CN103197069A - Enzyme linked immunosorbent assay (ELISA) kit for detecting sulfamethazine and enrofloxacin and application thereof - Google Patents
Enzyme linked immunosorbent assay (ELISA) kit for detecting sulfamethazine and enrofloxacin and application thereof Download PDFInfo
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Abstract
The present invention discloses an ELISA kit for detecting sulfamethazine and/or enrofloxacin and application thereof. The kit provided by the present invention includes an antibody 1, an antibody 2, a coating antigen 1, a coating antigen 2, an enzyme marker 1 and an enzyme marker 2. The antibody 1 is an anti-sulfamethazine antibody, the antibody 2 is an anti-enrofloxacin antibody, the coating antigen 1 is a conjugate of sulfamethazine hapten and a carrier protein, the coating antigen 2 is a conjugate of enrofloxacin hapten and a carrier protein, the enzyme marker 1 is an enzyme-labeled second antibody resisting the antibody 1, and the enzyme marker 2 is an enzyme-labeled second antibody resisting the antibody 2. The kit provided by the present invention can qualitatively or quantitatively detect sulfadimidine and enrofloxacin residue in a sample to-be-detected. Sample pretreatment process is simple. The detection method of the invention is simple, and high in specificity, sensitivity and accuracy.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit and application thereof that detects sulfamethazine and/or Enrofloxacin.
Background technology
Sulfamethazine is by artificial synthetic sulphanilamide derivant, is mainly used in prevention and treatment bacterial infection disease.Sulfamethazine can suppress gram-positive bacteria and some negative bacterium, can treat various bacteria and infect, and is widely used in treating the various livestock and poultries that infected by sensitive bacterial at veterinary clinic.
Because sulfamethazine effect and the metabolism time in vivo is longer, all might in human body, accumulate by the sulfanilamide (SN) that any approach is taken in.When accumulating concentration above certain value, will cause damage to human body.Short time, heavy dose of or long-time low dose of stimulation can cause acute respectively or slow poisoning, influence uropoiesis, the immune system of body, tissues such as destruction muscle, kidney and thyroid gland, as the thyroid cancer etc. of bringing out the people.In addition, long-term existence sulfanilamide (SN) can cause many bacteriums that sulfa drugs is developed immunity to drugs in the human body.Therefore, the international food code council (CAC) and many national regulations, the maximum residue limit of sulfamido total amount (MRL) is 0.1mg/kg in the food.
Enrofloxacin is afloqualone class antimicrobial, because of its antibiotic wide spectrum, antibacterial action is strong, oral absorption good, it is wide to distribute in the body, has now become one of most important anti-microbial type medicine in poultry industry and the culture fishery, is used for the treatment of in a large number, prevention and growth promotion.This product is the broad-spectrum sterilization medicine, and mycoplasma is had special efficacy.Escherichia coli, klebsiella bacillus, salmonella, proteus, Pseudomonas aeruginosa, haemophilus, pasteurella multocida, pasteurella haemolytica, golden Portugal bacterium, streptococcus etc. there is sterilization effectiveness.Enrofloxacin can be used as the animal-use drug product, long half time in animal body has the favorable tissue distributivity, belongs to the broad-spectrum bacteriostatic agent, have bacteriostasis for gram-positive bacteria, negative bacterium and mould slurry, once be used in the vibrios disease of cultured fishes and the control of Escherichia coli disease.Because its drug resistance and potential carcinogenicity, European Union and China have all formulated the maximum residue limit(MRL) (100 μ g/kg) in tissue.
At present, the chemical method that detects sulfamethazine and Enrofloxacin has high performance liquid chromatography and liquid chromatography-tandem mass spectrometry method, but because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.
Do not have at present commercial for the enzyme linked immunological kit that detects sulfamethazine and Enrofloxacin simultaneously as yet.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme linked immunological kit and application thereof that detects sulfamethazine and/or Enrofloxacin.
The enzyme linked immunological kit of detection sulfamethazine provided by the present invention and/or Enrofloxacin specifically can comprise antibody 1, antibody 2, coating antigen 1, coating antigen 2, enzyme labeling thing 1 and enzyme labeling thing 2; Described antibody 1 is the antibody of anti-sulfamethazine, described antibody 2 is the antibody of anti-Enrofloxacin, described coating antigen 1 is the conjugate of sulfamethazine haptens and carrier protein, described coating antigen 2 is the conjugate of Enrofloxacin haptens and carrier protein, described enzyme labeling thing 1 is the ELIAS secondary antibody of anti-described antibody 1, and described enzyme labeling thing 2 is the ELIAS secondary antibody of anti-described antibody 2.
In the present invention, described sulfamethazine haptens is specially sulfamethazine; Described Enrofloxacin haptens is specially Enrofloxacin.
Described enzyme linked immunological kit also comprises at least a in following: sulfamethazine standard solution, Enrofloxacin standard solution, substrate colour developing liquid, stop buffer, cleansing solution, bag are cushioned liquid, confining liquid and sample diluting liquid.
In one embodiment of the invention, described enzyme linked immunological kit specifically is cushioned liquid, described confining liquid and described sample diluting liquid and is formed by described antibody 1, described antibody 2, described coating antigen 1, described coating antigen 2, described enzyme labeling thing 1, described enzyme labeling thing 2, described sulfamethazine standard solution, described Enrofloxacin standard solution, described substrate colour developing liquid, described stop buffer, described cleansing solution, described bag.
In described enzyme linked immunological kit, the antibody of described anti-sulfamethazine can be the monoclonal antibody of anti-sulfamethazine, also can be the polyclonal antibody of anti-sulfamethazine.In one embodiment of the invention, the antibody of described anti-sulfamethazine is the monoclonal antibody of anti-sulfamethazine, is specifically produced by sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393.The antibody of described anti-Enrofloxacin can be the polyclonal antibody of anti-Enrofloxacin, also can be the monoclonal antibody of anti-Enrofloxacin.In one embodiment of the invention, the antibody of described anti-Enrofloxacin is the polyclonal antibody of anti-Enrofloxacin, specifically is that the conjugate with Enrofloxacin haptens and carrier protein obtains as immunogen immune vertebrate (as new zealand white rabbit).
