CN105759040A - Enzyme-linked immunosorbent reagent kit for detecting chloramphenicol residues - Google Patents
Enzyme-linked immunosorbent reagent kit for detecting chloramphenicol residues Download PDFInfo
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- CN105759040A CN105759040A CN201511015733.3A CN201511015733A CN105759040A CN 105759040 A CN105759040 A CN 105759040A CN 201511015733 A CN201511015733 A CN 201511015733A CN 105759040 A CN105759040 A CN 105759040A
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The invention provides an enzyme-linked immunosorbent reagent kit for detecting chloramphenicol residues.The enzyme-linked immunosorbent reagent kit is characterized by comprising enzyme-labeled plates coated with chloramphenicol antibodies, chloramphenicol antibodies, chloramphenicol standard solution, enzyme conjugate concentrated solution, enzyme conjugate diluent, substrate solution, stop solution and recombination solution.The enzyme-linked immunosorbent reagent kit has the advantages that the enzyme-linked immunosorbent reagent kit can be used for quickly detecting the residues of chloramphenicol in animal-origin foods and is high in detection speed, sensitivity, specificity and accuracy and low in detection cost, requirements of quick qualitative and quantitative detection in fields can be met, and the like.
Description
Technical field
The present invention relates to field of detection of food safety, specifically, relate to a kind of detecting the enzyme linked immunological kit of chloromycetin drug residue in food.
Background technology
Chloromycetin (Chloramphenicol, CAP) it is a kind of high-efficiency broad spectrum antibiotic, gram positive bacteria and gram negative bacteria can be suppressed, it is widely used in all kinds of poultry, domestic animal, infectious disease in aquatic products production process controls, but chloromycetin has serious toxic and side effects, health can be constituted potential hazard by the residual in food, the bone marrow hematogenesis causing people is in disorder, cause aplastic anemia, granular white blood cells deficiency disease and neonate, the diseases such as premature infant's Synthetic Grey disease, USA and EU has forbidden use chloromycetin in animal, and specify must not detect in animal-derived food.The Ministry of Agriculture of China announces No. 235 file and also specifies, chloromycetin and salt thereof, ester (including chloramphenicol succinate) must not detect in all Edible tissues of all food animals.At present, China's Determination of Chloramphenicol Residue In Food is still very serious, and the aquatic products of outlet, Mel are detected residual chloromycetin repeatedly, have a strong impact on the agricultural products in China export trade.
The most popular method detecting chloromycetin at present is mainly gas chromatogram, liquid chromatograph and chromatography mass spectrometry, these methods have the advantages such as highly sensitive, result is accurate, reproducible, false positive is few, but sample pretreatment process is complicated, expensive equipment, need specialty detection technique personnel, be difficult to meet actually detected requirement.Microbial method detection chloromycetin easily operates, expense is low, but sensitivity is low, poor specificity.Find a kind of method that sensitivity is higher, high specificity, sample treatment are simple, analysis time is short imperative, immunological assay method, it is suitable for the examination of on-site supervision and great amount of samples, is a kind of simple and easy to do method of inspection.
Summary of the invention
It is an object of the present invention to provide a kind of enzyme linked immunological kit detecting residual chloromycetin, this detection kit can reduce operating error and working strength to greatest extent.
Test kit provided by the present invention includes being coated with the ELISA Plate of chloramphenicol antibody, chloromycetin standard substance, chloromycetin high standard product, enzyme conjugates concentrated solution, enzyme combination diluent, substrate solution A liquid, substrate solution B liquid, stop buffer and concentration redissolution liquid.
Described chloramphenicol antibody is by chloromycetin hapten-carrier protein conjugate, immune mouse obtains, described chloromycetin hapten reacts with succinic anhydrides, prepare hapten, hapten directly and carrier protein carry out coupling, described carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, thyroprotein, human serum albumin.
