CN103018450B - A kind of preparation method of chemical luminescence ELISA detection kit of chloromycetin - Google Patents
A kind of preparation method of chemical luminescence ELISA detection kit of chloromycetin Download PDFInfo
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Abstract
The invention discloses a kind of chemical luminescence ELISA detection kit of chloromycetin, comprise box body, the reagent being located at the ELISA Plate in box body and being located in box body; It is characterized in that, each hole of described ELISA Plate is coated with the conjugate of envelope antigen and chloromycetin and carrier protein; 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; Chemical luminescence ELISA detection kit of the present invention have high sensitivity, easy fast, the high feature of accuracy, compare with traditional colorimetric ELISA method, the running time significantly reduces; Can be used as detecting animal tissue's (pork, chicken, pork liver, chicken gizzard), aquatic products (flesh of fish, shrimp) and Chloramphenicol Residue in Milk to detect.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit of chlorine detection mycin, for detecting chloromycetin content in animal tissue's (pork, chicken, pork liver, chicken gizzard), aquatic products (fish, shrimp), honey and milk or residual quantity.Belong to field of immunological detection.
Background technology
((Chloramphenicol, CAP) is a kind of broad-spectrum antibiotic of Cheap highly effective to chloromycetin, has good inhibiting effect to Gram-positive and negative bacteria, is therefore once being widely used in farming and animal husbandry.But animal derived food is taken in by human body for a long time along with food chain, can cause various diseases.The lighter destroys the equilibrium state of normal flora in human body, flora imbalance, makes human body produce drug-fast bacteria pearl, brings harmful effect to ill use antibiotic therapy from now on; The people of microbiotic allergic constitution there will be allergic reaction, jeopardizes health.The synthesis of bone marrow cell protein can be disturbed time serious, and suppress juvenile cell DNA to synthesize, cause granulocyte to reduce, cause the malignant diseases such as alpastic anemia, haemolysis, purple paralysis.
In view of the toxic and side effect of chloromycetin, international food educational circles is classified as banning drugs, European Union, the U.S. etc. all specify in regulation that residual chloromycetin limit standard is for " zero tolerance " (Zerotolerance), namely must not detect, according to European Union " 2002/657/EC " standard regulation, in animal derived food, the maximum of chloromycetin requires that detection limit is 0.3 μ g/kg.Soon U.S. FDA also makes corresponding regulation.The Ministry of Agriculture of China defines CAP and must not detect in the edible tissues of all food animals, and it is deleted from " Chinese veterinary pharmacopoeia ", is classified as banning drugs.And correspondingly formulated SN0219-93 and SCT3018-2004 industry standard, in line with international standards.
For adapting to higher examination criteria, detection technique level must improve accordingly, and nearly ten years chloromycetin detection techniques are one of popular projects of international expert scholar's 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 enzyme exempts from method (ELISA), and the sensitivity of detection all reaches ppb (μ g/kg) rank.The detection method of current main-stream is the method that LC-China industry standard uses, and ELISA.In recent years, Chinese scholars develops again the physical detection new methods such as liquid chromatography tandem mass spectrometry (LC-MS/MS), liquid chromatography electrospray ionization mass spectrometry (LC-EITMS), microbiological analysis, there has also been further research to the application of ELISA method.
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 advantages such as the term of validity long (6 ~ 18 months), its detectability is than euzymelinked immunosorbent assay (ELISA) and the high several order of magnitude of Physico-chemical tests 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 match, import reagent usually can not use in domestic equipment, causes chemiluminescence immunoassay method cannot popularize in basic unit.Chemoluminescent substrate is the key reagents of chemiluminescence enzyme immunity detection method, makes low cost and functional, be applicable to that domestic equipment uses luminous substrate liquid, can reduce the use cost of chemiluminescence method, what be conducive in basic unit is universal.Setting up stable chemiluminescence enzyme immunoassay method, is also the basis of the development carrying out commercial chemistry luminescence reagent box.
Summary of the invention
The object of this invention is to provide a kind of chemical luminescence ELISA detection kit of chloromycetin.This kit has that detection sensitivity is high, applying flexible, easily feature.
The chemical luminescence ELISA detection kit of chloromycetin of the present invention, comprise box body, the enzyme mark chloromycetin monoclonal antibody, chloromycetin series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the chemical luminescence for liquid that are located at the ELISA Plate in box body and are located in box body; It is characterized in that:
Each hole of described ELISA Plate is coated with the envelope antigen made with chloromycetin and ovalbumin coupling; Wherein said envelope antigen concentration is 10 μ g/mL preferably.
