residue, comprise the preparation of immunogen, coating antigen and antibody, its step is as follows:

(1) conjugate florfenicol amine (FFA) and succinyl oxide (HS) reacted florfenicol amine hemisuccinic acid ester (FFA-HS) and carrier protein couplet obtained is as immunogen;

(2) florfenicol amine (FFA) and carrier protein couplet are obtained coating antigen;

(3) prepare by the immunogen of step (1) the hybridoma cell strain TAP/5F4 that preserving number is CCTCCNO:C201549;

(4) monoclonal antibody is prepared with the hybridoma cell strain TAP/5F4 that preserving number is CCTCCNO:C201549;

(5) use the coating antigen bag of step (2) by solid phase carrier;

(6) process of sample and detection.

Preferably, immunogenic carrier albumen is hemocyanin (KLH), and coating antigen carrier proteins is oralbumin (OVA).

The invention has the beneficial effects as follows:

1, the present invention is when prepared by antibody, and using florfenicol amine hemisuccinic acid ester as haptens, using haptens and carrier protein couplet as immunogen, the monoclonal anti physical efficiency prepared by this immunogen identifies florfenicol and thiamphenicol specifically simultaneously.

2, enzyme linked immunological kit of the present invention and method are applicable to detect edible animal tissue as the florfenicol in pork, chicken, fish, pig liver and chicken liver and thiamphenicol drug residue, and sensitivity, the accuracy of detection are high, and precision is good, IC ₅₀value is only 0.35 ± 0.03 μ g/L.

3, detection method involved in the present invention is simple, and easy to operate, testing cost is low, relatively little to the healthy harm of operator.

Accompanying drawing explanation

Fig. 1 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and thiamphenicol standard substance, X-axis is thiamphenicol (TAP) concentration of standard solution logarithmic value, and Y-axis is that the optical density value of thiamphenicol standard solution is divided by " zero " hole optical density value (B/B ₀).

Embodiment

Below by embodiment, the invention will be further described, but do not limit the present invention.

The preparation of embodiment 1 immunogen and coating antigen

The preparation of 1.3 florfenicol amine hemisuccinic acid esters and limpet hemocyanin conjugate (FFA-HS-KLH)

Take florfenicol amine (FFA) 247mg (1mmol), BOC acid anhydrides 218mg (1mmol), Na ₂cO ₃106mg (1mmol) is in 50mL round-bottomed flask, and add methylene dichloride 10mL, room temperature reaction spends the night.Filter, remove Na ₂cO ₃, then add succinyl oxide 200mg (2mmol), triethylamine 1mL, under 60 DEG C of oil bath conditions, back flow reaction 1d.Evaporate to dryness removes methylene dichloride, add trifluoroacetic acid 2mL, room-temperature water bath reaction 2-4h, question response drips triethylamine completely and adjusts neutral, with ethyl acetate washing 2-3 time, merge organic phase, after anhydrous magnesium sulfate dehydration, evaporate to dryness obtains yellow oil, i.e. haptens florfenicol amine hemisuccinic acid ester (FFA-HS).Reaction formula is as follows:

Take florfenicol amine hemisuccinic acid ester 69.4mg, be dissolved in 1mLDMF, add DCC60mg and N-hydroxy-succinamide (NHS) 34.6mg, room temperature lucifuge reaction 8h, the filtration that reacts completely obtains A liquid.Getting 3mLKLH is dissolved in 3mLPBS, is B liquid.Under condition of ice bath, A liquid is slowly added drop-wise in B liquid, 4 DEG C of reaction 8-10h.Product is loaded in dialysis tubing, to dialyse 5d with PBS under 4 DEG C of conditions, change dialyzate every day 3 times.What obtain is FFA-HS-KLH.

