CN103018451A - Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof - Google Patents
Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof Download PDFInfo
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- CN103018451A CN103018451A CN2011102790985A CN201110279098A CN103018451A CN 103018451 A CN103018451 A CN 103018451A CN 2011102790985 A CN2011102790985 A CN 2011102790985A CN 201110279098 A CN201110279098 A CN 201110279098A CN 103018451 A CN103018451 A CN 103018451A
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Abstract
The present invention provides an enzyme-linked immunoassay kit for chloramphenicol detection. The enzyme-linked immunoassay kit comprises an enzyme label plate coated with coating antigen, an enzyme marker, a specific chloramphenicol antibody working solution (when the enzyme label plate is coated with antigen and the enzyme marker is enzyme-labeled anti-antibody or the enzyme label plate is coated with anti-antibody and the enzyme marker is enzyme-labeled antigen, the kit contains the specific chloramphenicol antibody working solution), chloramphenicol standard solutions, a substrate coloration solution, a termination solution, a concentration washing solution and a concentration reconstituted solution. The present invention further discloses a method for detecting chloramphenicol by using the enzyme-linked immunoassay kit. The method comprises: carrying out a sample pretreatment, adopting the kit to detect, and finally analyzing a detection result. With the enzyme-linked immunoassay kit, chloramphenicol residue in animal tissues (muscles, livers and the like), delicatessen, aquatic products, royal jelly, urine, feeds, milk and other samples can be detected, characteristics of simple operation, low cost and high sensitivity are provided, and the kit and the method can be applicable for screening and on-site monitoring of a large number of samples.
Description
Technical field
The present invention relates to the enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detection of chloromycetin, it is particularly suitable for the detection of residual chloromycetin in animal tissue's (muscle, liver etc.), prepared food, aquatic products, royal jelly, urine, feed and the milk equal samples.
Technical background
Chloromycetin (Chloramphenicol, CAP) be a kind of broad-spectrum antibiotic that 20 middle of century are found, Gram-negative bacteria and positive bacteria all there is good inhibiting effect, because its good antibiotic property, the stable property of medicine and cheap price, once within one period, be applied to humans and animals as the medicine of bacteriosis clinical, and be widely used in animal husbandry production as feed addictive.Yet, it is found that in use chloromycetin has toxicity, the residual meeting in animal food is by the health generation toxic action of the animal foods such as meat, egg, milk to the people.Therefore, China Ministry of Agriculture announces files specify No. 235, and chloromycetin and salt thereof, ester (comprising Chloramphenicol Succinate) must not detect in all Edible tissues of all food animals.But because the good antimicrobial effect of chloromycetin, price is low, still has the people to use it in poultry, domestic animals, the aquatic products.For the trade contacts that ensure the healthy of the people and enlarge food, set up highly sensitive, high specificity, quick, economic residual chloromycetin detection method is necessary.
At present, being used for the most popular method of chlorine detection mycin mainly is gas chromatography, liquid chromatography and chromatography mass spectrometry, that although these methods have is highly sensitive, the result accurately, the advantage such as good reproducibility, false positive be few, but still there is certain shortcoming, complicated such as sample pretreatment process, the instrumentation degree is high and expensive, and analysis speed is slow, can not satisfy gradually developed country and food processing enterprises to the requirement of detection method detectability.By comparison, immunological assay method sensitivity is higher, high specificity, sample pretreatment is simple, analysis time is short, is fit to the examination of on-site supervision and great amount of samples.
Summary of the invention
The object of the invention is to provides a kind of enzyme linked immunological kit for the chloromycetin detection for above-mentioned deficiency, and it is simple to operate, is fit to the screening of on-the-spot batch samples.
Kit of the present invention, it contains:
(1) is coated with the ELISA Plate of coating antigen;
(2) enzyme labeling thing;
(3) chloromycetin standard solution;
(4) substrate nitrite ion;
(5) stop buffer;
(6) concentrated cleaning solution;
(7) the concentrated liquid that redissolves.