The preferred milky of described ELISA Plate opaque polystyrene 96 hole chemiluminescence ELISA Plate.
Described chloromycetin series standard solution dilutes and obtains from chloromycetin sterling, dilution is the 0.05mmol/L containing 10% methyl alcohol, the PBS of pH=7.4, chloromycetin standard concentration is 0pg/mL respectively, 20pg/mL, 60pg/mL, 180pg/mL, 540pg/mL and 1620pg/mL, described number percent is percent by volume.
Described enzyme mark chloromycetin monoclonal antibody is the monoclonal antibody that the artificial immunogen immune animal of being made up of chloromycetin and bovine serum albumin coupling obtains, and its working concentration is preferably 1: 16000.
Three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Described concentrated phosphoric acid salt buffer is often liter and contains NaH
2pO
42H
2o5.74g, Na
2hPO
412H
2the aqueous solution of O32.6g.
Described thickening and washing solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
Described bag is the solution (CB) containing 1.59g sodium carbonate and 2.53g sodium bicarbonate in every premium on currency by solution, pH=9.5.
Described lock solution is containing 10g ovalbumin (OVA, ovalbumin, also claim chicken ovalbumin or chicken ovalbumin, be made up of 385 amino acid residues, molecular weight is about 45kDa) and to add quality be 5 ‰ NaN in often liter of wash solution
3solution, described number percent is mass percent.
The preparation of solution of the present invention:
The sensitivity impact that the chloromycetin standard solution related in kit of the present invention, enzyme mark chloromycetin monoclonal antibody solution, chemiluminescent solution and wash solution and formula thereof detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method thereof are:
1, chloromycetin standard solution: in conventional manner by the 0.05mmol/L of chloromycetin sterling containing 10% methyl alcohol, 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 is percent by volume.
2, enzyme mark chloromycetin monoclonal antibody solution: chloromycetin monoclonal antibody is by the obtained monoclonal antibody of artificial immunizing antigen immune animal, is diluted to the working concentration of 1: 16000 after gained chloromycetin monoclonal antibody horseradish peroxidase-labeled being marked with wash solution.
3, chemiluminescent solution: three (methylol) aminomethane solution that A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.
4, concentrated phosphoric acid salt buffer: NaH
2pO
42H
2o6.52g, Na
2hPO
412H
2o38.7g is dissolved in 1L deionized water.
5, thickening and washing solution: by volume Tween-20 is added into pH=7.5, in 0.1mol/L phosphate buffer by mark 0.05%.
6, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH=9.5.
7, lock solution preparation: 10gOVA is dissolved in 1L wash solution, then adds the NaN that weight ratio is 5 ‰
3.
The bag quilt of ELISA Plate of the present invention:
In the present invention, coated elisa plate adopts and CAP-OVA conjugate is placed in the bag of setting by solution, with the concentration set, reacts bag quilt in 37 DEG C of constant temperature ovens.
What the present invention adopted is the sodium carbonate-bicarbonate buffer solution of pH=9.5.In the present invention, in microwell plate, the CAP-OVA of institute's bag quilt can well be combined on microwell plate frosting under alkaline environment, can stand repeatedly to wash plate, and the envelope antigen concentration of employing is 10 μ g/mL.
Bag can be closed by lock solution by good microwell plate, and in confining liquid, the preferred OVA of inert protein, need add NaN
3prevent from going bad.
The preparation of enzyme mark chloromycetin monoclonal antibody solution:
In the present invention, enzyme mark chloromycetin monoclonal antibody solution is the key factor determining chloromycetin enzyme linked immunological test kit measurement range and sensitivity in the present invention.
The enzyme mark chloromycetin monoclonal antibody solution related in the present invention can be diluted to the working concentration of 1: 16000 with wash solution.
The kit prepared according to above-mentioned enzyme mark chloromycetin monoclonal antibody solution concentration can reach the good range of linearity (standard lines scope can reach 0pg/mL ~ 1620pg/mL) and good sensitivity (20pg/mL).
The preparation of chemiluminescent solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly luminol-hydrogen peroxide system.