It is a further object to provide a kind of method preparing mentioned reagent box, it includes step:
1) chloromycetin is reacted with succinic anhydrides, prepare chloromycetin hapten；
2) by chloromycetin hapten and carrier protein couplet, chloromycetin hapten-carrier protein conjugate is prepared；
3) with chloromycetin hapten-carrier protein conjugate immune mouse, pass through to merge, screen by mouse boosting cell and murine myeloma cell, obtain the hybridoma cell strain of secretion chloromycetin monoclonal antibody；
4) extract mouse IgG or rabbit igg immune health goat, obtain sheep anti mouse anti antibody；
5) chloromycetin specific antibody is coated on ELISA Plate hole；
6) test kit assembles；
7) residual of chloromycetin in test kit detection sample is utilized；
It is a further object to provide a kind of method applying mentioned reagent box detection Determination of Chloramphenicol Residue In Food, it includes step:
(1) sample treatment；
(2) detect with test kit；
(3) Analysis of test results.
The test kit principle of the present invention: adopt competitive ELISA method, pre-coated chloramphenicol antibody on ELISA Plate capillary strip, the chloromycetin remained in sample and chloromycetin enzyme combine the chloramphenicol antibody on competition ELISA Plate capillary strip, develop the color with tmb substrate, sample absorbance and the content of residue chloromycetin contained by it are negative correlation, compare with standard curve, then be multiplied by the extension rate of its correspondence, the residual quantity of chloromycetin in sample can be drawn.
The test kit of the present invention adopts the chloromycetin monoclonal antibody of high specific, it is ensured that the reliability of testing result；In detection process, the chloramphenicol antibody on micropore can be made to be fully contacted with measuring samples liquid, fully reflection, thus reducing error, increase the reaction sensitivity of whole system, have highly sensitive, high specificity, cost are low, simple to operate, the detection time is short, be suitable for various units uses, store advantage simple, long shelf-life.Wherein, detect the method for residual chloromycetin in sample with test kit of the present invention, easy, quick, directly perceived, accurate, applied widely, cost is low, easily promote the use of.
Accompanying drawing explanation
Fig. 1 chloromycetin hapten synthesis route.
Fig. 2 chloromycetin hapten synthesis qualification result.
Fig. 3 chloromycetin kit standard curve.
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention, without limiting the scope of the present invention.
The preparation of embodiment 1 reagent constituents
1, the haptenic preparation of chloromycetin and qualification
Weigh in the dichloromethane that 0.42g-3g chloromycetin is dissolved in after drying; logical nitrogen protection; add the intoxicated stirring at normal temperature of 0.12-0.8 succinic acid to dissolve; add 0.1-1gDMAP solid and carry out catalytic reaction; monitor reaction with TLC lamellae to carry out, until not having raw material or raw material point very shallow, stopped reaction; silicagel column purifies, and concentrates to obtain product.Structural analysis is carried out, hapten synthesis success through proton nmr spectra.
2, the preparation of antigen
Immunogen is prepared chloromycetin hapten and is obtained immunogen with bovine serum albumin (BSA) coupling.
Weigh that 26mg half Hangzhoupro is former to be dissolved in 4mLDMF solution, add each 60mgEDC and 60mgNHS(to be dissolved in 2mL water) carry out activation 30 minutes, join 110-264mg carrier protein BSA(and be dissolved in 5mL containing in 30%DMF aqueous solution) carry out coupling and prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, prepares antibody for animal immune after having dialysed.Subpackage, saves backup in-20 DEG C.
Immunogen is prepared chloromycetin hapten and is obtained immunogen with ovalbumin (OVA) coupling.
Weigh 26.5mg hapten and be dissolved in 2mLDMF solution, add each 60mgEDC and NHS(to be dissolved in 2mL water) carry out activation 30 minutes, join 110-264mg carrier protein OVA(to be dissolved in 5mL water) carry out coupling and prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, to remove unreacted small-molecule substance；Antibody is prepared for animal immune after having dialysed.Subpackage, saves backup in-20 DEG C.