The preparation of 1.4 florfenicol amine oralbumin conjugates (FFA-EDC-OVA)

Take florfenicol amine 24.7mg, being dissolved in 100 μ LDMF is A liquid.Taking 40mgOVA is dissolved in 6mLPBS, is B liquid.Slowly be added drop-wise in B liquid by A liquid, then add EDC16mg and NHS12mg, room temperature reaction spends the night, and is loaded by reactant in dialysis tubing, to dialyse 5d, changes dialyzate every day 3 times for 4 DEG C with PBS.What obtain is complete antigen FFA-EDC-OVA.Reaction formula is as follows:

The preparation of embodiment 2 monoclonal antibody

2.1 animal immune

The immune Balb/C mouse of immunogen (FFA-HS-KLH) (purchased from Hubei Prov. Academy of Medical Sciences's Experimental Animal Center) utilizing the national basic veterinary drug at contriver place to remain benchmarks room to prepare.Immune programme for children gets containing injecting in Mice Body after the protein solution of FFA-HS-KLH conjugate 50 ~ 100 μ g and adjuvant balanced mix, makes it produce specific serum.

2.2 cytogamy and cloning

With reference to Yang Hanchun " animal immunology ", utilize FF-HS-KLH immunogen immune Balb/C mouse (purchased from Disease Prevention Control Center, Hubei Prov's Experimental Animal Center) prepared by contriver the country one belongs to residue of veterinary drug benchmarks room, immune programme for children is: fundamental immunity is by after immunogen and isopyknic Freund's complete adjuvant emulsification, in the subcutaneous multi-point injection of mouse back, once at interval of 2 weeks booster immunizations, use Freund's incomplete adjuvant emulsification instead later.Finally in first three sky of fusion (be better than most immunity terminate rear rest and reorganization January) abdominal injection, reinforced immunological, antigen amount doubles, and does not add adjuvant.

During fusion, the Balb/C mouse of last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum), soaks 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen, isolates splenocyte, with the SP2/0 myeloma cell (SP2/0 myeloma cell is from this laboratory) of fresh preparation by 1 ~ 2 × 10 ⁷individual SP2/0 and 10 ⁸the ratio of individual immune spleen cell (1:10 ~ 1:15) is in 50mL centrifuge tube, and with 15mLRPMI-1640 basal liquid re-suspended cell, the centrifugal 5min of 1500r/min, washes cell 1 time.The substratum that temperature is bathed by centrifugal gap, the water of temperature bath, super clean bench put into by the polyoxyethylene glycol (polyethyleneglycol, PEG) etc. of temperature bath.Then take out the thieving paper of sterilizing, by the centrifuge tube that myeloma cell and immune spleen cell be housed emptying to the greatest extent, tipping upside down on and thieving paper controls solid carbon dioxide dripping, rapping at the bottom of pipe and cell is loosened.Open timing register, draw 0.8mLPEG, the hand-held centrifuge tube that cell mixing is housed with 1mL suction pipe, place it in a moment in water-bath, be added drop-wise to slowly on cell mixing by PEG, limit edged stirs gently, adds in 1min, Keep agitation 30s.Get 10mL basal liquid with suction pipe, be slowly added on fused cell along tube wall, limit edged shakes gently (can not blow and beat), adds 1mL respectively in 5min, 2mL, 3mL, 4mL, finally add basal liquid to 40mL, after adding, cover lid, puts upside down several times repeatedly, and cell is mixed.800r/min5min is centrifugal, abandons supernatant.Draw the HAT substratum containing feeder cell, stirred gently by the fused cell in centrifuge tube, be dropwise added dropwise in the serum bottle containing feeder cell, stirring and evenly mixing near liquid level with suction pipe, action is wanted gently to be stirred gently by cell, must not blow and beat.Put upside down mixing.Then be seeded on Tissue Culture Plate by cell, two, every hole, is placed in incubator and cultivates.Single cell fusion can inoculate 4 ~ 6 piece of 96 orifice plate.Also can plant less as required, the cell count of generally pressing SP2/0 calculates, and every hole inoculum size is about containing 10 ⁴a left and right SP2/0 cell.In 37 DEG C, 5%CO ₂cultivate in incubator.