In described enzyme linked immunological kit, described sulfamethazine standard solution and described Enrofloxacin standard solution can be the standard solution of the multiple concentration in the finite concentration scope, for example can between 0～100 μ g/L, be respectively 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L and 72.9 μ g/L as concentration.
In described enzyme linked immunological kit, described carrier protein can be human serum albumins (HSA), oralbumin (OVA), hemocyanin (KLH), bovine serum albumin(BSA) (BSA), mouse haemocyanin (MSA), thyroprotein (BCG) or rabbit anteserum albumen (RSA).In the present invention, be specially human serum albumins (HSA) with the carrier protein of described sulfamethazine hapten conjugation, be specially oralbumin (OVA) with the carrier protein of described Enrofloxacin hapten conjugation.
In described enzyme linked immunological kit, the marker enzyme of described enzyme labeling thing can be horseradish peroxidase, also can be alkaline phosphatase; Horseradish peroxidase can adopt several different methods of the prior art with it and two anti-carry out coupling, as glutaraldehyde method, and sodium periodate method etc.
When marker enzyme is horseradish peroxidase, described substrate colour developing liquid is made up of A liquid and B liquid, described A liquid can be urea peroxide solution or superoxol (specifically as volumn concentration be 2% urea peroxide solution), and described B liquid can be tetramethyl biphenyl amine aqueous solution or o-phenylenediamine solution (be as volumn concentration 1% tetramethyl biphenyl amine aqueous solution); Described stop buffer can be sulfuric acid solution, and its concentration can be 1～2mol/L(such as 2M).When marker enzyme was alkaline phosphatase, described substrate colour developing liquid was p-nitrophenyl phosphate damping fluid (concrete is the p-nitrophenyl phosphate solution of 0.1mg/mL as concentration).Colour developing liquid and stop buffer also can be this area other substrate colour developing liquid and stop buffers commonly used.
In described enzyme linked immunological kit, described cleansing solution is pH7.4, contain the phosphate buffer of the 0.02M of polysorbas20; The volume content of described polysorbas20 in described cleansing solution is 0.05%.
In described enzyme linked immunological kit, described bag is cushioned liquid and can be carbonate buffer solution, and is concrete as be sodium carbonate-sodium bicarbonate buffer liquid of pH9.6,0.05mol/L; The solvent of the sodium carbonate of described 0.05mol/L-sodium bicarbonate buffer liquid is water, and solute and concentration thereof are as follows: Na
2CO
31.59g/L and NaHCO
32.93g/L.
In described enzyme linked immunological kit, described confining liquid is phosphate buffer; In one embodiment of the invention, described confining liquid is specially pH7.4, contains calf serum, sucrose and caseic phosphate buffer; The volume content of described calf serum in described confining liquid is 0.5%, the content of described sucrose in described confining liquid is to contain the described sucrose of 5g in the described confining liquid of every 100ml, and the content of described casein in described confining liquid is to contain the described casein of 1g in the described confining liquid of every 100ml.
Certain described bag is cushioned liquid and described confining liquid also can be that this area other bag commonly used is cushioned liquid and confining liquid.
In described enzyme linked immunological kit, described sample diluting liquid is specially the phosphate buffer (pH7.4) of 0.02M.
More than the solvent of phosphate buffer of all described 0.02M be water, solute and concentration thereof are all as follows: NaCl 16.00g/L, KCl 0.40g/L, Na
2HPO
42.30g/L and KH
2PO
40.40g/L.
Another object of the present invention provides the preparation method of described enzyme linked immunological kit.
The preparation method of described enzyme linked immunological kit provided by the present invention specifically comprises the step of following (1) or (2):
(1) step that described antibody 1, described antibody 2, described coating antigen 1, described coating antigen 2, described enzyme labeling thing 1 and described enzyme labeling thing 2 are packed separately respectively;
(2) described antibody 1, described antibody 2, described coating antigen 1, described coating antigen 2, described enzyme labeling thing 1, described enzyme labeling thing 2, described sulfamethazine standard solution, described Enrofloxacin standard solution, described substrate colour developing liquid, described stop buffer, described cleansing solution, described bag are cushioned the step that liquid and described confining liquid are packed separately respectively.
Also purpose of the present invention provides the method that whether contains sulfamethazine and/or Enrofloxacin in a kind of detection or the auxiliary detection testing sample.
The method that whether contains sulfamethazine and/or Enrofloxacin in detection provided by the present invention or the auxiliary detection testing sample specifically is to utilize in described enzyme linked immunological kit detection or the auxiliary detection testing sample whether contain sulfamethazine and/or Enrofloxacin.
In described method, the coating antigen in the described enzyme linked immunological kit is the potpourri of described coating antigen 1 and described coating antigen 2; Antibody in the described enzyme linked immunological kit is the potpourri of described antibody 1 and described antibody 2; Enzyme labeling thing in the described enzyme linked immunological kit is the potpourri of described enzyme labeling thing 1 and described enzyme labeling thing 2.
More concrete, when described method is used for quantitatively detecting, can comprise the steps:
(1) pre-treatment of testing sample: described testing sample is milk sample; With sample diluting liquid (phosphate buffer of 0.02mol/L) to described milk sample according to volume ratio 1:10(1+9) dilution, for example 100 μ L milk samples add the sample diluting liquid of 900 μ L, to handle the back sample.
(2) with described enzyme linked immunological kit to the processing of step (1) after sample detect;
(3) analyzing and testing result:
With the absorbance mean value (B) of the standard solution of each concentration that the obtains absorbance (B divided by first standard solution (standard items concentration is 0 μ g/L)
0) multiply by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Concentration (μ g/L) with described sulfadimidine standard solution and described Enrofloxacin standard solution is X-axis respectively, and the percentage absorbance is Y-axis, the drawing standard curve map.With the percentage absorbance of sample solution after the processing of same way calculation procedure (1), the concentration of corresponding each processing back sample then can be read sulfadimidine described in the sample and described contents of enrofloxacin from typical curve.Also can adopt regression equation method, calculate sample solution concentration.Can also utilize computer professional software, the be more convenient for express-analysis of a large amount of samples of this method, whole testing process only needs the short period, namely can finish in the 1.5h.
