CN103018450A - Chloramphenicol chemiluminescence enzyme-linked immunodetection kit - Google Patents
Chloramphenicol chemiluminescence enzyme-linked immunodetection kit Download PDFInfo
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Abstract
The present invention discloses a chloramphenicol chemiluminescence enzyme-linked immunodetection kit, which comprises a kit body, an enzyme label plate placed inside the kit body, and reagents placed inside the kit body, and is characterized in that every hole of the enzyme label plate is coated with coating antigen, the coating antigen is a chloramphenicol and carrier protein conjugate, and the reagents comprise horseradish peroxidase-labeled chloramphenicol monoclonal antibody, a series of chloramphenicol standard solutions, a concentrated phosphate buffer, a concentrated washing solution and a chemiluminescence solution. The chloramphenicol chemiluminescence enzyme-linked immunodetection kit has characteristics of high sensitivity, simple and rapid detection, and high accuracy, provides a substantially reduced operation time compared to the conventional colorimetric ELISA method, and can be used for detection of chloramphenicol residues in animal tissues (pork, chicken, pork liver and chicken liver), aquatic products (fish and shrimp) and milk.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit of chlorine detection mycin, for detection of chloromycetin content or the residual quantity in animal tissue's (pork, chicken, pork liver, chicken gizzard), aquatic products (fish, shrimp), honey and the milk.Belong to the immunology detection field.
Background technology
((Chloramphenicol, CAP) is a kind of broad-spectrum antibiotic of Cheap highly effective to chloromycetin, and Gram-positive and negative bacteria are had good inhibiting effect, therefore once is widely used in the farming and animal husbandry.But animal derived food can cause various diseases along with food chain is taken in for a long time by human body.The lighter destroys the equilibrium state of normal flora in the human body, and flora imbalance makes human body produce the drug-fast bacteria pearl, and giving from now on, ill use antibiotic therapy brings harmful effect; Allergic reaction can appear in the people of microbiotic allergic constitution, jeopardizes health.Can disturb the synthetic of bone marrow cell protein when serious, and it is synthetic to suppress juvenile cell DNA, causes granulocyte to reduce, cause the malignant diseases such as alpastic anemia, haemolysis, purple paralysis.
Toxic and side effect in view of chloromycetin, international food educational circles classifies them as banning drugs, European Union, the U.S. etc. all in rules regulation residual chloromycetin limit standard be " zero tolerance " (Zerotolerance), namely must not detect, according to European Union " 2002/657/EC " standard code, to require detection limit be 0.3 μ g/kg to the maximum of chloromycetin in the animal derived food.Soon U.S. FDA is also made corresponding regulation.China Ministry of Agriculture has stipulated that CAP must not detect in the edible tissues of all food animals, and it is deleted from " Chinese veterinary pharmacopoeia ", classifies banning drugs as.And corresponding SN0219-93 and the SCT3018-2004 industry standard formulated, in line with international standards.
Be to adapt to higher examination criteria, the detection technique level must improve accordingly, one of popular project that nearly ten years chloromycetin detection techniques are international experts and scholars' research always.The detection method of chloromycetin is divided into physical-chemical process and immuno-chemical method, the former has liquid phase chromatography (LC), high performance liquid chromatography (HPLC), mass spectroscopy (MS) etc., the latter mainly is that enzyme is exempted from method (ELISA), and the sensitivity of detection all reaches ppb (μ g/kg) rank.The detection method of current main-stream is the employed method of LC-China industry standard, and ELISA.In recent years, Chinese scholars is developed again the physical detection new methods such as Liquid Chromatography-Tandem Mass Spectrometry method (LC-MS/MS), liquid chromatography electrospray ionization mass spectrometry (LC-EITMS), microbiological analysis, and the application of ELISA method has also been had further research.