The enzyme linked immunological kit of chlorine detection mycin provided by the present invention comprises ELISA Plate and the enzyme labeling thing working fluid of pre-coated coating antigen; Described coating antigen is chloromycetin antigen, antibody or antiantibody, described enzyme labeling thing is enzyme labeling chloromycetin haptens, enzyme labeling chloramphenicol antibody or enzyme labeling antiantibody, when envelope antigen on the ELISA Plate and enzyme labeling thing are also to contain chloromycetin specific antibody working fluid when coated antiantibody and enzyme labeling thing are enzyme-labelled antigen on enzyme labeling antiantibody or the ELISA Plate.
Described chloromycetin haptens is that the nitro in the chloromycetin molecular structure is obtained by catalytic hydrogenation reaction.
Described chloromycetin specific antibody is the chloromycetin monoclonal antibody; Described chloromycetin monoclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, is preferably the chloromycetin mouse monoclonal antibody; Described antiantibody is the sheep anti mouse antiantibody.
Above antibody all can prepare as immunogene with the conjugate of chloromycetin haptens and carrier protein.Described carrier protein can be the common carrier albumen such as mouse haemocyanin, thyroprotein (BCG), bovine serum albumin, rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin and fibrinogen; The conjugate of described chloromycetin haptens and carrier protein can obtain by chloromycetin haptens and carrier protein are carried out coupling with glutaraldehyde method.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; The sheep anti mouse antiantibody of enzyme labeling adopts glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out coupling and obtains; Enzyme labeling chloromycetin haptens adopts diazotising method or glutaraldehyde method that marker enzyme and chloromycetin hapten conjugation are obtained; The enzyme labeling specific antibody adopts glutaraldehyde method or sodium periodate method that marker enzyme and specific antibody coupling are obtained, horseradish peroxidase can adopt several different methods of the prior art that itself and antiantibody are carried out coupling, such as glutaraldehyde method, sodium periodate method etc., the present invention improves the sodium periodate method through long-term labor and creation, make it save time, reduce the concentration rate of horseradish peroxidase (HRP) and antiantibody, saved starting material.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises chloromycetin standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves.
Described chloromycetin standard solution: 6 bottles, concentration is 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.
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is comprised of nitrite ion A liquid and nitrite ion B liquid, A liquid is hydrogen peroxide or urea peroxide, and B liquid is o-phenylenediamine or tetramethyl benzidine, and described stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme was alkaline phosphatase, described substrate nitrite ion was to the nitro phosphate buffer, and described stop buffer is 1～2mol/L sodium hydroxide solution.
It is 7.1～7.5 that described concentrated cleaning solution is preferably the pH value, contains the phosphate buffer of 0.8%～1.2% Tween-20,0.01 ‰～0.03 ‰ thimerosal antiseptic, 0.01～0.03mol/L, and described number percent is percent weight in volume.
It is 7.2～7.7 that described concentrated redissolution liquid is preferably the pH value, contains the phosphate buffer of 8%～12% ovalbumin, 0.1～0.4mol/L, and described number percent is percent weight in volume.
Wherein used coated damping fluid is that the pH value is 9.6 in the ELISA Plate preparation process, 0.05mol/L carbonate buffer solution, used confining liquid is that the pH value is 9.1～9.5, the carbonate buffer solution that contains 3%～10% calf serum, 0.2% Tween-20,0.1～0.3mol/L, described number percent are percent weight in volume.
The preparation process of ELISA Plate is among the present invention: with coated damping fluid coating antigen is diluted to 0.1～0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines is with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150～200 μ l confining liquids, 37 ℃ of incubation 1～2h, inclining, liquid pats dry in the hole, preserves with the vacuum seal of aluminium film after dry.
Immunogenic building-up process is among the present invention:
1. the chloromycetin haptens synthesizes (synthetic route such as Fig. 2)
The chloromycetin haptens is that the nitro in chloromycetin (structural formula such as Fig. 1) molecular structure is obtained by catalytic hydrogenation reaction, and concrete reactions steps is as follows:
Chloromycetin is dissolved in the methyl alcohol, adds 5% palladium carbon catalyst (Pd/C), pass into hydrogen, keep certain pressure, room temperature reaction 2h removes by filter Pd/C, and solvent evaporated obtains faint yellow thick liquid, is the chloromycetin haptens; The mol ratio of wherein said Pd/C and chloromycetin is 1: 10.