Also purpose of the present invention provides a kind of hybridoma cell strain.
Hybridoma cell strain provided by the present invention is specially sulfamido monoclonal antibody hybridoma cell strain SAs, and its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.3393.
The monoclonal antibody that is produced by described sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393 also belongs to protection scope of the present invention.
Compared with prior art, the present invention has following beneficial effect:
Easy, the easy row of the inventive method, immune effect is good.The invention provides the enzyme linked immunosorbent detection pattern, can qualitative or quantitatively detect the residual of sulfamethazine and Enrofloxacin in the milk, sample pretreatment process is simple.In addition, agents useful for same of the present invention adopts independent packaging, and the method for inspection is simple and easy to do, has characteristics such as specificity height, highly sensitive, degree of accuracy height, will play a significant role in the detection of sulfamethazine and Enrofloxacin.
The preservation explanation
The biomaterial (strain) of ginseng a tree name: SAs
Scientific description: sulfamido monoclonal antibody hybridoma cell strain
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on November 3rd, 2009
The preservation center numbering of registering on the books: CGMCC No.3393
Description of drawings
Fig. 1 is the canonical plotting of sulfadimidine and Enrofloxacin.Wherein, SM2 is the typical curve that adopts the result of horseradish peroxidase chromogenic assay, and ENR is the result's of alkaline phosphatase assay typical curve.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Sulfamethazine standard items: German Dr.Ehrenstorfer GmbH company, catalog number C16990580;
Enrofloxacin standard items: German Dr.Ehrenstorfer GmbH company, catalog number C13170000;
Sulfamethazine analog (daimeton, sulfisomidine, the sulfanilamide (SN) quinoline is disliked beautiful jade, sulfadimethoxine, cistosulfa, 5-methoxysulfadiazine, sulfapryidine, sulfadimethoxine, sulfamethoxypyridazine, sulfadoxine, sulfacetamide, sulfamethyldiazine, NU-445, sulfanitran, Sulfamethoxazole, sulphadiazine, sulphathiazole sulfamoxole, ayerlucil and sulfabenzamide) standard items, Enrofloxacin analog (oxolinic acid, Enoxacin, flumequine, Lomefloxacin, Danofloxacin, Norfloxacin, Pefloxacin, Enrofloxacin, Orbifloxacin, Ofloxacin, marbofloxacin and Sparfloxacin) standard items: German Dr.Ehrenstorfer GmbH company;
Balb/c mouse: Beijing Vital River Experimental Animals Technology Co., Ltd.
New zealand white rabbit: Beijing Vital River Experimental Animals Technology Co., Ltd.
Freund's complete adjuvant, incomplete Freund: U.S. Sigma company, catalog number is respectively F-5881 and F-5506.
SP2/0 myeloma cell: Beijing Xin Xingtang bio tech ltd.
The detection principle of related enzyme linked immunological kit is the indirect competition method among the following embodiment: when after wrapping by sulfamethazine and Enrofloxacin hapten-carrier protein-coupled antigen on the capillary strip in advance, after adding testing sample solution or sulfamethazine and Enrofloxacin standard solution, add sulfamethazine and Enrofloxacin specific antibody solution again, the coupled antigen competition specific antibody of bag quilt on residual medicine (sulfamethazine or Enrofloxacin) or sulfamethazine and Enrofloxacin standard items and the ELISA Plate in the testing sample, add ELIAS secondary antibody and carry out amplification, with the colour developing of colour developing liquid, the sample light absorption value becomes negative correlation with the content of sample Chinese traditional medicine (sulfamethazine or Enrofloxacin), relatively can draw the content of sample Chinese traditional medicine (sulfamethazine or Enrofloxacin) with typical curve.Simultaneously also can be according to shade on the ELISA Plate, compare with the standard solution color of series concentration, thus the concentration range of rough judgement testing sample Chinese traditional medicine (sulfamethazine or Enrofloxacin).
Preparation and the application thereof of the enzyme linked immunological kit of embodiment 1, detection sulfamethazine and/or Enrofloxacin
One, detects the preparation of the enzyme linked immunological kit of sulfamethazine and/or Enrofloxacin
The enzyme linked immunological kit that detects sulfamethazine and/or Enrofloxacin comprises:
(1) pre-coated elisa plate: the ELISA Plate that is coated with coating antigen (conjugate of sulfamethazine and carrier protein, and the conjugate of Enrofloxacin and carrier protein);
(2) primary antibodie: the antibody working fluid of anti-sulfamethazine, and the antibody working fluid of anti-Enrofloxacin, it is 0.1 μ g/mL that the antibody that is about to the antibody of anti-sulfamethazine and anti-Enrofloxacin uses antibody diluent (the 0.02M phosphate buffer that contains the Proclin300 of 0.05% Triton X-100 and 0.02%) to be diluted to its protein concentration respectively.
(3) ELIAS secondary antibody: with the sheep anti mouse two anti-working fluids of alkali phosphatase enzyme mark, and the goat-anti rabbit two anti-working fluids of horseradish peroxidase-labeled, being about to the goat-anti rabbit two of the anti-and horseradish peroxidase-labeled of the sheep anti mouse two of alkali phosphatase enzyme mark, anti-to use enzyme labelled antibody dilution (the 0.02M phosphate buffer that contains the hyclone of 0.02% Proclin300 and 5%) to be diluted to its protein concentration respectively be 0.06 μ g/mL.
(4) sulfamethazine standard solution, and Enrofloxacin standard solution: respectively sulfamethazine standard items and Enrofloxacin standard items respectively are diluted to 10 bottles of standard solution with the 0.02M phosphate buffer, concentration is respectively 0 μ g/L, 0.01 μ g/L, 0.05 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L and 72.9 μ g/L;
(5) p-nitrophenyl phosphate (PNPP) solution of alkaline phosphatase substrate colour developing liquid: 0.1mg/mL.