Chemical luminous immune detection method have high specificity, stable fast, high, the stable reagent of wide, the simple to operate automaticity of sensing range and the long advantages such as (6～18 months) of the term of validity, its detectability is than euzymelinked immunosorbent assay (ELISA) and the high several orders of magnitude of physics and chemistry detection method.Chemiluminescence import instrument and reagent better performances, and external technical monopoly causes Chemiluminescence Apparatus, luminous substrate liquid and kit to hold at high price, and reagent or kit and instrument are complementary, import reagent usually can not use in domestic equipment, causes the chemiluminescence immunoassay method to popularize in basic unit.Chemoluminescent substrate is the key reagents of chemiluminescence enzyme immunity detection method, make low-cost and functional, be fit to that domestic equipment uses luminous substrate liquid, can reduce the use cost of chemiluminescence method, be conducive to popularizing in basic unit.Setting up stable chemiluminescence enzyme immunoassay method, also is the basis of carrying out the development of commercial chemistry luminescence reagent box.
Summary of the invention
The chemical luminescence ELISA detection kit that the purpose of this invention is to provide a kind of chloromycetin.This kit has that detection sensitivity is high, applying flexible, characteristics easily.
The chemical luminescence ELISA detection kit of chloromycetin of the present invention, comprise box body, be located at the ELISA Plate in the box body and be located at box body interior enzyme mark chloromycetin monoclonal antibody, chloromycetin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, chemical luminescence for liquid; It is characterized in that:
Each hole of described ELISA Plate is coated with the envelope antigen made from chloromycetin and ovalbumin coupling; The preferred 10 μ g/mL of wherein said envelope antigen concentration.
The opaque polystyrene 96 hole chemiluminescence ELISA Plate of the preferred milky of described ELISA Plate.
Described chloromycetin series standard solution dilutes from the chloromycetin sterling and obtains, dilution is the 0.05mmol/L that contains 10% methyl alcohol, the PBS of pH=7.4, chloromycetin standard items concentration is respectively 0pg/mL, 20pg/mL, 60pg/mL, 180pg/mL, 540pg/mL and 1620pg/mL, described number percent are percent by volume.
Described enzyme mark chloromycetin monoclonal antibody is the monoclonal antibody that is made by the artificial immunogen immune animal that chloromycetin and bovine serum albumin coupling are made, and its working concentration is preferably 1: 16000.
Described chemical luminescence for liquid A liquid is that luminol content is that 0.01M, p-cresol content are three (methylol) aminomethane solution of 0.001M pH=8.8, and B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2HPO
42.82g, the solution of 0.75% carbamide peroxide 0.64mL.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Described concentrated phosphoric acid salt buffer is every liter and contains NaH
2PO
42H
2O 5.74g, Na
2HPO
412H
2The aqueous solution of O 32.6g.
Described thickening and washing solution is the pH=7.4 that contains volume fraction 0.05% Tween-20, the 0.1mol/L phosphate buffer.
Described coated solution is the solution (CB) that contains 1.59g sodium carbonate and 2.53g sodium bicarbonate in every premium on currency, pH=9.5.
Described lock solution is that to contain 10g ovalbumin (OVA, ovalbumin also claim chicken ovalbumin or chicken ovalbumin, are comprised of 385 amino acid residues, and molecular weight is 45kDa approximately) in every liter of wash solution and add quality be 5 ‰ NaN
3Solution, described number percent is mass percent.
The preparation of solution of the present invention:
The chloromycetin standard solution that relates in the kit of the present invention, enzyme mark chloromycetin monoclonal antibody solution, chemiluminescence solution and wash solution and prescription thereof are very large on the sensitivity impact that kit of the present invention detects; Wherein the principal ingredient of each solution and compound method thereof are:
1, chloromycetin standard solution: the chloromycetin sterling is used the 0.05m mol/L that contains 10% methyl alcohol with conventional method, the PBS of pH=7.4 is mixed with concentration and is respectively 0pg/mL, 20pg/mL, 60pg/mL, 180pg/mL, the chloromycetin standard solution of 540pg/mL and 1620pg/mL, described number percent are percent by volume.