2. immunogenic synthetic
Adopt glutaraldehyde method to carry out coupling chloromycetin haptens and carrier protein and obtain immunogene, concrete preparation method is as follows:
Extracting chloromycetin haptens 30mg with the water-soluble solution of 1.5ml, obtains (I) liquid; Get 50% glutaraldehyde (GA) 10 μ l and add in (I), stirring reaction 18h under the room temperature obtains (II) liquid; Get bovine serum albumin(BSA) (BSA) 100mg and add in (II) liquid with the dilution of 1.5ml water is rear, react the rear adding 24mg NaBH that spends the night
4Reaction 3h; 48h namely gets immunogene with the tri-distilled water dialysis.
The preparation process of chloromycetin monoclonal antibody is among the present invention:
(1) animal immune program: adopt the Balb/c mouse as immune animal, take chloromycetin haptens and carrier protein couplet thing as immunogene, obtain preferably polyvalent antibody after, the taking-up spleen carries out Fusion of Cells.
(2) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell and SP2/0 myeloma cell and merge, the screening cell line is until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
The preparation process of sheep anti mouse antiantibody is among the present invention: as immune animal, as immunogene the pathogen-free domestic sheep is carried out immunity take mouse source antibody with sheep, obtain the sheep anti mouse antiantibody.
Detection principle of the present invention is:
When pre-coated chloromycetin coupled antigen on capillary strip, after adding sample solution or standard solution, add again enzyme labeling chloromycetin specific antibody solution, chloromycetin coupled antigen coated on chloromycetin in the sample and the ELISA Plate is competed the chloromycetin specific antibody, develop the color with nitrite ion, the content of sample absorbance and chloromycetin is negative correlation, relatively can draw the residual quantity of chloromycetin in the sample with typical curve; Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of chloramphenicol residue in the judgement sample of the comparison of the chloromycetin standard solution color of series concentration.
When pre-coated chloromycetin coupled antigen on capillary strip, after adding sample solution or standard solution, add again chloromycetin specific antibody solution, chloromycetin coupled antigen coated on chloromycetin in the sample and the ELISA Plate is competed the chloromycetin specific antibody, add the enzyme labeling antiantibody and carry out amplification, with nitrite ion colour developing, the content of sample absorbance and chloromycetin is negative correlation, relatively can obtain the residual quantity of chloromycetin in the sample with typical curve; Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of chloramphenicol residue in the judgement sample of the comparison of the chloromycetin standard solution color of series concentration.
When pre-coated chloromycetin specific antibody on capillary strip, after adding sample solution or standard solution, add again enzyme labeling chloromycetin haptens solution, chloromycetin in the sample and ENR-HRP competition are coated on the chloromycetin specific antibody on the ELISA Plate, develop the color with nitrite ion, the content of sample absorbance and chloromycetin is negative correlation, relatively can draw the residual quantity of chloromycetin in the sample with typical curve; Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of chloramphenicol residue in the judgement sample of the comparison of the chloromycetin standard solution color of series concentration.
When pre-coated antiantibody on capillary strip, after the adding chloramphenicol antibody is hatched, add sample solution or standard solution, add again enzyme labeling chloromycetin haptens solution, chloromycetin in the sample and enzyme labeling chloromycetin haptens competition chloramphenicol resistance specific antibody, with nitrite ion colour developing, the content of sample absorbance and chloromycetin is negative correlation, relatively can draw the residual quantity of chloromycetin in the sample with typical curve; Simultaneously according to the depth of color on the ELISA Plate, with the rough concentration range of chloramphenicol residue in the judgement sample of the comparison of the chloromycetin standard solution color of series concentration.
The present invention also provides a kind of method of using above-mentioned enzyme linked immunological kit chlorine detection mycin, and it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) analyzing and testing result.
Determination mainly is in order to obtain chloromycetin solution from sample, thereby is used for follow-up detection.The below is common several determination methods:
1. organize (chicken/liver, pork/liver, shrimp, fish etc.) pre-treating method
Behind homogenizer homogeneous structure sample, take by weighing the equal pledge of 3.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 6ml ethyl acetate, with the oscillator 5min that vibrates, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 4ml upper organic phase (sample that approximately is equivalent to 2g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.