(6) horseradish peroxidase substrate colour developing liquid: be made up of A liquid and B liquid, A liquid is the 2%(volumn concentration) the urea peroxide aqueous solution, B liquid is the 1%(volumn concentration) the tetramethyl biphenyl amine aqueous solution;
(7) HRP colour developing stop buffer: 2mol/L sulfuric acid; (2mol/L sulfuric acid is the stop buffer of corresponding HRP, and corresponding alkaline phosphatase does not need stop buffer)
(8) cleansing solution: pH7.4, contain the 0.05%(volumn concentration) the 0.02M phosphate buffer of polysorbas20;
(9) phosphate buffer of sample diluting liquid: 0.02M (pH7.4).
The solvent of the phosphate buffer of above 0.02M is water, and solute and concentration thereof are all as follows: NaCl 16.00g/L, KCl 0.40g/L, Na
2HPO
42.30g/L and KH
2PO
40.40g/L.
Wherein, the preparation method of pre-coated elisa plate, primary antibodie, ELIAS secondary antibody is specific as follows:
(1) preparation of pre-coated elisa plate
1, the preparation of the conjugate of sulfamethazine and carrier protein
Employing diazotising legal system is equipped with the conjugate of sulfamethazine and carrier protein, and is specific as follows:
(1) 104mg sulfamethazine (SM
2), add 8mL0.25mol L
-1Sulfuric acid in, place 4 ℃ of refrigerators, form the solution I;
(2) human serum albumins of 200mg (HSA) is dissolved in the Na of 8mL
2CO
3In the aqueous solution (pH=10), place 4 ℃ of refrigerators, form the solution II;
(3) 38mg NaNO
2, add the 2mL pure water, form NaNO
2Aqueous solution;
(4) with NaNO
2Aqueous solution slowly joins in the solution I, in this process, with SM
2Place trash ice, general 15min forms SM
2-NaNO
2
(5) with SM
2-NaNO
2Slowly join in the solution II, and shake, the red generation arranged this moment; Behind the 6min, carry out magnetic agitation (room temperature stirs at a slow speed), continue slowly to add SM
2-NaNO
2In whipping process, detect the pH value, make it to remain between the 9-10 and (regulate with 1M NaOH), final solution becomes kermesinus liquid; Room temperature continues to stir 4h; Reacted solution is packed in the bag filter, and normal saline dialysis was changed a dislysate every 12 hours, dialysed continuously three days, obtained the conjugate (SM of sulfamethazine and carrier protein
2-HAS).
2, the preparation of the conjugate of Enrofloxacin and carrier protein
The employing active ester method prepares the conjugate of Enrofloxacin and carrier protein, and is specific as follows:
(1) 15mg Enrofloxacin (ENR) is dissolved in 5mL N, and in the N dimethyl formamide (DMF), ultrasonic making it dissolved fully, and cools off in precooled ethanol, obtains ENR solution;
(2) 20mg carbodiimides (EDC) and 20mg N-hydroxy-succinamide (NHS) join in the above-mentioned ENR solution, and room temperature reaction spends the night;
(3) 15mg oralbumin (OVA) is dissolved in (pH8.0) in the 5mL carbon acid solution, dropwise joins in above-mentioned steps (2) the gained solution, continues to stir 4h; The bag filter of then reactant liquor being packed into, 4 ℃ with normal saline solution dialysis 48 hours, change water 6 times.With dislysate filter membrane by 0.2 μ m under aseptic condition, obtain the Enrofloxacin of purifying and the conjugate of carrier protein (ENR-OVA), be sub-packed in the ampere bottle-20 ℃ of preservations.
3, coated elisa plate
Bag is cushioned sodium carbonate-sodium bicarbonate buffer liquid (pH9.6) of liquid: 0.05mol/L, and solvent is water, and solute and concentration thereof are as follows: Na
2CO
31.59g/L and NaHCO
32.93g/L.
Cleansing solution: contain the 0.05%(volumn concentration) the 0.02M phosphate buffer of polysorbas20, pH7.4.
Confining liquid: contain the 0.5%(volumn concentration) calf serum, 5%(5g/100ml) sucrose, 1%(1g/100ml) phosphate buffer of caseic 0.02M, pH7.4.Wherein, the solvent of the phosphate buffer of 0.02M is water, and solute and concentration thereof are all as follows: NaCl 16.00g/L, KCl 0.40g/L, Na
2HPO
42.30g/L and KH
2PO
40.40g/L.
Be cushioned the conjugate SM that liquid prepares step 1 and step 2 respectively with bag
2-HAS and ENR-OVA are diluted to 1 μ g/mL, every hole adds 100 μ L, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the bag that inclines is cushioned liquid, with ELISA Plate with cleansing solution after washing 3 times, each 30s, pat dry, in every hole, add 200 μ L confining liquids, 37 ℃ of incubation 2h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
(2) preparation of primary antibodie
1, the preparation of sulfamethazine specific antibody (monoclonal antibody)
(1) the immunogenic preparation of sulfamethazine
Preparation process is with the preparation process of the conjugate of above-mentioned sulfamethazine and carrier protein, the SM behind the final purifying that obtains
2-HAS is the sulfamethazine immunogene.
(2) animal immune
The sulfamethazine immunogene that step (1) is obtained is injected in the Balb/c mouse body, and immunizing dose is 75 μ g//times.Immunization ways is the subcutaneous multi-point injection in back; The immunity time interval is 15 days; Immune time is 7-8 time.
(3) Fusion of Cells and cloning
Immune Balb/c mouse boosting cell is got in back 10 days of last immunity, merges in 5:1 ratio and SP2/0 myeloma cell, adopts indirect ELISA to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains a strain stably excreting sulfamethazine monoclonal antibody-sulfamido monoclonal hybridoma strain, called after SAs.This hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 3rd, 2009 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCCNo.3393.