2, enzyme mark chloromycetin monoclonal antibody solution: the chloromycetin monoclonal antibody is the monoclonal antibody that makes with artificial immunizing antigen immune animal, will be diluted to 1: 16000 working concentration with wash solution behind the gained chloromycetin monoclonal anti body and function horseradish peroxidase-labeled mark.
3, chemiluminescence solution: A liquid is that luminol content is that 0.01M, p-cresol content are three (methylol) aminomethane solution of 0.001M pH=8.8, and B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2HPO
42.82g, the solution of 0.75% carbamide peroxide 0.64mL.
4, concentrated phosphoric acid salt buffer: NaH
2PO
42H
2O 6.52g, Na
2HPO
412H
2O 38.7g is dissolved in the 1L deionized water.
5, thickening and washing solution: by volume mark 0.05% is with the pH=7.5 that is added into of Tween-20, in the 0.1mol/L phosphate buffer.
6, coated solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in the 1L water, regulate pH=9.5.
7, lock solution preparation: 10g OVA is dissolved in the 1L wash solution, adds weight ratio again and be 5 ‰ NaN
3
Being coated with of ELISA Plate of the present invention:
Coated elisa plate adopts the CAP-OVA conjugate is placed the coated solution of setting among the present invention, and with the concentration of setting, reaction is coated in 37 ℃ of constant temperature ovens.
What the present invention adopted is sodium carbonate-sodium bicarbonate buffer solution of pH=9.5.The CAP-OVA that is coated with in the microwell plate among the present invention can well be combined under alkaline environment on the microwell plate frosting, can stand repeatedly to wash plate, and the envelope antigen concentration of employing is 10 μ g/mL.
Coated good microwell plate can seal with lock solution, and the preferred OVA of inert protein in the confining liquid needs to add NaN
3Antiseptic.
The preparation of enzyme mark chloromycetin monoclonal antibody solution:
Enzyme mark chloromycetin monoclonal antibody solution is the key factor that determines chloromycetin enzyme linked immunological test kit measurement range and sensitivity among the present invention among the present invention.
The enzyme mark chloromycetin monoclonal antibody solution that relates among the present invention can be diluted to wash solution 1: 16000 working concentration.
(the standard lines scope can reach 0pg/mL～1620pg/mL) and well sensitivity (20pg/mL) can to reach the good range of linearity according to the kit of above-mentioned enzyme mark chloromycetin monoclonal antibody solution concentration preparation.
The preparation of chemiluminescence solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly is luminol-hydrogen peroxide system.
Described chemical luminescence for liquid A liquid is that luminol content is that 0.01M, p-cresol content are three (methylol) aminomethane solution of 0.001M pH=8.8, and B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2HPO
42.82g, the solution of 0.75% carbamide peroxide 0.64mL.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Principle of the present invention is that antibody-antigen reactive high degree of specificity and enzymatic high sensitivity are combined, and utilizes the chemiluminescence reaction of substrate for enzymatic activity to detect production concentration.
Chemical luminescence ELISA detection kit of the present invention has highly sensitive, easy fast and accurately characteristics, and with traditional colorimetric ELISA method comparison, sensitivity can improve an order of magnitude.Be expected to play a significant role in the residual chloromycetin detection in animal food (such as animal tissue, aquatic products, honey, milk).
Description of drawings
Fig. 1 is chloromycetin haptens synthetic reaction formula.
Fig. 2 is chemiluminescence reaction formula of the present invention.
Fig. 3 is the working curve of chloramphenicol antibody of the present invention.