2. prepared food pre-treating method
Behind homogenizer homogeneous sample, take by weighing the equal pledge of 3.0 ± 0.05g to 50ml polystyrene centrifuge tube, add the 3ml deionized water, add again 6ml ethyl acetate, with the oscillator 10min that vibrates, 4000rpm, the centrifugal 10min of room temperature (20-25 ℃/68-77 °F); Pipette 4ml upper organic phase (sample that approximately is equivalent to 2g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.
3. urine pre-treating method
Get the limpid urine specimen of 100 μ l, add 900 μ l wash operating solutions, mixing is got 50 μ l and is used for analyzing (if urine specimen is not limpid, needing with urine 4000rpm the centrifugal 5min of room temperature).
4. royal jelly pre-treating method
Take by weighing 1.0 ± 0.05g sample to 50ml polystyrene centrifuge tube, add 1ml 0.1mol/L CB (taking by weighing 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adding 500ml deionized water dissolving mixing), 8ml ethyl acetate, add again the 2g natrium carbonicum calcinatum, with the oscillator 5min that vibrates, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 2ml upper organic phase (sample that approximately is equivalent to 0.25g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, whirling motion 1min adds 1ml again and redissolves working fluid, with vortex instrument whirling motion 15s, 4000rpm, and the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, get 50 μ l liquid and be used for analyzing.
5. milk pre-treating method
Pipette 500 μ l milk, add 500 μ l wash operating solutions, fully shake mixing, get 50 μ l and be used for analyzing.
6. feed pre-treating method
Take by weighing 1.0 ± 0.05g feed to 50ml polystyrene centrifuge tube, add 10ml ethyl acetate vibration 5min, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette the 2ml upper organic phase to the clean glass tube of 10ml, flow down in 50～60 ℃ of nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, get 50 μ l and be used for analyzing.
7. egg pre-treating method
Behind homogenizer homogeneous sample, take by weighing 1 ± 0.05g egg to 50ml polystyrene centrifuge tube, add 8ml ethyl acetate, 1ml 0.1mol/L CB (taking by weighing 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adding 500ml deionized water dissolving mixing), with the oscillator 5min that vibrates, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 4ml upper organic phase (sample that approximately is equivalent to 0.5g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.
When detecting with kit among the present invention: when coating antigen is the chloromycetin coupled antigen, add standard solution in the ELISA Plate micropore or sample solution adds enzymic-labelled antibody again, washing pats dry behind the incubation, colour developing, stops, and measures absorbance with microplate reader; When coating antigen is the chloromycetin coupled antigen, add standard solution in the ELISA Plate micropore or sample solution adds antibody again, washing pats dry behind the incubation, adds enzyme mark antiantibody again, and washing pats dry behind the incubation, colour developing, stops, and measures absorbance with microplate reader; When coating antigen is the chloromycetin specific antibody, add standard solution in the ELISA Plate micropore or sample solution adds enzyme labeling chloromycetin haptens again, washing pats dry behind the incubation, colour developing, stops, and measures absorbance with microplate reader; When coating antigen is antiantibody, in the ELISA Plate micropore, add chloramphenicol antibody, washing pats dry behind the incubation, add enzyme mark chloromycetin haptens after adding again standard solution or sample solution, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
The Analysis of test results process is among the present invention: use the absorbance mean value (B) of standard solution of each concentration that obtains divided by the absorbance (B of first standard solution (0 standard)
0) multiply by again 100%, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Take the logarithm value of the concentration (μ g/L) of chloromycetin standard solution as X-axis, the percentage absorbance is Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the chloromycetin content of corresponding each sample then can be read from typical curve.
The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.
The analysis of testing result can also utilize computer professional software among the present invention, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process at most only needed to finish in 75 minutes.
The enzyme linked immunological kit of chlorine detection mycin of the present invention mainly adopts the content of chloromycetin in the qualitative or quantitative test sample of competitive ELISA method; Low to the determination requirement, sample pretreatment process is simple, simultaneously the fast detecting batch samples; Adopt the chloromycetin monoclonal antibody of high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, the accuracy high.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.Kit of the present invention will play a significant role in the detection of chloromycetin.
