CN105566493A - Monoclonal antibody and enzyme-linked immunosorbent assay method and kit for detecting florfenicol - Google Patents

Abstract

The invention discloses a specific monoclonal antibody of florfenicol. The monoclonal antibody is secreted by a hybridoma cell strain FF/7A8, the hybridoma cell strain FF/7A8 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO:C201548. The invention further discloses an enzyme-linked immunosorbent assay method and kit for detecting the florfenicol. Compared with the prior art, the prepared monoclonal antibody can specifically identify the florfenicol, and the enzyme-linked immunosorbent assay method and kit have the advantages of being high in detection sensitivity and accuracy, good in precision and the like.

Description

For detecting the monoclonal antibody of 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 florfenicol, and for the enzyme-linked immunoassay method (ELISA) that detects florfenicol and test kit.

Background technology

Florfenicol belongs to chloromycetin compound, is mainly used in treatment animal respiratory and intestinal tract infections.Florfenicol has certain embryotoxicity, and many countries such as China, the U.S., Japan and European Union and organizing all define its maximum residue limit in animal tissues.Although the method for current instrumental analysis can reach the testing requirement to florfenicol, expensive equipment, sample pre-treatments are loaded down with trivial details, also very high to operator's requirement.Immunochemical analyses method particularly enzyme-linked immunosorbent analytical technique has the advantages such as quick, highly sensitive, simple to operate, strong adaptability, and be applicable to the screening of high-throughput sample, compared with instrument detection method, ELISA method has more advantage.

Enzyme-linked immunosorbent assay carries out with the specific reaction principle of antigen-antibody the biotechnology that detects, because it is a kind of simple, highly sensitive, detection method that specificity is good, is therefore widely used.At present both at home and abroad the existing report about florfenicol preparation method for antibody: Luo (2009) establishes the residual method detecting florfenicol in pork and florfenicol amine, take florfenicol amine as haptens, adopt formaldehyde coupling method to carry out immunogen synthesis, obtain the polyclonal antibody that a strain can identify florfenicol and florfenicol amine.Sun Faliang etc. (2009) establish the method for detecting residue of florfenicol in the middle of chicken, with florfenicol succinyl oxide for haptens, by succinyl oxide method synthetic immunogen, obtain a strain polyclonal antibody, to florfenicol IC ₅₀be 79.3 μ g/L.Feng Caimao etc. (2012) take florfenicol amine as haptens, and adopt carbodlimide method complete antigen synthesis, the polyclonal antibody lowest detection of preparation is limited to 0.5 μ g/L, is 150% to florfenicol cross reacting rate.Zhu Airong etc. (2015) take florfenicol amine as haptens, by glutaraldehyde method synthetic antigen, finally obtain the polyclonal antibody that can identify florfenicol.Shen Jianzhong etc. (2008) apply for a kind ofly detecting the method for florfenicol and florfenicol amine and the patent of special ELISA reagent kit thereof, take florfenicol amine as haptens, by EDC method synthetic immunogen, finally obtain the polyclonal antibody that simultaneously can identify florfenicol and florfenicol amine.Lv Shiyuan bio tech ltd of Shenzhen has applied for a kind of patent of fluorobenzene niekau series medicament fast detecting reagent kit in 2009, wherein using florfenicol succinyl oxide as haptens, by mixed anhydride method synthetic immunogen, obtain can identify monoclonal antibody or the polyclonal antibody of florfenicol by immune mouse or new zealand white rabbit.Therefore, current bibliographical information mostly be polyclonal antibody, although polyclonal antibody sensitivity is higher, batch variation is large, does not have monoclonal antibody stable in properties.In addition, specificity and the sensitivity of existing mono-clonal or polyclonal antibody identification are lower, cannot identify florfenicol specifically, and the IC identified ₅₀too high.

Summary of the invention

Object of the present invention is:

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

(2) described monoclonal antibody is provided to detect the application in the enzyme linked immunological kit of florfenicol drug residue in preparation;

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

(4) application of described enzyme linked immunological kit in florfenicol drug residue non-diagnostic object detects is provided;

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

(6) application of a kind of described ELISA method in florfenicol drug residue non-diagnostic object detects is provided.

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

One can identify florfenicol monoclonal antibody, secreted by the hybridoma cell strain FF/7A8 that it is is CCTCCNO:C201548 by preserving number.

Described hybridoma cell strain FF/7A8, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201548.

Described monoclonal antibody is that the conjugate obtained using the florfenicol amine hemisuccinic acid ester (FFA-HS) obtained after florfenicol amine (FFA) and succinyl oxide (HS) reacts 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 florfenicol.

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

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

The present invention further provides a kind of non-diagnostic object enzyme-linked immune detection method of florfenicol drug residue, the method comprises the following steps:

(1) by florfenicol amine (FFA) and succinyl oxide (HS) reacted florfenicol amine hemisuccinic acid ester (FFA-HS) and carrier protein couplet, using the conjugate that obtains 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 FF/7A8 that preserving number is CCTCCNO:C201548;

(4) monoclonal antibody is prepared with the hybridoma cell strain FF/7A8 that preserving number is CCTCCNO:C201548;

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

(6) sample preparation 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, florfenicol amine and succinyl oxide is adopted to react, obtain florfenicol amine hemisuccinic acid ester, it can be used as haptens and with carrier protein couplet after as immunogen, the monoclonal antibody prepared by this immunogen can identify florfenicol specifically.