Merge and be designated as 0d the same day, front 3d tries not migratory cell, and 3d adds in every hole 1 1%HAT perfect medium, observes colony growth situation.5d every hole sucking-off 100 μ L supernatant, then add 2 0.5%HAT perfect mediums, continue tracing observation fused cell.

Cell colony to be fused grows to culture hole 1/10 ~ 1/5, screens with the indirect ELISA method set up simultaneously.Compared with zero medicine hole, medicine hole OD value repressedly can be judged to the positive.According to inhibiting rate and cell colony upgrowth situation, select the cell hole only having 1-2 single colony of 2 ~ 6 strong positives, adopt limited dilution method to carry out cloning.

Through 3 ~ 4 time clonings, finishing screen selects the monoclonal hybridoma strain of secreting anti-thiamphenicol drug antibody, applicant is by its called after TAP/5F4, and China typical culture collection center (CCTCC) preservation being positioned at Wuhan City, Hubei Province Wuhan University is sent on April 24th, 2015, deposit number is CCTCCNO:C201549.Chromosome counting has been carried out to this clone, result shows, and the chromosome number of SP2/0 is 62 ~ 68, and splenocyte karyomit(e) is 40, and the chromosome number mean value of hybridoma is 104.5, the cell of SP2/0 really of fused cell and the hybrid product of splenocyte are described.By this cell strain through abdominal injection Balb/C mouse, manufacture order clonal antibody.Adopt and identify the hypotype of the monoclonal antibody that the present invention obtains and light chain purchased from mouse monoclonal antibody (Monoclonalantibody, Mab) the Rapid ELISA isotyping kit of ThermoSxientific company, result is mouse IgG ₁hypotype.

The foundation of embodiment 3 thiamphenicol racing ELISA detecting method

The preparation (reagent that the present embodiment uses all adopts following methods preparation except another indicating) of 3.1 reagent

Phosphate buffered saline buffer: NaCl8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o2.9g, KCl0.2g, add distilled water to 1000mL, regulates pH to 7.4;

Coating buffer: get Na ₂cO ₃1.59g, NaHCO ₃2.93g, adds tri-distilled water to 1000mL, adjust ph to 9.6;

Washings: NaCl8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o2.9g, KCl0.2g, Tween200.5mL, add distilled water to 1000mL, regulates pH to 7.4;

Confining liquid: ovalbumin 1g is dissolved in 100mL phosphate buffered saline buffer;

Substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;

Substrate solution B:Na ₂hPO ₄14.6g, citric acid 9.3g, 0.75% Urea Peroxide 6.4mL, adds distilled water to 1000mL;

Substrate cocktail: by A liquid and B liquid by volume 1:1 mix and get final product, now with the current;

Stop buffer: 2mol/L sulphuric acid soln.

Tentatively determining of 3.2 coating antigen concentration and antibody working concentration

Select the FFA-EDC-OVA of above-mentioned synthesis as coating antigen, 16mg/L, 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L8 concentration is diluted to coating buffer, at 96 hole enzyme plates, the from first to the 8th leu adds, and 4 DEG C are spent the night; Wash 3 times, pat dry, add confining liquid 200 μ L, 37 DEG C of closed 60min; Wash 3 times, pat dry, the first row to the 8th row of enzyme plate add successively 100 μ L phosphate buffered saline buffers dilution extension rate be 1000,2000,4000,8000,16000,32000,64000,128000 monoclonal antibody, hatch 30min for 37 DEG C, wash 3 times, pat dry; The sheep anti-mouse igg antibody that each hole adds the HRP mark of 1:5000 times of phosphate buffered saline buffer dilution (is called for short two to resist, the anti-sheep anti-mouse igg antibody being HRP mark of following indication two, purchased from Wuhan Fei Yi Bioisystech Co., Ltd) 100 μ L, hatch 30min for 37 DEG C, wash 5 times, pat dry; Each hole adds 100 μ L Substrate cocktail, and lucifuge colour developing 15min, adds 50 μ L stop buffers, measure optical density value (OD value), the results are shown in Table 1 by automatic microplate reader at 450nm wavelength place.