Description of drawings
Fig. 1: chloromycetin structural drawing
Fig. 2: chloromycetin haptens synthetic route chart
Fig. 3: take the conjugate of chloromycetin haptens and carrier protein as coating antigen, enzymic-labelled antibody is the canonical plotting of the kit of enzyme labeling thing
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used for explanation the present invention, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 kit components
1. antigen is synthetic
A. the chloromycetin haptens is synthetic
Chloromycetin is dissolved in the methyl alcohol, adds 5% palladium carbon catalyst (Pd/C), pass into hydrogen, keep certain pressure, room temperature reaction 2h removes by filter Pd/C, and solvent evaporated obtains faint yellow thick liquid, is the chloromycetin haptens; The mol ratio of wherein said Pd/C and chloromycetin is 1: 10.
B. immunogene is synthetic
Adopt glutaraldehyde method to carry out coupling chloromycetin haptens and bovine serum albumin(BSA) and obtain immunogene.
Immunogenic preparation process:
Extracting chloromycetin haptens 30mg with the water-soluble solution of 1.5ml, obtains (I) liquid; Get 50% glutaraldehyde (GA) 10 μ l and add in (I), stirring reaction 18h under the room temperature obtains (II) liquid; Get bovine serum albumin(BSA) (BSA) 100mg and add in (II) liquid with the dilution of 1.5ml water is rear, react the rear adding 24mg NaBH that spends the night
4Reaction 3h; 48h namely gets immunogene with the tri-distilled water dialysis.
C. coating antigen is synthetic
Chloromycetin haptens and ovalbumin coupling are obtained coating antigen.
The preparation process of coating antigen:
Extracting chloromycetin haptens 30mg with the water-soluble solution of 1.5ml, obtains (I) liquid; Get 50% glutaraldehyde (GA) 10 μ l and add in (I), stirring reaction 18h under the room temperature obtains (II) liquid; Get ovalbumin (OVA) 30mg and add in (II) liquid with the dilution of 1.5ml water is rear, react the rear adding 24mg NaBH that spends the night
4Reaction 3h; 48h namely gets coating antigen with the tri-distilled water dialysis.
2. monoclonal antibody is synthetic
Animal immune: immunogene is injected in the Balb/c Mice Body, and immunizing dose is 100 μ g/, makes it produce polyclonal antibody.
Fusion of Cells and cloning: after the mice serum measurement result is higher, get its splenocyte, merge in 8: 1 ratios and SP2/0 myeloma cell, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
Obtain the monoclonal hybridoma strain of chloromycetin through screening, generation chloromycetin specific antibody that can be endless, and this antibody specificity is for chloromycetin, and sensitivity can reach 0.025 μ g/L.
Cell cryopreservation and recovery: the monoclonal hybridoma strain of chloromycetin is made 1 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
The production of monoclonal antibody and purifying: the Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, the stable chloromycetin monoclonal hybridoma strain 1 * 10 of pneumoretroperitoneum injection in 7 days
6Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method ,-20 ℃ of preservations.
3. the preparation of enzyme labeling chloromycetin monoclonal antibody
Adopt the sodium periodate method after improveing to carry out coupling chloromycetin monoclonal antibody and horseradish peroxidase (HRP).The molar concentration rate of enzyme and antibody is 4: 1 in traditional sodium periodate method requirement reaction system, because horseradish peroxidase produces many sites of being combined with antibody under strong oxidation, the horseradish peroxidase molecule of activation has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, made in the conjugate of preparation and be mixed with many condensates.In order to address this problem, we improve traditional method, that is:
(1) saved amino closed process, because can produce self amino amino reality that connects seldom;
(2) reduced horseradish peroxidase: the volumetric molar concentration ratio to 2 of antibody: 1, the method after the improvement is easier than traditional method, to the loss minimizing of enzymatic activity.