3, the preparation of chloromycetin monoclonal antibody
Animal immune: immunogen above-mentioned steps obtained is injected in Balb/c Mice Body, immunizing dose is 150 μ g/ so that it is produce antiserum.
Cell fusion and cloning: after mice serum measurement result is higher, take its splenocyte, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, the positive hole of screening.Utilize limiting dilution assay that positive hole is carried out cloning, until obtaining the hybridoma cell strain of secretion chloromycetin monoclonal antibody.
Cell cryopreservation and recovery: monoclonal hybridoma strain frozen stock solution is made the cell suspension of 1 × 106/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
The production of monoclonal antibody and purification: Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5ml/ only, the monoclonal hybridoma strain 5 × 10 that pneumoretroperitoneum injection in 7 days is stable5Individual/only, gather ascites after 7 days.Carry out ascites by sad-saturated ammonium sulfate method to purify ,-20 DEG C of preservations.
4, the preparation of sheep anti mouse anti antibody
With sheep for immune animal, with Mus source antibody for immunogen immune pathogen-free domestic sheep, obtain sheep anti mouse anti antibody.
5, the preparation of ELIAS secondary antibody
Over-voltage protection after sheep anti mouse anti antibody is adopted improvement with horseradish peroxidase (HRP) carries out coupling.In reaction system after improvement, enzyme is 2.0-2.5:1 with the molar concentration rate of antibody.
6, the preparation of ELISA Plate
With being coated buffer, coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 DEG C of lucifuges hatch 2h, liquid in hole of inclining, and wash 1 time with cleaning mixture, stop 30s, patting dry, then add 150 μ l confining liquids in every hole, 37 DEG C of lucifuges hatch 2h, the liquid in hole that inclines pats dry, and dried sealing by aluminum film vacuum preserves.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of chloromycetin
Set up the enzyme linked immunological kit of detection chloromycetin so that it is comprise following component:
(1) ELISA Plate of chloromycetin coupled antigen it is coated；
(2) chloromycetin standard solution 6 bottles, concentration respectively 0 μ g/L, 0.025 μ g/L, 0.075 μ g/L, 0.3 μ g/L, 1.2 μ g/L, 4.8 μ g/L；
(3) chloromycetin monoclonal antibody working solution；
(4) with the sheep anti mouse anti antibody of horseradish peroxidase-labeled；
(5) substrate nitrite ion is made up of substrate nitrite ion A liquid and substrate nitrite ion B liquid, and substrate nitrite ion A liquid is urea peroxide, and substrate nitrite ion B liquid is tetramethyl benzidine；
(6) stop buffer is 1mol/L sulphuric acid；
(7) cleaning mixture is pH value 7.2-7.5, containing 1.0%-1.5% tween 20,0.03 ‰-0.05 ‰ sodium azide preservatives, 0.1-0.3mol/L phosphate buffer, described percentage ratio is percent weight in volume.
(8) redissolve liquid to be pH value be 7.0, the phosphate buffer of 0.02mol/L, described percentage ratio is percent weight in volume.
The detection of residual chloromycetin in embodiment 3 tissue sample
Sample pre-treatments
1. tissue (Carnis Gallus domesticus/liver, Carnis Sus domestica/liver, shrimp, fish etc.) Sample pretreatment method
(1) with homogenizer homogeneous structure sample；
(2) weigh the equal pledge of 3.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 6ml ethyl acetate, vibrate 5min, more than 3000g, 20-25 DEG C of centrifugal 5min with agitator；
(3) pipette 4ml upper organic phase to 10ml clean dried teat glass, flow down in 50-60 DEG C of water-bath nitrogen and dry up；
(4) add 1ml normal hexane, with vortex instrument whirling motion 2min, add 1ml redissolution working solution, with vortex instrument whirling motion 30s, more than 3000g, 20-25 DEG C centrifugal 5min；
(5) remove upper organic phase, take off layer 50ul for analyzing.
2. the configuration of solution
0.1MCB: weigh 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adds the mixing of 500ml deionized water dissolving.