Embodiment
Embodiment 1: the preparation of immunogene, coating antigen and monoclonal antibody linked with peroxidase
(1) chloromycetin haptens preparation
A is dissolved in chloromycetin in the methyl alcohol, adds 5% Pd/C, passes into hydrogen, keeps certain pressure, and room temperature reaction 2h removes by filter Pd/C, and solvent evaporated obtains faint yellow thick liquid, has both got the chloromycetin haptens.The mol ratio of Pd/C and chloromycetin is 1: 10.Under 0～5 ℃, be 1～2 with the hydrochloric acid adjust pH with object obtained above, stir the NaNO of the lower 0.1～1mol/L of dropping
2Solution makes starch potassium iodide paper become blue, drips 0.1～1mol/L urea liquid again, makes the starch potassium iodide paper indigo plant that shoals, and the NaOH solution adjust pH that adds again 0.1～1mol/L is 7～9, obtains clear liquid for subsequent use as solution A.
B measures solution A 21.8mL and adds among the DMF 3mL, and 4 ℃ of precooling 10min add tri-n-butylamine, mixing; And the adding isobutyl chlorocarbonate, 4 ℃ are stirred 20min.Take by weighing 71.5mg BSA and be dissolved among 50% the DMF 10mL 4 ℃ of precoolings.Transfer the pH to 8 of BSA with 1M NaOH.The solution A liquid for preparing is added rapidly among the people BSA 4 ℃ of stirring reaction 4h.
(2) immunogene is synthetic
A, the extracting chloromycetin haptens 30mg water-soluble solution of 1.5ml; Get 50% GA10 μ l and add in 1 stirring reaction 18h under the room temperature;
B gets BSA100mg and adds in 1 with the dilution of 1.5ml water is rear; React the rear adding 24mgNaBH that spends the night
4Reaction 3h; With tri-distilled water dialysis 48 hours, namely get immunogene.
By the above-mentioned steps reaction, extracting chloromycetin haptens 30mg, OVA100mg, synthetic CAP-OVA is for coated.
(3) preparation of enzyme mark chloromycetin monoclonal antibody
A, animal immune: with the above-mentioned immunogene of preparing (CAP-BSA) by 150 μ g/ only, with physiological saline solution immune complex and Freund's complete adjuvant equal-volume mixing, the female mouse of nape section hypodermic injection immunity 6～8 week Balb/c in age, behind the initial immunity the 7th, 14,28 day with immune complex and incomplete Freunds adjuvant equal-volume mixing, each supplementary immunization once with immune complex 100 μ g/ only merges front 3 days, and supplementary immunization is once more not add Freunds adjuvant.
B, Fusion of Cells: carry out according to a conventional method, the splenocyte of getting immune mouse mixes with the murine myeloma cell that is in exponential phase (SP2/0), then the fusion agent (PEG4000) that slowly added preheating in 45 seconds merges, suspend evenly with the HAT nutrient culture media, add again an amount of feeder cells, be incubated at 96 well culture plates, in 37 ℃, 5%CO
2Cultivate in the incubator, partly changed liquid with the HT nutrient culture media afterwards in 5 days, entirely change liquid in the time of 9 days.
C, the screening of hybridoma: after the Fusion of Cells, treat long 1/4 o'clock of arriving the culture hole area of cell, adopt a minute step screening method screening hybridoma.Indirect ELISA method is adopted in primary election, with envelope antigen (in advance with square formation method conventional titration its best coated concentration and positive serum dilutability) coated elisa plate, add the measured hole culture supernatant, hatch, add sheep anti-mouse igg-HRP and IgM-HRP after cleaning, o-phenylenediamine carries out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening that filters out mixes cell conditioned medium first with the chloromycetin equal-volume of 100pg/mL, 37 ℃ of water-bath effect 30min join in the coated good ELISA Plate again.Replace chloromycetin with PBS simultaneously and compare, all the other steps are the same.If the OD450nm value after chloromycetin blocking-up drops to below 50% of control wells, then be judged to the positive, through 2～3 detections positive hole all, carry out subcloning with limiting dilution assay immediately.