4. the preparation of ELISA Plate
With coated damping fluid the chloromycetin coupled antigen is diluted to 0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, and coating buffer inclines, concentrated cleaning solution with 20 times of dilutions washs 2 times, each 30s pats dry, and then adds 200 μ l confining liquids in every hole, 37 ℃ of incubation 2h, the liquid in the hole that inclines is preserved with the vacuum seal of aluminium film after dry
The establishment of the enzyme linked immunological kit of embodiment 2 chlorine detection mycins
Set up the enzyme linked immunological kit of chlorine detection mycin, make it comprise following component:
(1) ELISA Plate of coated chloromycetin coupled antigen;
(2) the chloromycetin standard solution is 6 bottles, and concentration is 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 high concentration standard solution is 1 bottle, and concentration is 100 μ g/L;
(4) the chloromycetin monoclonal antibody working fluid of usefulness horseradish peroxidase-labeled;
(5) the substrate nitrite ion is comprised of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L sulfuric acid;
(7) concentrated cleaning solution is that the pH value is 7.1～7.5, contains the phosphate buffer of 0.8%～1.2% Tween-20,0.01 ‰～0.03 ‰ thimerosal antiseptic, 0.01～0.03mol/L, and described number percent is percent weight in volume;
(8) the concentrated liquid that redissolves is that the pH value is 7.2～7.7, contains the phosphate buffer of 8%～12% ovalbumin, 0.1～0.4mol/L, and described number percent is percent weight in volume.
The detection of chloromycetin in embodiment 3 samples
1. sample pre-treatments
(1) tissue (chicken/liver, pork/liver, shrimp, fish etc.) pre-treating method
Behind homogenizer homogeneous structure sample, take by weighing the equal pledge of 3.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 6ml ethyl acetate, with the oscillator 5min that vibrates, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 4ml upper organic phase (sample that approximately is equivalent to 2g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.
(2) prepared food pre-treating method
Behind homogenizer homogeneous sample, take by weighing the equal pledge of 3.0 ± 0.05g to 50ml polystyrene centrifuge tube, add the 3ml deionized water, add again 6ml ethyl acetate, with the oscillator 10min that vibrates, 4000rpm, the centrifugal 10min of room temperature (20-25 ℃/68-77 °F); Pipette 4ml upper organic phase (sample that approximately is equivalent to 2g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.
(3) urine pre-treating method
Get the limpid urine specimen of 100 μ l, add 900 μ l wash operating solutions, mixing is got 50 μ l and is used for analyzing (if urine specimen is not limpid, needing with urine 4000rpm the centrifugal 5min of room temperature).
(4) royal jelly pre-treating method
Take by weighing 1.0 ± 0.05g sample to 50ml polystyrene centrifuge tube, add 1ml 0.1mol/L CB (taking by weighing 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adding 500ml deionized water dissolving mixing), 8ml ethyl acetate, add again the 2g natrium carbonicum calcinatum, with the oscillator 5min that vibrates, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 2ml upper organic phase (sample that approximately is equivalent to 0.25g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, whirling motion 1min adds 1ml again and redissolves working fluid, with vortex instrument whirling motion 15s, 4000rpm, and the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, get 50 μ l liquid and be used for analyzing.
(5) milk pre-treating method
Pipette 500 μ l milk, add 500 μ l wash operating solutions, fully shake mixing, get 50 μ l and be used for analyzing.
(6) feed pre-treating method
Take by weighing 1.0 ± 0.05g feed to 50ml polystyrene centrifuge tube, add 10ml ethyl acetate vibration 5min, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette the 2ml upper organic phase to the clean glass tube of 10ml, flow down in 50～60 ℃ of nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, get 50 μ l and be used for analyzing.
(7) egg pre-treating method
Behind homogenizer homogeneous sample, take by weighing 1 ± 0.05g egg to 50ml polystyrene centrifuge tube, add 8ml ethyl acetate, 1ml 0.1mol/L CB (taking by weighing 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate adding 500ml deionized water dissolving mixing), with the oscillator 5min that vibrates, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 4ml upper organic phase (sample that approximately is equivalent to 0.5g) to the clean glass test tube of 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml and redissolve working fluid, with vortex instrument whirling motion 15s, 4000rpm, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Remove upper organic phase, take off layer 50 μ l and be used for analyzing.