The configuration of redissolution liquid: be diluted by 1:1 volume ratio by concentration redissolution liquid with deionized water, for the redissolution of sample, the working solution that redissolves can preserve one month at 4 DEG C of environment.
3. test kit detection operating procedure:
(1) required reagent is taken out from cold storage environment, rise again according to the method described above, notice that every kind of liquid reagent must shake up before using.
(2) take out the microwell plate needing quantity, no microwell plate is put back to aluminium foil bag vacuum again and seals, be stored in 2-8 DEG C of environment.
(3) concentration redissolution liquid and urine sample diluent also need to rise again as stated above before use.
4. numbering: being numbered according to the order of sequence by corresponding with standard substance for sample micropore, it is parallel that each sample does 2 holes with standard substance, and the position at record standard hole and sample aperture place.
5. enzyme conjugates working solution preparation: measure as required and enzyme conjugates concentrated solution enzyme combination diluent is diluted by 1:10 volume.
6. adding standard substance/sample and enzyme conjugates working solution: add standard substance/sample 50ml in corresponding micropore, be subsequently adding enzyme conjugates working solution 50ul/ hole, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
7, wash plate: carefully open cover plate film, liquid in hole is dried, add deionized water 250ul/ hole, fully washing 4-5 time, every minor tick 10s, sprinkles liquid in board falling hole, pats dry with absorbent paper.
8, colour developing: add substrate solution A liquid 50ul/ hole, adds substrate solution B liquid 50ul/ hole, and mixing of vibrating gently, with the rearmounted 25 DEG C of light protected environment reaction 20min of cover plate membrane cover plate.
9, measure: add stop buffer 50ul/ hole, mixing of vibrating gently, set microplate reader dual wavelength 450/630nm detection, in 5min, please run through data, measure every hole OD value.If without microplate reader, then being not added with stop buffer ocular estimate can judge.
4, Analysis of test results
The mean absorbance values (diplopore) of standard substance or sample is divided by the mean absorbance values of first standard substance (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard substance or sample.With standard substance percentage absorptance for vertical coordinate, with the logarithm of chloromycetin standard concentration (μ g/L) for abscissa, drawing standard curve chart.The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the actual concentrations that the extension rate of its correspondence is in sample chloromycetin.
The mean absorbance values of B standard substance or sample solution
The mean absorbance values of B0 0ppb standard solution
The detection of residual chloromycetin in embodiment 4 prepared food sample
Sample pre-treatments
1. prepared food Sample pretreatment method
(1) with homogenizer homogeneous structure sample；
(2) weigh the equal pledge of 3.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 3ml deionized water, add 6ml ethyl acetate, vibrate 10min, more than 3000g, 20-25 DEG C of centrifugal 10min with agitator；
(3) pipette 4ml upper organic phase to 10ml clean dried teat glass, flow down in 50-60 DEG C of water-bath nitrogen and dry up；
(4) add 1ml normal hexane, with vortex instrument whirling motion 1min, add 1ml redissolution working solution, with vortex instrument whirling motion 15s, more than 3000g, 20-25 DEG C centrifugal 5min；
(5) remove upper organic phase, take off layer 50ul for analyzing.
2. the configuration of solution
0.1MCB: weigh 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adds the mixing of 500ml deionized water dissolving.
The configuration of redissolution liquid: be diluted by 1:1 volume ratio by concentration redissolution liquid with deionized water, for the redissolution of sample, the working solution that redissolves can preserve one month at 4 DEG C of environment.
3. test kit detection operating procedure:
(1) required reagent is taken out from cold storage environment, rise again according to the method described above, notice that every kind of liquid reagent must shake up before using.
(2) take out the microwell plate needing quantity, no microwell plate is put back to aluminium foil bag vacuum again and seals, be stored in 2-8 DEG C of environment.
(3) concentration redissolution liquid and urine sample diluent also need to rise again as stated above before use.