Result shows, tentatively determine that the bag of coating antigen FFA-EDC-OVA is 1mg/L or 0.5mg/L by concentration, antibody working concentration is 1:16000 or 1:8000.

Tentatively determining of table 1 coating antigen concentration and antibody working concentration

The determination of 3.3 best coating antigen concentration and antibody working concentration

Wrap by concentration with the coating antigen FFA-EDC-OVA tentatively determined, 1mg/L, 0.5mg/L2 concentration coated elisa plate is set.Thiamphenicol phosphate buffered saline buffer is diluted to 1.6 μ g/L, 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, a 0 μ g/L6 concentration, the monoclonal antibody of being diluted by 1:16000 and 1:8000 phosphate buffered saline buffer is respectively (because when making indirect competitive ELISA, the injection volume of monoclonal antibody reduces half, and correspondingly monoclonal antibody extent of dilution reduces half) and above-mentioned thiamphenicol solution respectively add 50 μ L and carry out indirect competitive ELISA.Using thiamphenicol log concentration value as X-coordinate, with the ratio (B/B of the OD value of thiamphenicol standardized solution with " zero " hole OD value ₀) draw suppression curve as ordinate zou, thiamphenicol concentration (IC when select linear is better, generation 50% suppresses ₅₀) junior as bag by concentration.With best coating antigen concentration coated elisa plate, thiamphenicol is diluted to 1.6 μ g/L, 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, a 0 μ g/L6 concentration, antibody is arranged 4 extent of dilution with centre concentration 1:16000 equal difference, monoclonal antibody and series concentration thiamphenicol standardized solution respectively add 50 μ L and carry out indirect competitive ELISA, draw and suppress curve, select linear is better, IC ₅₀junior is as optimum antibody working concentration for value.The results are shown in Table 2 and table 3.

The best coating antigen concentration optimization of table 2

Coating antigen concentration	Antibody dilution multiple	0 hole OD	IC 50Value
				0.5	8000	2.233	0.51
1	16000	2.167	0.37

Table 3 optimum antibody extent of dilution is optimized

Antibody dilution multiple	0 hole OD	IC 50(μg/
			12000	2.396	0.51
14000	2.236	0.47
			16000	1.978	0.35
18000	1.629	0.41

Result shows, wrapping by concentration is 1mg/L, when antibody dilution is 1:16000, and its IC ₅₀minimum.

The foundation of 3.4 typical curves

Thiamphenicol phosphate buffered saline is become 1.6 μ g/L, 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, a 0 μ g/L6 series concentration, each concentration repeats 5 holes, measures, replication 5 times according to indirect competitive ELISA method.With the logarithmic value of thiamphenicol strength of solution for X-coordinate, B/B0 is ordinate zou drawing standard curve, obtains IC ₅₀.The IC of this test kit ₅₀value is 0.35 ± 0.03 μ g/L.

3.5 cross reaction tests

Chloromycetin medicine phosphate buffered saline is become proper concn, measures the IC of each medicine by the ELISA method set up ₅₀value, each medicine 3 repeating holes, with monoclonal antibody to the cross reacting rate of thiamphenicol for 100%, utilize formula 1 to calculate the cross reacting rate of monoclonal antibody to each medicine, the results are shown in Table 4.

Table 4 test kit of the present invention is to the cross reacting rate of various chloromycetin medicine

Result shows, monoclonal antibody only has higher cross reacting rate to thiamphenicol and florfenicol, can detect this two kinds of medicines simultaneously.