2. detect with kit
In the ELISA Plate micropore that is coated with the chloromycetin coupled antigen, add chloromycetin standard solution or sample solution 50 μ l, the chloromycetin monoclonal antibody working fluid 50 μ l/ holes that add again horseradish peroxidase-labeled, behind cover plate membrane cover plate, put lucifuge reaction 30min in 25 ℃ of constant temperature ovens, pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole behind the 30s, repeat operation and wash plate 5 times, pat dry with thieving paper; Every hole adds substrate nitrite ion A liquid urea peroxide 50 μ l, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ l, mixing gently vibrates, with lucifuge colour developing 20min in the rearmounted 25 ℃ of constant temperature ovens of cover plate membrane cover plate, every hole adds stop buffer 2mol/L sulfuric acid 50 μ l, the mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the microplate reader wavelength set.
3. Analysis of test results
With the absorbance mean value (B) of the standard solution of each concentration that the obtains absorbance (B divided by first standard solution (0 standard)
0) multiply by again 100%, obtain the percentage absorbance.Take the logarithm value of chloromycetin standard items concentration (μ g/L) as X-axis, the percentage absorbance is Y-axis, the drawing standard curve map, as shown in Figure 3.With the percentage absorbance of same way calculation sample solution, the chloromycetin content of corresponding each sample then can be read from typical curve.
Definite test of embodiment 4 kit technical parameters
1. standard items precision test
Respectively extract a collection of ELISA Plate out respectively from the ELISA Plate of three different time period preparations, every batch is extracted 10 kits, and every plate is extracted 20 micropores out, measures the absorbance of 0.3 μ g/L standard solution, calculates the coefficient of variation.
The repeatable test of table 1 standard (CV%)
Can draw by above-mentioned test findings, every batch of each 10 standard items coefficient of variation of kit meet precision and are less than or equal to 25% regulation between 3.8%～10.2%.
2. sample preci-sion and accuracy test
(1) sample precision test
Chloromycetin with 0.2 μ g/kg concentration adds mensuration to chicken, pork, pork liver, shrimp sample, chloromycetin with 1 μ g/kg (L) concentration adds mensuration to egg, milk sample, chloromycetin with 2 μ g/kg concentration adds mensuration to royal jelly, feed sample, chloromycetin with 5 μ g/L concentration adds mensuration to the pig urine sample, get respectively three different batches kit each three, each concentration repeats 5 times, calculates the coefficient of variation, the results are shown in Table 2～10.
The repeatable test of table 2 chicken sample
The repeatable test of table 3 pork sample
The repeatable test of table 4 pork liver sample
The repeatable test of table 5 shrimp sample
The repeatable test of table 6 egg sample
The repeatable test of table 7 milk sample
The repeatable test of table 8 royal jelly sample
The repeatable test of table 9 feed sample
The repeatable test of table 10 pig urine sample
The result shows, the variation lines number average of chicken, pork, pork liver, shrimp, egg, milk, royal jelly, feed, pig urine sample has met [2005] No. 17 annex 2 kits of " Ministry of Agriculture's file " agricultural doctor's method and has put on record with reference to the 4th precision standard in the judgment criteria between 4.7%～12.1%.
(2) sample accuracy test
Chloromycetin with 0.2,0.4 μ g/kg concentration adds mensuration to chicken, pork, pork liver, shrimp sample, chloromycetin with 1,2 μ g/kg (L) concentration adds mensuration to egg, milk sample, chloromycetin with 2,4 μ g/kg concentration adds mensuration to royal jelly, feed sample, chloromycetin with 5,10 μ g/L concentration adds mensuration to the pig urine sample, each concentration do 4 parallel, accuracy in computation the results are shown in Table 11 respectively.
The accuracy of table 11 kit
The result shows, chicken, pork, pork liver, shrimp sample add the recovery between 76.5%～98.3%, the egg sample adds the recovery between 83.9%～102.4%, the milk sample adds the recovery between 81.5%～97.4%, the royal jelly sample adds the recovery between 86.2%～103.0%, the feed sample adds the recovery between 84.2%～98.7%, and the pig urine sample adds the recovery between 80.7%～95.6%.
3. cross reacting rate test
Select the drug monitoring cross reacting rate that has similar structures and similar functions with chloromycetin, the typical curve by various medicines obtains respectively its 50% inhibition concentration, calculates kit to the cross reacting rate of other medicines with following formula.Cross reacting rate is larger, and this kit is just better to the specificity of the detection of chloromycetin so.