4. numbering: being numbered according to the order of sequence by corresponding with standard substance for sample micropore, it is parallel that each sample does 2 holes with standard substance, and the position at record standard hole and sample aperture place.
5. enzyme conjugates working solution preparation: measure as required and enzyme conjugates concentrated solution enzyme combination diluent is diluted by 1:10 volume.
6. adding standard substance/sample and enzyme conjugates working solution: add standard substance/sample 50ul in corresponding micropore, be subsequently adding enzyme conjugates working solution 50ul/ hole, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
7, wash plate: carefully open cover plate film, liquid in hole is dried, add deionized water 250ul/ hole, fully washing 4-5 time, every minor tick 10s, sprinkles liquid in board falling hole, pats dry with absorbent paper.
8, colour developing: add substrate solution A liquid 50ul/ hole, adds substrate solution B liquid 50ul/ hole, and mixing of vibrating gently, with the rearmounted 25 DEG C of light protected environment reaction 20min of cover plate membrane cover plate.
9, measure: add stop buffer 50ul/ hole, mixing of vibrating gently, set microplate reader dual wavelength 450/630nm detection, in 5min, please run through data, measure every hole OD value.If without microplate reader, then being not added with stop buffer ocular estimate can judge.
4, Analysis of test results
The mean absorbance values (diplopore) of standard substance or sample is divided by the mean absorbance values of first standard substance (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard substance or sample.With standard substance percentage absorptance for vertical coordinate, with the logarithm of chloromycetin standard concentration (μ g/L) for abscissa, drawing standard curve chart.The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the actual concentrations that the extension rate of its correspondence is in sample chloromycetin.
The mean absorbance values of B standard substance or sample solution
The mean absorbance values of B0 0ppb standard solution
The detection of residual chloromycetin in embodiment 5 tissue sample
Sample pre-treatments
1. Lac regis apis Sample pretreatment method
(1) 1.0 ± 0.05g Lac regis apis is weighed to 50ml polystyrene centrifuge tube；
(2) add 1ml0.1MCB, 8ml ethyl acetate, add 2g natrium carbonicum calcinatum；
(3) vibrate 5min, more than 3000g, 20-25 DEG C of centrifugal 5min with agitator；
(4) pipette 2ml upper organic phase to 10ml clean dried teat glass, flow down in 50-60 DEG C of water-bath nitrogen and dry up；
(5) add 1ml normal hexane, whirling motion 1min, add 1ml redissolution working solution, with vortex instrument whirling motion 15s, more than 3000g, 20-25 DEG C centrifugal 5min；
(6) remove upper organic phase, take off layer 50ul for analyzing.
2. the configuration of solution
0.1MCB: weigh 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adds the mixing of 500ml deionized water dissolving.
The configuration of redissolution liquid: be diluted by 1:1 volume ratio by concentration redissolution liquid with deionized water, for the redissolution of sample, the working solution that redissolves can preserve one month at 4 DEG C of environment.
3. test kit detection operating procedure:
(1) required reagent is taken out from cold storage environment, rise again according to the method described above, notice that every kind of liquid reagent must shake up before using.
(2) take out the microwell plate needing quantity, no microwell plate is put back to aluminium foil bag vacuum again and seals, be stored in 2-8 DEG C of environment.
(3) concentration redissolution liquid and urine sample diluent also need to rise again as stated above before use.
4. numbering: being numbered according to the order of sequence by corresponding with standard substance for sample micropore, it is parallel that each sample does 2 holes with standard substance, and the position at record standard hole and sample aperture place.
5. enzyme conjugates working solution preparation: measure as required and enzyme conjugates concentrated solution enzyme combination diluent is diluted by 1:10 volume.
6. adding standard substance/sample and enzyme conjugates working solution: add standard substance/sample 50ul in corresponding micropore, be subsequently adding enzyme conjugates working solution 50ul/ hole, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
7, wash plate: carefully open cover plate film, liquid in hole is dried, add deionized water 250ul/ hole, fully washing 4-5 time, every minor tick 10s, sprinkles liquid in board falling hole, pats dry with absorbent paper.