Embodiment 4 thiamphenicol of the present invention and fluoride protector detect the assembling of ELISA kit

4.1 ELISA kit of the present invention are made up of following part:

1) solid phase carrier (enzyme plate) of coating antigen FFA-EDC-OVA is coated with;

2) 6 bottles, thiamphenicol standardized solution, concentration is respectively 1.6 μ g/L, 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0 μ g/L;

3) preserving number is the monoclonal antibody of the hybridoma cell strain TAP/5F4 secretion of CCTCCNO:C201549;

4) the sheep anti-mouse igg antibody working fluid that marks of horseradish peroxidase (HRP);

5) concentrated phosphoric acid salt buffer: NaCl80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄.12H ₂o29.0g, KCl2.0g, add distilled water to 1000mL;

6) concentrated cleaning solution: NaCl80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄.12H ₂o29.0g, KCl2.0g, Tween205mL, add distilled water to 1000mL;

7) substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;

8) substrate solution B:Na ₂hPO ₄14.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, adds distilled water to 1000mL, regulates pH to 5.0 ~ 5.4;

9) stop buffer: 2mol/L sulphuric acid soln.

The preparation of 4.2 enzyme plates

With coating buffer, FFA-EDC-OVA is diluted to 1mg/L, every hole adds 100 μ L, and 4 DEG C are spent the night, and incline coating buffer, every hole adds 250 μ L washingss and washs 3 times, pat dry, then every hole adds confining liquid 250 μ L, hatches 60min for 37 DEG C, incline liquid in hole, washings washs 3 times, pats dry, and preserves with masking foil vacuum-sealing.

The mensuration program of embodiment 5 enzyme linked immunological kit of the present invention

The preparation of 5.1 reagent

1) sample diluting liquid: use after the concentrated phosphoric acid salt buffer tri-distilled water provided in test kit is diluted 10 times.

2) washings: use after the washings tri-distilled water provided in test kit is diluted 10 times.

3) Substrate cocktail: according to each institute expense, by the substrate solution A of preparation and substrate solution B by volume 1:1 mixing, now with the current.

5.2 sample pre-treatments

The pre-treatment of pork, chicken, the flesh of fish, pork liver and chicken gizzard:

1) take the homogeneous sample of 2.00 ± 0.02g edibility animal tissues in 50mL centrifuge tube, add 8mL ethyl acetate, the centrifugal 5min of concussion 5min, room temperature 4000r/min;

2) get supernatant 2mL nitrogen to dry up, redissolve with normal hexane 2mL, vortex 30s, then add PBS1mL mixing, the centrifugal 5min of room temperature 4000r/min, take off layer aqueous phase for kit measurement, present treatment is 2 to the extension rate of tissue sample.

5.3 determination step

1) application of sample: add a series of concentration thiamphenicol standardized solution or sample solution 50 μ L in enzyme plate micropore, then add monoclonal antibody working fluid 50 μ L, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;

2) wash: pour out the liquid in hole, add washings 250 μ L in every hole and wash 3 times and pat dry;

3) the sheep anti-mouse igg antibody working fluid that horseradish peroxidase (HRP) marks is added: in every hole, add the sheep anti-mouse igg antibody working fluid 100 μ L that horseradish peroxidase (HRP) marks, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;

4) wash: pour out the liquid in hole, in every hole, add washings 250 μ L, wash 3 times and pat dry;

5) substrate is added: add Substrate cocktail 100 μ L in every hole, be placed in wet box, 37 DEG C of constant-temperature incubation 15min;

6) stop buffer is added: in every hole, add stop buffer 50 μ L;

7) measure: the optical density value (OD value) measuring every hole by microplate reader at 450nm place.

5.4 results judge

Typical curve:

With measured standard substance OD value divided by " zero " hole OD value (B/B0) for ordinate zou, the logarithmic value of thiamphenicol concentration is that X-coordinate makes typical curve, line linearity of going forward side by side return, provide regression equation.

In tissue, thiamphenicol drug level calculates:

The inhibiting rate (the OD value of the sample obtained is divided by " zero " hole OD value) of calculation sample, substitutes in the regression equation of typical curve, calculates thiamphenicol concentration in tissue (μ g/kg).