D, monoclonal antibody preparation: 2～3 subclones are built hybridoma after the strain enlarge and cultivate, collect supernatant and measure with indirect ELISA and tire, frozen; And get 8～10 ages in week Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/ only, lumbar injection hybridoma 1～2 * 10 after 7～10 days
6/ only, extract mouse ascites after 7～10 days, the centrifuging and taking supernatant, mensuration is tired, and frozen for subsequent use.
E, monoclonal antibody linked with peroxidase preparation: take by weighing the 5mg horseradish peroxidase and be dissolved in the 0.5mL deionized water, add freshly prepared 0.06M NaIO
4Aqueous solution 0.5mL, mixing is put refrigerator 30min, take out and add 0.16M glycol water 0.5mL, after placing 30min, room temperature adds the aqueous solution 1mL that contains the 5mg monoclonal antibody purification, mixing and fill bag filter to 0.05M, pH=9.5 carbonic acid buffer slowly stir the dialysis 6h make it combination, then sucking-off adds NaBH
4Solution 0.2mL puts refrigerator 2h.
Above-mentioned bond mixed liquor is added the saturated sulfuric acid of equal-volume to be pressed, refrigerator is put after 30 minutes centrifugal, the gained sediment is dissolved among a little 0.02M, the pH=7.4PBS, and to dialysed overnight, next day is the centrifugal insolubles of removing again, namely get enzyme-antibody conjugates, add to 5mL with 0.02M, pH=7.4PBS and measure the postlyophilization preservation.
The foundation of embodiment 2:ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Vertically press 160.0 μ g/mL, 80.0 μ g/mL, 40.0 μ g/mL, 20.0 μ g/mL with every kind of envelope antigen, 10.0 μ g/mL, 5.0 μ g/mL, 2.5 μ g/mL, the serial dilution degree coated elisa plate of 1.25 μ g/mL, 100 μ L/ holes, place 37 ℃ of constant temperature oven 2h after, pat dry; With 150 μ L/ hole lock solution sealings, 37 ℃ of constant temperature ovens were placed 2 hours, washed plate once, patted dry; Add the enzyme mark chloromycetin monoclonal antibody (1: 1000 to 1: 512000) of the 50 a series of dilutions in μ L/ hole, room temperature (20～25 ℃) is hatched 15min, washes plate five times, pats dry for the last time; The chemical luminescence for liquid that adds 100 μ L/ holes is measured luminous value.There are the envelope antigen concentration of obvious graded and antibody dilution to carry out specific assay as optium concentration take luminous value with the concentration of envelope antigen.
(2) mensuration of antibody sensitivity
According to the optimization experiment of above-mentioned antagonist and envelope antigen concentration, the applicant selects and determines that antibody concentration is 1: 16000, and envelope antigen concentration is the mensuration that 10 μ g/mL carry out the sensitivity of antibody:
A, coated: as envelope antigen to be made into the solution of 10 μ g/mL with the coated solution of the carbonate of 0.05M pH=9.6, to add 100 μ L in the reacting hole of each polystyrene board, 37 ℃ of constant temperature oven 2h.Discard solution in the hole, pat dry.
B, sealing: with the above-mentioned coated ELISA Plate of lock solution sealing, 150 μ L/ holes, then 37 ℃ of constant temperature oven 2h wash plate once, pat dry.
C, application of sample: ((1: 16000) 50 μ L/ holes are in the above-mentioned reacting hole that has sealed to add the chloromycetin solution 50 μ L/ holes of variable concentrations and the enzyme mark chloromycetin monoclonal antibody of dilution, room temperature (20～25 ℃) lucifuge is hatched 15min, then washes plate five times, pats dry for the last time.
D, luminous: as in each reacting hole, to add the chemiluminescence solution 100 μ L/ holes of interim preparation, detect with chemical illumination immunity analysis instrument behind the reaction 3min.
E, testing result is calculated with inhibiting rate:
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution are measured, RLU
0It is the luminous intensity values of blank (concentration is 0 standard solution).