8, colour developing: add substrate solution A liquid 50ul/ hole, adds substrate solution B liquid 50ul/ hole, and mixing of vibrating gently, with the rearmounted 25 DEG C of light protected environment reaction 20min of cover plate membrane cover plate.
9, measure: add stop buffer 50ul/ hole, mixing of vibrating gently, set microplate reader dual wavelength 450/630nm detection, in 5min, please run through data, measure every hole OD value.If without microplate reader, then being not added with stop buffer ocular estimate can judge.
4, Analysis of test results
The mean absorbance values (diplopore) of standard substance or sample is divided by the mean absorbance values of first standard substance (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard substance or sample.With standard substance percentage absorptance for vertical coordinate, with the logarithm of chloromycetin standard concentration (μ g/L) for abscissa, drawing standard curve chart.The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the actual concentrations that the extension rate of its correspondence is in sample chloromycetin.
The mean absorbance values of B standard substance or sample solution
The mean absorbance values of B0 0ppb standard solution
The detection of residual chloromycetin in embodiment 6 Feed Sample
1. feedstuff Sample pretreatment method
(1) 1.0 ± 0.05g feedstuff is weighed to 50ml polystyrene centrifuge tube；
(2) add 10ml ethyl acetate agitator to vibrate 5min, more than 3000g, 20-25 DEG C of centrifugal 5min；
(3) pipette 2ml upper organic phase to 10ml clean dried teat glass, flow down in 50-60 DEG C of nitrogen and dry up；
(4) add 1ml normal hexane, with vortex instrument whirling motion 1min, add 1ml redissolution working solution, with vortex instrument whirling motion 15s, more than 3000g, 20-25 DEG C centrifugal 5min；
(5) remove upper organic phase, take off layer 50ul for analyzing.
2. the configuration of solution
0.1MCB: weigh 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adds the mixing of 500ml deionized water dissolving.
The configuration of redissolution liquid: be diluted by 1:1 volume ratio by concentration redissolution liquid with deionized water, for the redissolution of sample, the working solution that redissolves can preserve one month at 4 DEG C of environment.
3. test kit detection operating procedure:
(1) required reagent is taken out from cold storage environment, rise again according to the method described above, notice that every kind of liquid reagent must shake up before using.
(2) take out the microwell plate needing quantity, no microwell plate is put back to aluminium foil bag vacuum again and seals, be stored in 2-8 DEG C of environment.
(3) concentration redissolution liquid and urine sample diluent also need to rise again as stated above before use.
4. numbering: being numbered according to the order of sequence by corresponding with standard substance for sample micropore, it is parallel that each sample does 2 holes with standard substance, and the position at record standard hole and sample aperture place.
5. enzyme conjugates working solution preparation: measure as required and enzyme conjugates concentrated solution enzyme combination diluent is diluted by 1:10 volume.
6. adding standard substance/sample and enzyme conjugates working solution: add standard substance/sample 50ul in corresponding micropore, be subsequently adding enzyme conjugates working solution 50ul/ hole, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
7, wash plate: carefully open cover plate film, liquid in hole is dried, add deionized water 250ul/ hole, fully washing 4-5 time, every minor tick 10s, sprinkles liquid in board falling hole, pats dry with absorbent paper.
8, colour developing: add substrate solution A liquid 50ul/ hole, adds substrate solution B liquid 50ul/ hole, and mixing of vibrating gently, with the rearmounted 25 DEG C of light protected environment reaction 20min of cover plate membrane cover plate.
9, measure: add stop buffer 50ul/ hole, mixing of vibrating gently, set microplate reader dual wavelength 450/630nm detection, in 5min, please run through data, measure every hole OD value.If without microplate reader, then being not added with stop buffer ocular estimate can judge.