Wherein, the indirect elisa method operation steps is specific as follows:
1) bag quilt: the SM that in 96 hole ELISA Plate, adds the 1 μ g/mL of 100 μ L
2-HAS solution (being diluted by dilution with the same bag of prescription) arranges the not contrast of envelope antigen simultaneously, and 4 ℃ of bags are spent the night, with PBS damping fluid washing 3 times.
2) sealing: add the confining liquid (it is same as above to fill a prescription) in 150 μ L/ holes, hatch 2h at 37 ℃, abandon confining liquid, wash 3 times, pat dry.It is standby to place 4 ℃ of refrigerators to preserve.
3) add testing sample: draw cell conditioned medium 100 μ l, add in the corresponding ELISA Plate, hatch 30min for 37 ℃, wash plate 4 times, pat dry.
Contrast without the mice immunized cell conditioned medium is set simultaneously; Replace the contrast (negative control hole) of detected sample with PBS.
4) add ELIAS secondary antibody: (Jackson Immunosearch company, products catalogue: number 115-475-003), after 1:5000 doubly diluted by volume, 30min was hatched for 37 ℃ in 100 μ l/ holes, washed 4 times, patted dry to get HRP mark goat anti-mouse igg antibody.
5) colour developing: horseradish peroxidase substrate colour developing liquid is added (each 50 μ l of A liquid and B liquid use behind the mixing) by 100 μ l/ holes, 37 ℃ of colour developing 15-30min.
6) stop: add stop buffer (2M H
2SO
4) 50 μ l/ holes.
7) reading: measure each hole OD value with the single wavelength of 450nm, be limited greater than 2.1 with the ratio (P/N) with negative control hole (replacing the contrast of testing sample with PBS) OD value, as being judged as positive critical point.
ELISA is decision method as a result: if P/N〉2.1, then differentiate positive cell; If 1＜P/N＜2.1, then strengthen bag and detected P/N after the concentration again〉2.1 still be judged to the positive.
(4) cell cryopreservation and recovery
The sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393 that step (3) is obtained makes 1 * 10 with cryopreserving liquid
6The cell suspension of individual/mL is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
(5) sulfamethazine MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
The preparation method can be following two kinds:
A increment cultivation
Sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393 is placed the DEME nutrient culture media, cultivated the collecting cell supernatant 3 days for 37 ℃.
The preparation of B ascites
The Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.4mL/, 7 days assorted sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393 cells 5 * 10 of pneumoretroperitoneum injection
5Individual/as only, to gather ascites after 7 days.
Collecting cell supernatant or ascites, adopt indirect elisa method to measure it and tire that (assay method is same as above, when mensuration is tired with P/N 2.1 cell conditioned medium or ascites maximum dilution multiple represent), the result shows that tiring of cell conditioned medium is 1:10000, tiring of ascites is 1:50000.Then, with sad-saturated ammonium sulfate method it is carried out purifying, put into-20 ℃ of environment behind the purifying and preserve.
2, the preparation of the antibody of anti-Enrofloxacin (how anti-)
(1) the immunogenic preparation of Enrofloxacin
Preparation process is with the preparation process of the conjugate of above-mentioned Enrofloxacin and carrier protein, and the ENR-OVA behind the final purifying that obtains is the Enrofloxacin immunogene.
(2) animal immune
With the Enrofloxacin immunogen immune new zealand white rabbit that step (1) obtains, immunizing dose be the 1.5mg/kg body weight/time, immunization ways is the subcutaneous multi-point injection of nape portion.When head exempts from immunogene and isopyknic Freund's complete adjuvant are mixed and made into emulsifying agent, get in 3～4 weeks of every interval the same dose immunogene add isopyknic incomplete Freund mixing and emulsifying after booster immunization once, after adopting this mode to add altogether to exempt from 3 times, get the same dose immunogene at interval 3～4 weeks again and do not add adjuvant and carry out the last immunity.The last immunity is the heart blood sampling after 10 days, adopts indirect elisa method to measure serum antibody titer (assay method is same as above), and the result shows that serum titer is 1:10000.Then, obtain the Enrofloxacin polyclonal antibody of purifying with ammonium sulfate precipitation.
(3) preparation of ELIAS secondary antibody
1, the anti-preparation of the sheep anti mouse two of alkali phosphatase enzyme mark
The acquisition that sheep anti mouse two resists: adopt the pathogen-free domestic goat as immune animal, to be that 1mg/kg carries out immunity according to immunizing dose as immunogenic mouse endogenous antibody (Beijing Wdwkbio Biotechnology Co., Ltd), when head exempts from immunogene and isopyknic Freund's complete adjuvant are mixed and made into emulsifying agent, get in 3～4 weeks of every interval the same dose immunogene add isopyknic incomplete Freund mixing and emulsifying after booster immunization once, after adopting this mode to add altogether to exempt from 3 times, get the same dose immunogene at interval 3～4 weeks again and do not add adjuvant and carry out the last immunity.The last immunity is the heart blood sampling after 10 days, adopts indirect elisa method to measure serum antibody titer (assay method is same as above, and coating antigen is non-immune with mouse source antibody), obtains the goat anti-mouse antibody of purifying with ammonium sulfate precipitation.
Sheep anti mouse two resists the coupling with alkaline phosphatase: with alkaline phosphatase (the Sigma company of 10mg, catalog number SIGMA-P7640) pH that is dissolved in 2mL is acetic acid-sodium acetate buffer solution (0.2M sodium acetate of 0.2M acetic acid+19.5ml of 30.5ml of 5.0,200 times of uses of dilute with water before using) in, add 1.0% glutaraldehyde water solution 0.1mL in this solution; According to enzyme: it is anti-that the mass ratio of antibody is that the ratio of 1:2 adds sheep anti mouse to be marked two, stirring at room reaction 4 hours.According to NaBH4: the alkaline phosphatase mass ratio is that the ratio of 1:10 adds NaBH4, stirring at room reaction 4 hours.The sheep anti mouse two of purification storage gained alkali phosphatase enzyme mark is anti-.