The concentration of medicine is the sensitivity of this antibody when calculating 50% inhibiting rate.
Embodiment 3: the chemiluminescence enzyme linked immunoassay reagent kit of chlorine detection mycin
(1) composition of the chemiluminescence enzyme linked immunoassay reagent kit of chlorine detection mycin
A is coated with the solid phase carrier (ELISA Plate) of coating antigen (CAP-OVA);
B, chloromycetin standard solution: 0pg/mL, 20pg/mL, 60pg/mL, 180pg/mL, 540pg/mL and 1620pg/mL.
C, enzyme mark chloramphenicol antibody solution: prepare the monoclonal antibody of gained with artificial immunizing antigen (CAP-BSA) immune animal, the gained chloramphenicol antibody is diluted to 1: 16000 working concentration with wash solution after with horseradish peroxidase-labeled.
D, luminous solution: A liquid is that luminol content is that 0.01M, p-cresol content are three (methylol) aminomethane solution of 0.001M pH=8.8, and B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2HPO
42.82g, the solution of 0.75% carbamide peroxide 0.64mL.A liquid and B liquid were by 1: 1 mixing before using.
E, concentrated phosphoric acid salt buffer are every liter and contain NaH
2PO
42H
2O 5.74g, Na
2HPO
412H
2The aqueous solution of O 32.6g.
F, thickening and washing solution: contain the pH=7.4 of volume fraction 0.05% Tween-20, the 0.1mol/L phosphate buffer.
(2) preparation of ELISA Plate
With coating buffer envelope antigen is diluted to 10 μ g/mL, every hole adds 100 μ L, and 37 ℃ of constant temperature ovens are placed 2h, and coating buffer inclines, pat dry, then every hole adds confining liquid 150 μ L, and 37 ℃ of constant temperature ovens are placed 2h, liquid in the hole of inclining, the cleansing solution washing once pats dry, and preserves with masking foil vacuum seal.
Embodiment 4: the application of the chemiluminescence enzyme linked immunoassay reagent kit of chlorine detection mycin
(1) preparation of reagent
A, wash solution: the concentrated cleaning solution that provides in the kit is used after by 1: 19 times of dilution with deionized water.
B, phosphate buffer: the concentrated phosphoric acid salt buffer that provides in the kit is spent ionized water use after by 1: 1 times of dilution.
C, chemiluminescence solution: use front with A liquid and 1: 1 by volume mixing of B liquid.
(2) sample pre-treatments
A, animal tissue's (pork, chicken, pork liver, chicken gizzard), aquatic products (fish, shrimp etc.):
---with 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 10min that vibrates, more than the 3000g, the centrifugal 10min of room temperature (20～25 ℃);
---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 30s, add the 1mL phosphate buffer again, 1min changes in the 2mL centrifuge tube with the whirling motion of vortex instrument, more than the 3000g, and the centrifugal 15min of room temperature (20～25 ℃);
---remove upper organic phase, take off layer 50 μ L and be used for analyzing.
B, milk:
---get the milk sample, more than the 3000g, 10 ℃ of centrifugal 10min absorb upper strata fat;
---get 5mL and remove fatty milk sample to 10mL polystyrene centrifuge tube, add 250 μ L0.36M, two hydration sodium nitroprusside solution and 250 μ L1M Zinc vitriol solution fully mix, more than the 3000g, 4～12 ℃ of centrifugal 10min;
---pipette 2.2mL upper strata liquid to another 10mL polystyrene centrifuge tube, add the 4mL ethyl acetate 10min that vibrates up and down;
---more than the 3000g, the centrifugal 10min of room temperature (20～25 ℃);
---pipette 2mL ethyl acetate upper organic phase, to the clean glass test tube of 10mL, flow down in 50～60 ℃ of water-bath nitrogen and to dry up;
---add the 0.5mL phosphate buffer, the dry residue of whirling motion 2min dissolving;
---remove upper organic phase, get 50 μ L and be used for analyzing.