4, Analysis of test results
The mean absorbance values (diplopore) of standard substance or sample is divided by the mean absorbance values of first standard substance (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard substance or sample.With standard substance percentage absorptance for vertical coordinate, with the logarithm of chloromycetin standard concentration (μ g/L) for abscissa, drawing standard curve chart.The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the actual concentrations that the extension rate of its correspondence is in sample chloromycetin.
The mean absorbance values of B standard substance or sample solution
The mean absorbance values of B0 0ppb standard solution
The detection of residual chloromycetin in embodiment 7 egg sample
Egg Sample pretreatment method
(1) with homogenizer low speed homogenizing sample, Ovum Gallus domesticus album and egg yolk is made to be sufficiently mixed；
(2) the egg sample after 1.0 ± 0.05g homogenizing is weighed to 50ml polystyrene centrifuge tube；
(3) add 8ml ethyl acetate, add 1ml0.1MCB, vibrate 5min, more than 3000g, 20-25 DEG C of centrifugal 5min with agitator；
(4) pipette 4ml upper organic phase to 10ml clean dried teat glass, flow down in 50-60 DEG C of water-bath nitrogen and dry up；
(5) add 1ml normal hexane, with vortex instrument whirling motion 1min, add 1ml redissolution working solution, with vortex instrument whirling motion 15s, more than 3000g, 20-25 DEG C centrifugal 5min；
(6) remove upper organic phase, take off layer 50ml for analyzing.
2. the configuration of solution
0.1MCB: weigh 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adds the mixing of 500ml deionized water dissolving.
The configuration of redissolution liquid: be diluted by 1:1 volume ratio by concentration redissolution liquid with deionized water, for the redissolution of sample, the working solution that redissolves can preserve one month at 4 DEG C of environment.
3. test kit detection operating procedure:
(1) required reagent is taken out from cold storage environment, rise again according to the method described above, notice that every kind of liquid reagent must shake up before using.
(2) take out the microwell plate needing quantity, no microwell plate is put back to aluminium foil bag vacuum again and seals, be stored in 2-8 DEG C of environment.
(3) concentration redissolution liquid and urine sample diluent also need to rise again as stated above before use.
4. numbering: being numbered according to the order of sequence by corresponding with standard substance for sample micropore, it is parallel that each sample does 2 holes with standard substance, and the position at record standard hole and sample aperture place.
5. enzyme conjugates working solution preparation: measure as required and enzyme conjugates concentrated solution enzyme combination diluent is diluted by 1:10 volume.
6. adding standard substance/sample and enzyme conjugates working solution: add standard substance/sample 50ul in corresponding micropore, be subsequently adding enzyme conjugates working solution 50ul/ hole, mixing of vibrating gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
7, wash plate: carefully open cover plate film, liquid in hole is dried, add deionized water 250ul/ hole, fully washing 4-5 time, every minor tick 10s, sprinkles liquid in board falling hole, pats dry with absorbent paper.
8, colour developing: add substrate solution A liquid 50ul/ hole, adds substrate solution B liquid 50ul/ hole, and mixing of vibrating gently, with the rearmounted 25 DEG C of light protected environment reaction 20min of cover plate membrane cover plate.
9, measure: add stop buffer 50ul/ hole, mixing of vibrating gently, set microplate reader dual wavelength 450/630nm detection, in 5min, please run through data, measure every hole OD value.If without microplate reader, then being not added with stop buffer ocular estimate can judge.
4, Analysis of test results
The mean absorbance values (diplopore) of standard substance or sample is divided by the mean absorbance values of first standard substance (0 standard), then is multiplied by 100%, obtains the percentage absorbance of standard substance or sample.With standard substance percentage absorptance for vertical coordinate, with the logarithm of chloromycetin standard concentration (μ g/L) for abscissa, drawing standard curve chart.The percentage absorptance of sample is substituted in standard curve, from standard curve, read the concentration corresponding to sample, be multiplied by the actual concentrations that the extension rate of its correspondence is in sample chloromycetin.