2, the anti-preparation of the goat-anti rabbit two of horseradish peroxidase-labeled
Goat-anti rabbit two is anti-: the preparation that the preparation method resists with sheep anti mouse in the step 1 two, difference is to adopt rabbit endogenous antibody (Beijing Wdwkbio Biotechnology Co., Ltd) as immunogene.
Adopt the sodium periodate method that improves that goat-anti rabbit two is resisted with horseradish peroxidase (HRP) and carry out coupling, specific as follows: 8mg horseradish peroxidase (Sigma company, catalog number: SU3472) be dissolved in the 2mL distilled water.The 100mmol/L NaIO that adds existing preparation
4Aqueous solution 0.4mL, stirring at room reaction 20min.In 4 ℃ of dialysed overnight, remove unnecessary NaIO with 1mmol/L pH=5.0 acetic acid-sodium acetate buffer solution (the 0.2M sodium acetate of 0.2M acetic acid+19.5ml of 30.5ml, 200 times of uses of dilute with water before using)
4, make the enzyme reduction of self coupling simultaneously.Add phosphate buffer (pH8.6,0.5mol/L) 40 μ L and phosphate buffer (pH8.6, the 5mol/L) 2.0mL that contains goat-anti rabbit two anti-IgG16mg, stirring at room reaction 4h.The NaBH that adds existing preparation
4Aqueous solution (1mol/L) 0.1mL is at 4 ℃ of reaction 4h, with reduction Schiff alkali.The goat-anti rabbit two of purification storage gained horseradish peroxidase-labeled is anti-.
Two, utilize the kit of step 1 to detect sulfamethazine and/or Enrofloxacin
1, sample pre-treatments
Milk sample: with sample diluting liquid (phosphate buffer of 0.02M) to milk sample according to volume ratio 1:10(1+9) dilute; For example, 100 μ L milk samples add the sample diluting liquid of 900 μ L.Get sample on the 50 μ L.
2, the kit with step 1 detects
(1) in the micropore of the pre-coated elisa plate of the conjugate potpourri of the conjugate that is coated with sulfamethazine and carrier protein and Enrofloxacin and carrier protein, adds standard solution (sulfamethazine standard solution or Enrofloxacin standard solution) or handle back sample solution 50 μ L, add two kinds of primary antibodies (sulfamethazine monoclonal antibody and the Enrofloxacin polyclonal antibody of preparation in the step 1 (two)) each 50 μ L of working fluid again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens;
(2) washing: pour out liquid in the hole, every hole adds 250 μ L cleansing solutions, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper;
(3) add ELIAS secondary antibody (the sheep anti mouse two goat-anti rabbits two anti-and horseradish peroxidase-labeled of the alkali phosphatase enzyme mark of preparation are anti-in the step 1 (three)) working fluid 100 μ L, react 30min in 37 ℃ of constant temperature ovens;
(4) pour out liquid in the hole, repeat as the described washing step of step (2);
(5) alkaline phosphatase colour developing: every hole adds alkaline phosphatase substrate colour developing liquid 100 μ L, the mixing that vibrates gently, and 37 ℃ of constant temperature oven lucifuge colour developing 15min use microplate reader, measure every hole absorbance (OD value), and the detection wavelength is 405nm.
(6) washing: pour out liquid in the hole, every hole adds 250 μ L cleansing solutions, pours out liquid in the hole behind the 30s, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper;
(7) every hole adds HRP substrate colour developing liquid A liquid urea peroxide, substrate colour developing liquid B liquid tetramethyl benzidine (TMB), wherein the volume ratio of A liquid and B liquid is each 50 μ L of 1:1(); The mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuges colour developing 15min, every hole adds stop buffer 2mol/L sulfuric acid 50 μ L, and the mixing that vibrates is gently used microplate reader, measures every hole absorbance (OD value), and the detection wavelength is 450nm.
3, testing result analysis
The absorbance mean value (B) of the standard solution of each concentration of gained is divided by the absorbance mean value (B of first standard solution (0 μ g/L standard solution)
0) multiply by 100% again, obtain the percentage absorbance.
Percentage absorbance (%)=(B/B
0) * 100%
Concentration (μ g/L) with described sulfadimidine standard solution and described Enrofloxacin standard solution is X-axis respectively, and the percentage absorbance is Y-axis, the drawing standard curve map, as shown in Figure 1.With the percentage absorbance of sample solution after the processing of same way calculation procedure 1, the concentration of corresponding each processing back sample then can be read sulfadimidine described in the sample and described contents of enrofloxacin from typical curve.Also can adopt regression equation method, calculate sample solution concentration.Can also utilize computer professional software, the be more convenient for express-analysis of a large amount of samples of this method, whole testing process only needs the short period, namely can finish in the 1.5h.
The enzyme linked immunological kit precision of embodiment 2, embodiment 1 preparation, accuracy, specificity and storage life experiment
Present embodiment will carry out the detection of precision, accuracy, specificity and storage life to the enzyme linked immunological kit of embodiment 1 preparation, and the using method of related kit, interpretation of result etc. are referring to embodiment 1 step 2 correlation step therebetween.
One, kit precision experiment
1, standard items precision test
From the enzyme linked immunological kit of embodiment 1 preparation, get three batches and carry out the precision detection, every batch is extracted 10 kits, every plate is extracted 20 micropores out, measure the 20 μ g/L standard solution (mixed solutions of sulfadimidine standard items and Enrofloxacin standard items, both concentration are 20 μ g/L) absorbance, calculate the coefficient of variation (CV%), the results are shown in Table 1.
Table 1 standard items Precision test result (CV%)
Can draw by the test findings in the table 1, every batch of kit, each measures 10 standard items coefficient of variation between 5.5%～16.3%, meet precision less than 20%, meet " Ministry of Agriculture's file " farming doctor and send out the precision standard that [2005] No. 17 annex 2 kits are put on record and stipulated with reference in the 4th precision in the judgment criteria and the accuracy.
(2) testing sample precision test
Get sulfadimidine standard items and Enrofloxacin standard items respectively, be added in the milk sample, be 10 μ g/L to the final concentration of two standard items.Get each three of the kits of three different batches of embodiment 1 preparation respectively, each kit repeats 5 times, calculates the coefficient of variation respectively.The testing result of three batches of kits sees Table 2.