2, test kit of the present invention and enzyme-linked immune detection method can be used for detecting animal food as the florfenicol drug residue in pork, chicken, fish, pig liver and chicken liver, and detection sensitivity, accuracy are high, and precision is good, IC ₅₀value is only 0.21 ± 0.02 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 figure of monoclonal antibody of the present invention and florfenicol standard substance, X-axis is florfenicol (FF) concentration of standard solution logarithmic value, and Y-axis is that the optical density value of florfenicol 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.A liquid is slowly added drop-wise in B liquid, then adds EDC16mg and NHS12mg.Room temperature reaction spends the night.Reactant is loaded in dialysis tubing, to dialyse 5d with PBS, change dialyzate every day 3 times for 4 DEG C.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 FFA-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 (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-florfenicol drug antibody, applicant is by its called after FF/7A8, 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:C201548.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 102.8, 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 florfenicol 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 ₄.12H 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, 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L, 0.0625mg/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 15000,30000,60000,120000,240000,480000,960000,1920000 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 1:240000 or 1:120000.

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.Florfenicol phosphate buffered saline buffer is diluted to 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0.05 μ g/L, a 0 μ g/L6 concentration, the monoclonal antibody of being diluted by 1:240000 and 1:120000 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 florfenicol solution respectively add 50 μ L and carry out indirect competitive ELISA.Using florfenicol log concentration value as X-coordinate, with the ratio (B/B of the OD value of florfenicol standardized solution with " zero " hole OD value ₀) draw suppression curve as ordinate zou, florfenicol concentration (IC when select linear is better, generation 50% suppresses ₅₀) junior as bag by concentration.With best coating antigen concentration coated elisa plate, florfenicol is diluted to 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0.05 μ g/L, a 0 μ g/L6 concentration, antibody is arranged 4 extent of dilution with centre concentration 1:240000 equal difference, monoclonal antibody and series concentration florfenicol 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	160000	2.236	0.37
1	240000	2.147	0.23

Table 3 optimum antibody extent of dilution is optimized

Antibody dilution multiple	0 hole OD	IC 50(μg/
			230000	2.398	0.33
240000	2.133	0.21
			250000	1.995	0.31
260000	1.634	0.42

Result shows, wrapping by concentration is 1mg/L, when antibody dilution is 1:240000, and its IC ₅₀minimum.

The foundation of 3.4 typical curves

Florfenicol phosphate buffered saline is become 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0.05 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 florfenicol solution concentration for X-coordinate, B/B0 is ordinate zou drawing standard curve, obtains IC ₅₀.The IC of this test kit ₅₀value is 0.21 ± 0.02 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, the multiple hole of each medicine 3, with monoclonal antibody to the cross reacting rate of florfenicol 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 recognition rate to florfenicol, very low to other chloromycetin medicine recognition rate, shows that specificity is better.

Embodiment 4 fluoride protector of the present invention detects 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, florfenicol standardized solution, concentration is respectively 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0.05 μ g/L, 0 μ g/L;

3) preserving number is the monoclonal antibody of the hybridoma cell strain FF/7A8 secretion of CCTCCNO:C201548;

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 ₄12H ₂o29.0g, KCl2.0g, add distilled water to 1000mL;

6) concentrated cleaning solution: NaCl80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄12H 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 edible tissue 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 florfenicol 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 florfenicol concentration is that X-coordinate makes typical curve, line linearity of going forward side by side return, provide regression equation.

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, calculates florfenicol concentration in tissue (μ g/kg).

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 florfenicol standard substance is become 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0.05 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 florfenicol concentration, then calculate the mean value of florfenicol 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 0.21 ± 0.02 μ g/L, and the lowest detectable limit of florfenicol 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

Florfenicol standard substance are diluted to 0.8 μ g/L, 0.4 μ g/L, 0.2 μ g/L, 0.1 μ g/L, 0.05 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 florfenicol 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 florfenicol of three concentration is added to respectively in pork, chicken, the flesh of fish, pig liver and chicken liver sample, its TIANZHU XINGNAO Capsul is between 78.0% ~ 111.8%, 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 (9)

1. can identify a monoclonal antibody for florfenicol, it is characterized in that: secreted by the hybridoma cell strain FF/7A8 that it is is CCTCCNO:C201548 by preserving number.

2. the hybridoma cell strain FF/7A8 described in claim 1, is deposited in China typical culture collection center, and preserving number is CCTCCNO:C201548.

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 detects the application in the enzyme linked immunological kit of florfenicol in preparation.

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 florfenicol drug residue.

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

8. a non-diagnostic object enzyme-linked immune detection method for florfenicol drug residue, comprises the following steps:

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

(2) monoclonal antibody is prepared with the hybridoma cell strain FF/7A8 that preserving number is CCTCCNO:C201548;

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

(4) sample preparation and detection.

9. the application of enzyme-linked immunoassay method according to claim 8 in the non-diagnostic object of florfenicol drug residue detects.