In tissue, florfenicol drug level calculates:

The inhibiting rate (the OD value of the sample obtained is divided by " zero " hole OD value) of calculation sample, substitutes in the regression equation of typical curve, is converted to florfenicol drug level according to formula 2.

The sensitivity of embodiment 6 test kit of the present invention, precision, accuracy, replica test

The sensitivity test of 6.1 test kits of the present invention

With the IC of typical curve ₅₀value and organize lowest detectable limit (LOD) as the sensitivity index of detection kit of the present invention.The dilution of thiamphenicol standard substance is become 1.6 μ g/L, 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, a 0 μ g/L6 concentration, the multiple hole of each concentration 5, according to indirect competitive ELISA method replication 5 times, get the IC of 5 mensuration ₅₀mean value.LOD is determined by following steps, measures the OD value of 20 parts of blank tissue samples, goes out corresponding thiamphenicol concentration, then calculate the mean value of thiamphenicol concentration according to the regression equation calculation of typical curve with standard deviation (SD), according to formula calculate the lowest detectable limit in tissue.IC of the present invention ₅₀value is 0.35 ± 0.03 μ g/L, and thiamphenicol and the florfenicol lowest detection line in animal tissues refers to table 5.

Lowest detectable limit in table 5 animal tissues

The precision test of 6.2 test kits of the present invention

Thiamphenicol standard substance are diluted to 1.6 μ g/L, 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, a 0 μ g/L6 concentration, every concentration 5 repetition, according to indirect competitive ELISA method replication 5 times, the regression equation calculation of application standard curve goes out the measured value of each concentration thiamphenicol standardized solution, the variation coefficient in computing board and between plate, the results are shown in Table 6.

The variation coefficient in the plate of table 6 typical curve and between plate

The accuracy of 6.3 test kits of the present invention, replica test

The thiamphenicol of three concentration and florfenicol add in pork, chicken, the flesh of fish, pig liver and chicken liver sample by this research respectively, its TIANZHU XINGNAO Capsul is between 72.8% ~ 113.4%, with interassay coefficient of variation < 15% in batch, measurement result is in table 7 ~ 11.

TIANZHU XINGNAO Capsul in table 7 pork

TIANZHU XINGNAO Capsul in table 8 chicken

TIANZHU XINGNAO Capsul in table 9 flesh of fish

TIANZHU XINGNAO Capsul in table 10 pig liver

TIANZHU XINGNAO Capsul in table 11 chicken liver

Claims (8)

1. can identify a monoclonal antibody for thiamphenicol and florfenicol, it is characterized in that: secreted by the hybridoma cell strain TAP/5F4 that it is is CCTCCNO:C201549 by preserving number.

2. the hybridoma cell strain TAP/5F4 described in claim 1, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201549.

3. monoclonal antibody according to claim 1, is characterized in that: it prepares as immunogen after the florfenicol amine hemisuccinic acid ester obtained after florfenicol amine and succinyl oxide react and carrier protein couplet.

4. the monoclonal antibody described in claim 1 or 3 is preparing the application in the enzyme linked immunological kit detecting thiamphenicol and florfenicol.

5. comprise the test kit of monoclonal antibody described in claim 1 or 3.

6. test kit according to claim 5, this test kit is the enzyme linked immunological kit detecting thiamphenicol and florfenicol drug residue.

7. the application of test kit according to claim 5 in thiamphenicol and florfenicol drug residue non-diagnostic object detect.

8. a non-diagnostic object enzyme-linked immune detection method for thiamphenicol and florfenicol drug residue, its step is as follows:

(1) florfenicol amine and carrier protein couplet are obtained coating antigen;

(2) monoclonal antibody is prepared with the hybridoma cell strain TAP/5F4 that preserving number is CCTCCNO:C201549;

(3) use the coating antigen bag of step (1) by solid phase carrier;

(4) process of sample and detection.