The Precision test result of table 2 milk sample
The result shows, three batches of kits add sample to milk, the plate within variance coefficient is less than 10%, the variation within batch coefficient is less than 15%, interassay coefficient of variation is less than 20%, meets " Ministry of Agriculture's file " farming doctor and sends out the precision standard that [2005] No. 17 annex 2 kits are put on record and stipulated with reference in the 4th precision in the judgment criteria and the accuracy.
Two, kit accuracy experiment
Get sulfadimidine standard items and Enrofloxacin standard items, respectively the milk sample that does not contain sulfadimidine and Enrofloxacin is added recovery test, two standard items are all respectively established two and are added concentration (10 μ g/L and 20 μ g/L), each concentration is done 5 repetitions, accuracy in computation the results are shown in Table 3 respectively.
The sample accuracy determination of table 3 kit
Can find out that from table 3 the interpolation recovery of milk sample is between 76.1%～97.9%; The bioassay standard that meets accuracy.
Three, kit sensitivity experiment
Have multiplely about the index of the sensitivity of ELISA, generally adopt by 50% inhibition concentration (IC
50) sensitivity of the ELISA that sets up of value evaluation, wherein IC
50Value is generally calculated by four parametric equations.General procedure is as follows: the OD value of the standard solution of the zero-dose when detecting the inhibition of thing standard items not have is B
0Value, the OD value during respective concentration standard solution inhibitory reaction is the B value.Being ordinate with the OD value, is horizontal ordinate with the logarithm of the standard solution concentration of serial gradient concentration, and the drawing standard curve can draw the linear equation of typical curve simultaneously.By describing absorptivity the logarithm of analyte concentration is obtained competition curve, meets logstic four parametric equations:
Here the maximum light absorption value of A when not having analyte, B is the slope of curve deformation point, C is the concentration (IC of inhibition analysis thing 50%
50), D is the minimum light absorption value when analyte is infinitely great.
With the absorbance mean value (B) of the standard solution of each concentration that the obtains absorbance (B divided by first standard solution (standard items concentration is 0 μ g/L)
0) multiply by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
The sensitivity of kit adopts the standard solution with sulfamethazine, Enrofloxacin and each analog to pass through standard curve determination 50% inhibition concentration IC separately
50Estimate.Under above-mentioned top condition, it is a series of gradient concentration that standard items are made into concentration, presses the sensitivity that the indirect competitive ELISA method is measured curve.Concentration (μ g/L) with described sulfadimidine standard solution and described Enrofloxacin standard solution is X-axis respectively, and the percentage absorbance is Y-axis, the drawing standard curve map.With the percentage absorbance of sample solution after the processing of same way calculation procedure (1), the concentration of corresponding each processing back sample then can be read sulfadimidine described in the sample and described contents of enrofloxacin from typical curve.Also can adopt regression equation method, calculate sample solution concentration.Can also utilize computer professional software, the be more convenient for express-analysis of a large amount of samples of this method, whole testing process only needs the short period, namely can finish in the 1.5h.
According to the typical curve among Fig. 1, calculate 50% inhibition concentration (IC of sulfamethazine according to the method described above
50) be 0.48ng/mL, 50% inhibition concentration (IC of Ciprofloxacin
50) be 0.72ng/mL.
Four, kit specificity experiment
Select to have with sulfamethazine 20 kinds of medicines of similar structures and similar functions, and with Enrofloxacin the cross reacting rate of 12 kinds of drug monitoring kits of similar structures and similar functions is arranged.Standard solution with sulfamethazine, Enrofloxacin and each analog passes through standard curve determination 50% inhibition concentration (IC separately
50), calculate kit to the cross reacting rate of each analog with following formula.The cross reacting rate of analog is more little, illustrates that kit is more high to the specificity of sulfamethazine and Enrofloxacin detection.
Wherein, the method by standard curve determination 50% inhibition concentration is as follows:
(1) typical curve is drawn: according to the described method operation of embodiment 1 step 2, respectively with the absorbance mean value (B) of the standard solution of sulfamethazine, Enrofloxacin and each analog divided by the absorbance mean value (B of corresponding first standard solution (0 μ g/L standard solution) separately
0) multiply by 100% again, obtain the percentage absorbance.Be ordinate with the percentage absorbance, with the standard items concentration (ng/mL) in each standard solution as horizontal ordinate drawing standard curve, drawing standard curve.
Determining of (2) 50% inhibition concentrations: by typical curve, obtain each material concentration (ng/mL) that Y value equals 50% correspondence, i.e. IC
50Value.
Testing result is as shown in table 4.As can be seen from Table 4, the kit of embodiment 1 preparation to the cross reacting rate of sulfamethazine analog and Enrofloxacin analog all less than 1%, the kit of this explanation embodiment 1 preparation has high specificity to sulfamethazine and Enrofloxacin, satisfy the confessed cross reacting rate of those skilled in the art and think to have than this saying of high specific (notice-2003 of The Ministry of Agriculture of the People's Republic of China, MOA's residue of veterinary drug analytical technology standard) less than 10%, the interference of analog can be effectively got rid of, the detection of these two kinds of materials of sulfamethazine and Enrofloxacin can be specifically designed to.