(3) detecting step
A, application of sample: in the ELISA Plate micropore, add chloromycetin series standard concentration solution or sample solution 50 μ L, then add enzyme mark chloramphenicol antibody solution 50 μ L, room temperature (20～25 ℃) constant-temperature incubation 15min;
B, washing: the middle liquid that portals that inclines, every hole adds wash solution 250 μ L, washs 5 times, pats dry;
C adds luminous solution: every hole adds luminous solution 100 μ L, reaction 3min;
D detects: the luminous intensity of measuring every hole with chemical illumination immunity analysis instrument.
(4) result judges
(luminous value of (0 standard) multiply by 100 to the mean value of the standard items that obtain and sample luminous value again divided by first standard, take inhibiting rate as ordinate, the logarithm of chloramphenicol concentration is that horizontal ordinate is made typical curve, and the concentration of each sample can be read from typical curve.
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution are measured, RLU
0It is the luminous intensity values of blank (concentration is 0 standard solution).
Embodiment 5: the kit specific test
The medicine that is used for the antibody cross reaction Journal of Sex Research is and chloromycetin structure or intimate medicine: chloromycetin-glucosiduronate, chloramphenicol palmitate, thiamphenicol, Florfenicol, caproyl chloride mycin.Press the kit procedure operation, but the competition thing that adds is respectively different chloromycetin analogs, makes and suppress curve, calculate according to linear equation and respectively compete thing 50% inhibition concentration (IC50).Cross reacting rate (%CR) is antibody to the IC of chloromycetin
50With the IC of antibody to the competition thing
50The percentage of ratio, calculate by following formula:
The results are shown in table 1:
Table 1 chloromycetin kit specific test
| The competition thing | IC 50(pg/mL) | Cross reacting rate (%) |
| Chloromycetin | 59.911 | 100 |
| Chloromycetin-glucosiduronate | 66567.778 | ＜0.1 |
| Chloramphenicol palmitate | 85587.143 | ＜0.1 |
| Thiamphenicol | 119822.000 | ＜0.1 |
| Florfenicol | 74888.750 | ＜0.1 |
| The caproyl chloride mycin | 299555.000 | ＜0.1 |
Embodiment 6: the test of kit preci-sion and accuracy
Chloromycetin interpolation pork, chicken, pork liver, chicken gizzard, the flesh of fish, shrimp and milk sample with variable concentrations adds recovery test respectively, calculate different pharmaceutical and in different samples, get the recovery, thereby determine the accuracy of kit, each sample adds two concentration, each concentration is added 6 samples, extracts 3 batches of kits and tests.
Linear equation according to the typical curve of formulating carries out the quantitative calculating of the recovery, the results are shown in following table 2.
Table 2 chloromycetin pharmaceutical kit accuracy test
From the said determination result, the recovery of pork sample between 84.1～96.8%, the recovery of chicken sample between 88.6～95.2%, the recovery of pork liver sample between 86.6～106.8%, the recovery of chicken gizzard sample between 80.2～99.1%, the recovery of flesh of fish sample between 83.9～102.8%, the recovery of shrimp sample between 87.0～94.5%, the recovery of milk sample is between 77.4～99.2%.The overall recovery shows that this kit has good accuracy between 75～110%.
Claims (9)
1. the chemical luminescence ELISA detection kit of a chloromycetin comprises box body, is located at the ELISA Plate in the box body and is located at the interior reagent of box body; It is characterized in that, each hole of described ELISA Plate is coated with the envelope antigen made from chloromycetin and ovalbumin coupling; Described reagent comprises: the chloromycetin monoclonal antibody of horseradish peroxidase-labeled, chloromycetin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, chemical luminescence for liquid.
2. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: described ELISA Plate is the opaque polystyrene 96 hole chemiluminescence ELISA Plate of milky.
3. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: described envelope antigen concentration is 10 μ g/mL.
4. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: the working concentration of described chloromycetin monoclonal antibody is 1: 16000.
5. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: the monoclonal antibody of described chloromycetin is to be prepared as immunogen immune Balb/c mouse by the conjugate that chloromycetin and bovine serum albumin coupling are made.
6. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: described chloromycetin series standard solution concentration is respectively: 0pg/mL, 20pg/mL, 60pg/mL, 180pg/mL, 540pg/mL and 1620pg/mL.
7. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: described concentrated phosphoric acid salt buffer is every liter and contains NaH
2PO
42H
2O 5.74g, Na
2HPO
412H
2The aqueous solution of O 32.6g.
8. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: described thickening and washing solution is the pH=7.4 that contains volume fraction 0.05% Tween-20, the 0.1mol/L phosphate buffer.
9. the chemical luminescence ELISA detection kit of described chloromycetin according to claim 1, it is characterized in that: described chemical luminescence for liquid A liquid is that luminol content is that 0.01M, p-cresol content are three (methylol) aminomethane solution of 0.001M pH=8.8; B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2HPO
42.82g the solution of 0.75% carbamide peroxide 0.64mL, described number percent are mass percent.
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| CN201110279084.3A CN103018450B (en) | 2011-09-20 | 2011-09-20 | A kind of preparation method of chemical luminescence ELISA detection kit of chloromycetin |
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| CN201110279084.3A Active CN103018450B (en) | 2011-09-20 | 2011-09-20 | A kind of preparation method of chemical luminescence ELISA detection kit of chloromycetin |
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| CN104165990A (en) * | 2014-07-24 | 2014-11-26 | 广州万联生物科技有限公司 | Chemiluminescent enzyme-linked immunosorbent assay kit for chloramphenicol and use method thereof |
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| CN105572367A (en) * | 2014-10-13 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Chemiluminescent enzyme-linked immunoassay method for detecting pretilachlor in rice |
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| CN110146694A (en) * | 2019-04-26 | 2019-08-20 | 山东省食品药品检验研究院 | A kind of chloramphenicol direct competitive chemiluminescence high specific immunoassay method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4489156A (en) * | 1981-04-23 | 1984-12-18 | Syva Company | Chloramphenicol derivatives |
| CN1690708A (en) * | 2004-04-30 | 2005-11-02 | 中国农业大学 | Kit for detecting chloromycetin |
| CN101446589A (en) * | 2008-12-29 | 2009-06-03 | 山东大学 | Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting salbutamol |
-
2011
- 2011-09-20 CN CN201110279084.3A patent/CN103018450B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4489156A (en) * | 1981-04-23 | 1984-12-18 | Syva Company | Chloramphenicol derivatives |
| CN1690708A (en) * | 2004-04-30 | 2005-11-02 | 中国农业大学 | Kit for detecting chloromycetin |
| CN101446589A (en) * | 2008-12-29 | 2009-06-03 | 山东大学 | Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting salbutamol |
Non-Patent Citations (6)
| Title |
|---|
| HAMBURGER RN ET AL: "Chloramphenicol-Specific Antibody II. REACTIVITY TO ANALOGUES OF CHLORAMPHENICOL", 《IMMUNOLOGY》 * |
| HAMBURGER RN: "Chloramphenicol-specific antibody", 《SCIENCE》 * |
| SUXIA ZHANG ET AL: "Development of a Chemiluminescent ELISA for Determining Chloramphenicol in Chicken Muscle", 《J. AGRIC. FOOD CHEM.》 * |
| VAN DE WATER C ET AL: "Sensitive streptavidin-biotin enzyme-linked immunosorbent assay for rapid screening of chloramphenicol residues in swine muscle tissue.", 《J ASSOC OFF ANAL CHEM.》 * |
| XU CHUANLAI ET AL: "Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues", 《LUMINESCENCE》 * |
| 胥传来等: "化学发光酶免疫法测定水产品中残留氯霉素", 《分析化学》 * |
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