Three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Principle of the present invention is combined with enzymatic high sensitivity by the high degree of specificity that antibody-antigene reacts, 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 feature fast and accurately, compares with traditional colorimetric ELISA method, and sensitivity can improve an order of magnitude.Play a significant role during the residual chloromycetin be expected in animal food (as animal tissue, aquatic products, honey, milk) detects.
Accompanying drawing explanation
Fig. 1 is chloromycetin hapten synthesis reaction equation.
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 methyl alcohol, adds the Pd/C of 5%, passes into hydrogen, keeps certain pressure, room temperature reaction 2h, and cross and filter Pd/C, solvent evaporated obtains faint yellow thick liquid, both obtains chloromycetin haptens.The mol ratio of Pd/C and chloromycetin is 1: 10.By object obtained above at 0 ~ 5 DEG C, be 1 ~ 2 with hydrochloric acid adjust pH, stir the NaNO of lower dropping 0.1 ~ 1mol/L
2solution, makes starch potassium iodide paper become blue, then drips 0.1 ~ 1mol/L urea liquid, starch potassium iodide paper is shoaled indigo plant, then the NaOH solution adjust pH adding 0.1 ~ 1mol/L is 7 ~ 9, obtains clear liquid for subsequent use as solution A.
B, measures solution A 21.8mL and adds in DMF3mL, and 4 DEG C of precooling 10min, add tri-n-butylamine, mixing; And add isobutyl chlorocarbonate, 4 DEG C are stirred 20min.Taking 71.5mgBSA is dissolved in the DMF10mL of 50%, 4 DEG C of precoolings.The pH to 8 of BSA is adjusted with 1MNaOH.The solution A liquid prepared is added rapidly in people BSA, 4 DEG C of stirring reaction 4h.
(2) immunogene synthesis
The water-soluble solution of A, extracting chloromycetin haptens 30mg 1.5ml; The GA10 μ l getting 50% adds in 1, stirred at ambient temperature reaction 18h;
B, adds in 1 after getting the dilution of BSA100mg 1.5ml water; Reaction adds 24mgNaBH after spending the night
4reaction 3h; Dialyse 48 hours with tri-distilled water, obtain immunogene.
By above-mentioned steps reaction, extracting chloromycetin haptens 30mg, OVA100mg, synthesis CAP-OVA, for bag by.
(3) preparation of enzyme mark chloromycetin monoclonal antibody
A, animal immune: by the above-mentioned immunogene (CAP-BSA) prepared by 150 μ g/, mix with physiological saline solution immune complex and Freund's complete adjuvant equal-volume, the female mouse of neck dorsal sc injection immunity Balb/c in 6 ~ 8 week age, within after initial immunity the 7th, 14,28 day, mix with immune complex and incomplete Freund's adjuvant equal-volume, each supplementary immunization once, with immune complex 100 μ g/ only merges first 3 days, and supplementary immunization is once more not add Freund's adjuvant.
B, Fusion of Cells: carry out according to a conventional method, the splenocyte getting immune mouse mixes with the murine myeloma cell being in exponential phase (SP2/0), then the fusion agent (PEG4000) slowly adding preheating in 45 seconds merges, suspend evenly with HAT nutrient culture media, then add appropriate feeder cells, be incubated at 96 well culture plates, in 37 DEG C, 5%CO
2cultivate in incubator, within 5 days, partly change liquid with HT nutrient culture media afterwards, when 9 days, entirely change liquid.
C, the screening of hybridoma: after Fusion of Cells, when cell grows to 1/4 of culture hole area, adopts a point step screening method screening hybridoma.Primary election adopts indirect ELISA method, with envelope antigen (wrapping by concentration and positive serum dilutability by its best of square formation method conventional titration in advance) coated elisa plate, add 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 filtered out, first mixes cell conditioned medium with the chloromycetin equal-volume of 100pg/mL, 37 DEG C of water-bath effect 30min, then joins bag by good ELISA Plate.Replace chloromycetin with PBS to compare, all the other steps are the same simultaneously.If the OD450nm value after chloromycetin blocks drops to less than 50% of control wells, be then judged to the positive, detecting through 2 ~ 3 times is all positive hole, carries out subcloning immediately with limiting dilution assay.
D, monoclonal antibody preparation: 2 ~ 3 subclones are built the hybridoma after strain and expands cultivation, collects supernatant indirect ELISA mensuration and tires, frozen; And get 8 ~ 10 week age Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, lumbar injection hybridoma 1 ~ 2 × 10 after 7 ~ 10 days
6/ only, extract mouse ascites after 7 ~ 10 days, centrifuging and taking supernatant, mensuration is tired, and frozen for subsequent use.