The specificity of table 4 kit
| The sulfa drugs title | Cross reacting rate (%) | The QNS title | Cross reacting rate (%) |
| Sulfamethazine | 100 | Ciprofloxacin | 100 |
| Daimeton | <1 | Oxolinic acid | <1 |
| Sulfisomidine | <1 | Enoxacin | <1 |
| The sulfanilamide (SN) quinoline is disliked beautiful jade | <1 | Flumequine | <1 |
| Sulfadimethoxine | <1 | Lomefloxacin | <1 |
| Cistosulfa | <1 | Danofloxacin | <1 |
| 5-methoxysulfadiazine | <1 | Norfloxacin | <1 |
| Sulfapryidine | <1 | Pefloxacin | <1 |
| Sulfadimethoxine | <1 | Enrofloxacin | <1 |
| Sulfamethoxypyridazine | <1 | Orbifloxacin | <1 |
| Sulfadoxine | <1 | Ofloxacin | <1 |
| Sulfacetamide | <1 | Marbofloxacin | <1 |
| Sulfamethyldiazine | <1 | Sparfloxacin | <1 |
| NU-445 | <1 | ? | ? |
| Sulfanitran | <1 | ? | ? |
| Sulfamethoxazole | <1 | ? | ? |
| Sulphadiazine | <1 | ? | ? |
| Sulphathiazole | <1 | ? | ? |
| Sulfamoxole | <1 | ? | ? |
| Ayerlucil | <1 | ? | ? |
| Sulfabenzamide | <1 | ? | ? |
Five, detectability determines
Measure 20 parts of blank milk samples, the mean value of the measurement result of 20 portions of blank milk is added the detectability of 3 times of these kits of standard deviation calculation.Wherein the detection of Enrofloxacin is limited to 4.6ng/mL, and the detection of sulfamethazine is limited to 5.2ng/mL.
Six, kit storage life experiment
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, sulfamethazine and Enrofloxacin added the practical measurement value all within normal range.Consider in transportation and the use, have improper preservation condition and occur, kit was placed 6 days that carry out the accelerated deterioration experiment, the result shows that every index of this kit meets the requirements fully under the condition of 37 ℃ of preservations.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at least at 2-8 ℃.
Claims (10)
1. an enzyme linked immunological kit that detects sulfamethazine and/or Enrofloxacin comprises antibody 1, antibody 2, coating antigen 1, coating antigen 2, enzyme labeling thing 1 and enzyme labeling thing 2; Described antibody 1 is the antibody of anti-sulfamethazine, described antibody 2 is the antibody of anti-Enrofloxacin, described coating antigen 1 is the conjugate of sulfamethazine haptens and carrier protein, described coating antigen 2 is the conjugate of Enrofloxacin haptens and carrier protein, described enzyme labeling thing 1 is the ELIAS secondary antibody of anti-described antibody 1, and described enzyme labeling thing 2 is the ELIAS secondary antibody of anti-described antibody 2.
2. enzyme linked immunological kit according to claim 1 is characterized in that: described enzyme linked immunological kit also comprises at least a in following: sulfamethazine standard solution, Enrofloxacin standard solution, substrate colour developing liquid, stop buffer, cleansing solution, bag are cushioned liquid, confining liquid and sample diluting liquid.
3. enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: the antibody of described anti-sulfamethazine is the monoclonal antibody of anti-sulfamethazine, is produced by sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393; Or
The antibody of described anti-Enrofloxacin is the polyclonal antibody of anti-Enrofloxacin.
4. according to arbitrary described enzyme linked immunological kit among the claim 1-3, it is characterized in that: described carrier protein is human serum albumins, oralbumin, hemocyanin, bovine serum albumin(BSA), mouse haemocyanin, thyroprotein or rabbit anteserum albumen.
5. according to arbitrary described enzyme linked immunological kit among the claim 1-4, it is characterized in that: the marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase; Or
Described substrate colour developing liquid is made up of A liquid and B liquid; Described A liquid is urea peroxide or hydrogen peroxide, and described B liquid is tetramethyl benzidine or o-phenylenediamine; Described stop buffer is sulfuric acid; Or
Described substrate colour developing liquid is p-nitrophenyl phosphate solution.
6. according to arbitrary described enzyme linked immunological kit among the claim 2-5, it is characterized in that: described cleansing solution is pH7.4, contain the phosphate buffer of the 0.02M of polysorbas20; The volume content of described polysorbas20 in described cleansing solution is 0.05%; Or
Described bag is cushioned the sodium carbonate that liquid is pH9.6,0.05mol/L-sodium bicarbonate buffer liquid;
Described confining liquid is pH7.4, contains calf serum, sucrose and caseic phosphate buffer; The volume content of described calf serum in described confining liquid is 0.5%, the content of described sucrose in described confining liquid is to contain the described sodium azide of 5g in the described confining liquid of every 100ml, and the content of described casein in described confining liquid is to contain the described casein of 1g in the described confining liquid of every 100ml;
Described sample diluting liquid is the phosphate buffer of 0.02mol/L.
7. the preparation method of arbitrary described enzyme linked immunological kit among the claim 1-6 comprises the step of following (1) or (2):
(1) step that described antibody 1, described antibody 2, described coating antigen 1, described coating antigen 2, described enzyme labeling thing 1 and described enzyme labeling thing 2 are packed separately respectively;
(2) described antibody 1, described antibody 2, described coating antigen 1, described coating antigen 2, described enzyme labeling thing 1, described enzyme labeling thing 2, described sulfamethazine standard solution, described Enrofloxacin standard solution, described substrate colour developing liquid, described stop buffer, described cleansing solution, described bag are cushioned the step that liquid and described confining liquid are packed separately respectively.
8. detect or the auxiliary detection testing sample in whether contain the method for sulfamethazine and/or Enrofloxacin, be utilize among the claim 1-6 arbitrary described enzyme linked immunological kit to detect or the auxiliary detection testing sample in whether contain sulfamethazine and/or Enrofloxacin.
9. method according to claim 8 is characterized in that: in the described method, the coating antigen in the described enzyme linked immunological kit is the coating antigen 1 described in arbitrary among the claim 1-6 and the potpourri of described coating antigen 2; Antibody in the described enzyme linked immunological kit is the antibody 1 described in arbitrary among the claim 1-6 and the potpourri of described antibody 2; Enzyme labeling thing in the described enzyme linked immunological kit is the enzyme labeling thing 1 described in arbitrary among the claim 1-6 and the potpourri of described enzyme labeling thing 2.
10. sulfamido monoclonal antibody hybridoma cell strain SAs, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.3393; Or
Monoclonal antibody by described sulfamido monoclonal antibody hybridoma cell strain SAs CGMCC No.3393 generation.
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