Cross reacting rate (%)=(cause 50% chloramphenicol concentration that suppresses/cause the 50% analog concentration that suppresses) * 100%
The specificity of table 12 kit
4. kit stability test
The kit preservation condition is 2～8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, chloromycetin added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use procedure, and kit was placed 15 days under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that this kit indices meets the requirements fully.Consider that the freezing situation of kit occurs, kit was put into-20 ℃ of refrigerator freezings 15 days, measurement result shows that also the kit indices is fully normal.Can draw kit from above result can preserve more than 12 months at least at 2～8 ℃.
Claims (9)
1. the enzyme linked immunological kit of a chlorine detection mycin is characterized in that it contains:
(1) is coated with the ELISA Plate of coating antigen;
(2) enzyme labeling thing;
(3) chloromycetin standard solution;
(4) substrate nitrite ion;
(5) stop buffer;
(6) concentrated cleaning solution;
(7) the concentrated liquid that redissolves,
Described coating antigen is chloromycetin antigen, antibody or antiantibody, and described enzyme labeling thing is enzyme labeling chloromycetin antigen, enzyme labeling chloramphenicol antibody or enzyme labeling antiantibody,
When envelope antigen on the ELISA Plate and enzyme labeling thing are also to contain chloromycetin specific antibody working fluid when coated antiantibody and enzyme labeling thing are enzyme-labelled antigen on enzyme labeling antiantibody or the ELISA Plate.
2. kit as claimed in claim 1, it is characterized in that described chloromycetin antigen is to be obtained by chloromycetin haptens and carrier protein couplet, described chloramphenicol antibody is to be prepared by described coupled antigen, and wherein said chloromycetin haptens is that the nitro in the chloromycetin molecular structure is obtained by catalytic hydrogenation reaction.
3. kit as claimed in claim 2 is characterized in that described carrier protein is mouse haemocyanin, thyroprotein, bovine serum albumin, rabbit anteserum albumen, human albumin, ovalbumin, hemocyanin or fibrinogen.
4. kit as claimed in claim 1 or 2 is characterized in that described chloramphenicol antibody is monoclonal antibody.
5. kit as claimed in claim 1 or 2 is characterized in that used coated damping fluid is that the pH value is the carbonate buffer solution of 9.6,0.05mol/L; Used confining liquid is that the pH value is 9.1～9.5, contains the carbonate buffer solution of 3%～10% calf serum, 0.2% Tween-20,0.1～0.3mol/L, and described number percent is percent weight in volume.
6. kit as claimed in claim 1 or 2, the marker enzyme that it is characterized in that described enzyme labeling thing is that horseradish peroxidase or bacterium are extracted alkaline phosphatase, when marker enzyme is horseradish peroxidase, substrate nitrite ion A liquid is hydrogen peroxide or urea peroxide, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme was bacterium extraction alkaline phosphatase, the substrate nitrite ion was to the nitro phosphate buffer, and stop buffer is 1～2mol/L NaOH.
7. kit as claimed in claim 1 or 2 is characterized in that used concentrated cleaning solution is that the pH value is 7.1～7.5, contains the phosphate buffer of 0.8%～1.2% Tween-20,0.01 ‰～0.03 ‰ thimerosal antiseptic, 0.01～0.03mol/L; Used concentrated redissolution liquid is that the pH value is 7.2～7.7, contains the phosphate buffer of 8%～12% ovalbumin, 0.1～0.4mol/L, and described number percent is percent weight in volume.
8. kit as claimed in claim 1 or 2 is characterized in that the concentration of chloromycetin standard solution is 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.
9. the method for residual chloromycetin in the test sample comprises step:
(1) sample pre-treatments;
(2) detect with each described kit of claim 1～8;
(3) analyzing and testing result.
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| CN106932566A (en) * | 2017-04-17 | 2017-07-07 | 四川精卫食品检测科技有限公司 | A kind of carbendazim and the two-in-one detection enzyme linked immunological kit of thiophanate-methyl |
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| CN109724930A (en) * | 2018-12-27 | 2019-05-07 | 北京普赞生物技术有限公司 | Edible oil and detection kit and detection method containing dibutyl phthalate in fatty foods |
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