The mean absorbance values of B standard substance or sample solution
The mean absorbance values of B0 0ppb standard solution
The determination test of embodiment 8 chloromycetin technical parameter
1, test kit sensitivity and detection limit
Conventionally measure test kit sensitivity, kit standard curve minimum point is 0.025 μ g/L, standard curve range for 0.025-4.8 μ g/L, IC50(50% inhibition concentration) domain of walker is 0.3-1.2 μ g/L；20 parts of samples are detected, the concentration corresponding to each percentage absorbance is found from standard curve, detection limit is represented plus 3 times of standard deviations with the meansigma methods of 20 parts of concentration of specimens, result obtains the method detection to tissue and is limited to 0.0125 μ g/kg, the detection of prepared food is limited to 0.0125 μ g/kg, and the detection of Lac regis apis is limited to 0.25 μ g/kg, and the detection of feedstuff is limited to 0.0125 μ g/kg, the detection of egg is limited to 0.05 μ g/kg, and sensitivity is 0.025 μ g/kg.
2, sample preci-sion and accuracy test
Using the response rate as accuracy estimating index, the testing result relative standard deviation (RSD%) of a certain concentration samples of replication is as precision evaluation index.Computing formula is: the response rate (%)=practical measurement value/theoretical value × 100%, and wherein theoretical value is the interpolation concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, and X is the meansigma methods of determination data.
Respectively by 0.1 μ g/g, 0.3 μ g/g, tri-concentration chloromycetin of 0.6 μ g/g Chicken Tissues, prepared food, egg sample are added reclaiming and measure, each sample do 4 parallel, being measured with three batches of different reagent, the average recovery rate and the precision result that calculate sample are shown in following table.
Table 1 Chicken Tissues precision and accuracy test
Table 2 prepared food precision and accuracy test
Table 3 egg precision and accuracy test
Respectively tissue, prepared food and egg sample being added with the chloromycetin of 0.1,0.3,0.6 tri-concentration of μ g/g, average recovery rate is between 88.1%-100.3%；Batch in, batch between relative standard deviation be respectively less than 10%.
3, stabilization of kit test
Test kit preservation condition is 2-8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of test kit, 50% inhibition concentration, chloromycetin added practical measurement value all within normal range.Considering in transport and use procedure, have improper preservation condition and occur, being placed 7 days under 37 DEG C of preservation conditions by test kit, be accelerated senile experiment, result shows that this test kit indices complies fully with requirement.Considering that test kit freezing situation occurs, test kit is put into-20 DEG C of refrigerator freezings 7 days, measurement result also indicates that test kit indices is completely normal.Can show that test kit at least can preserve more than 12 months at 2-8 DEG C from the result above.
4, test kit cross reacting rate
Table 4 chloromycetin and the contrast of other antibiotic cross reacting rates
In from the data above, it is found that the enzyme linked immunological kit specificity that the present invention detects residual chloromycetin is good.
Claims (2)
1. the enzyme linked immunological kit being used for detecting residual chloromycetin, by the ELISA Plate being coated chloramphenicol antibody, chloramphenicol antibody, chloromycetin standard solution, enzyme conjugates concentrated solution, enzyme combination diluent, substrate solution, stop buffer and multiple solution composition, wherein said chloramphenicol antibody is to be prepared by described chloromycetin antigen, described chloromycetin antigen is by being reacted with succinic anhydrides by the hydroxyl in chloromycetin molecular structure, obtain the hapten with chloromycetin functional group, obtain with carrier protein couplet again, carrier protein can be bovine serum albumin, ovalbumin, hemocyanin, human albumin, thyroprotein.
2. test kit as claimed in claim 1, it is characterised in that described chloromycetin standard solution concentration is 0ug/L, 0.025ug/L, 0.075ug/L, 0.3ug/L, 1.2ug/L, 4.8ug/L.
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| CN106866454A (en) * | 2017-02-27 | 2017-06-20 | 海南大学 | A kind of chloramphenicol haptens and its preparation method and application |
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