E, prepared by monoclonal antibody linked with peroxidase: take 5mg horseradish peroxidase and be dissolved in 0.5mL deionized water, add freshly prepared 0.06MNaIO
4aqueous solution 0.5mL, refrigerator 30min is put in mixing, taking-up adds 0.16M glycol water 0.5mL, the aqueous solution 1mL containing 5mg monoclonal antibody purification is added after room temperature places 30min, mix and fill bag filter to 0.05M, pH=9.5 carbonic acid buffer slowly stir dialysis 6h make it combine, 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 by, refrigerator is put after 30 minutes centrifugal, gained sediment is dissolved in a little 0.02M, pH=7.4PBS, and to dialysed overnight, next day is centrifugal removing insolubles again, obtain enzyme-antibody conjugates, add to 5mL with 0.02M, pH=7.4PBS and carry out the preservation of mensuration postlyophilization.
The foundation of embodiment 2:ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Longitudinally press 160.0 μ g/mL, 80.0 μ g/mL, 40.0 μ g/mL, 20.0 μ g/mL with often kind of envelope antigen, 10.0 μ g/mL, 5.0 μ g/mL, 2.5 μ g/mL, the dilution series coated elisa plate of 1.25 μ g/mL, 100 μ L/ holes, after being placed in 37 DEG C of constant temperature oven 2h, pat dry; Close with 150 μ L/ hole lock solution, 37 DEG C of constant temperature ovens place 2 hours, wash plate once, pat dry; Add 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 DEG C) hatches 15min, washes plate five times, pats dry for the last time; Add the chemical luminescence for liquid in 100 μ L/ holes, measure luminous value.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with 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, 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, bag quilt: the solution by solution, envelope antigen being made into 10 μ g/mL with the carbonate bag of 0.05MpH=9.6, adds 100 μ L, 37 DEG C of constant temperature oven 2h in the reacting hole of each polystyrene board.Discard solution in hole, pat dry.
B, closes: close above-mentioned ELISA Plate of having wrapped quilt by lock solution, 150 μ L/ holes, then 37 DEG C of constant temperature oven 2h wash plate once, pat dry.
C, application of sample: add the chloromycetin solution 50 μ L/ hole of variable concentrations and dilution enzyme mark chloromycetin monoclonal antibody ((1: 16000) 50 μ L/ hole in the above-mentioned reacting hole closed, room temperature (20 ~ 25 DEG C) lucifuge hatches 15min, then wash plate five times, pat dry for the last time.
D, luminous: the chemiluminescent solution 100 μ L/ hole adding Extemporaneous in each reacting hole, detect with chemical illumination immunity analysis instrument after reaction 3min.
E, testing result calculates with inhibiting rate:
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution measure, RLU
0it is the luminous intensity values of blank (concentration is the standard solution of 0).
The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
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: the monoclonal antibody preparing gained with artificial immunizing antigen (CAP-BSA) immune animal, is diluted to 1: 16000 working concentration by after gained chloramphenicol antibody horseradish peroxidase-labeled with wash solution.
D, luminescent solution: three (methylol) aminomethane solution that A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.Front A liquid and B liquid is used to mix by 1: 1.
E, concentrated phosphoric acid salt buffer is often liter and contains NaH
2pO
42H
2o5.74g, Na
2hPO
412H
2the aqueous solution of O32.6g.
F, thickening and washing solution: the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/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 2h placed by 37 DEG C of constant temperature ovens, and incline coating buffer, pat dry, then every hole adds confining liquid 150 μ L, and 37 DEG C of constant temperature ovens place 2h, liquid in hole of inclining, cleansing solution washing once, pats dry, 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 deionized water provided in kit is used by after 1: 19 times of dilution.
B, phosphate buffer: the concentrated phosphoric acid salt buffer provided in kit is spent ionized water and use by after 1: 1 times of dilution.
C, chemiluminescent solution: before using, A liquid and B liquid are mixed by volume at 1: 1.
(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 the equal pledge of 3.0 ± 0.05g in 50mL polystyrene centrifuge tube, add 6mL ethyl acetate, use oscillator vibrates 10min, more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 10min;
---pipette 4mL upper organic phase (being about equivalent to the sample of 2g) in the clean glass test tube of 10mL, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up;
---add 1mL normal hexane, with vortex instrument whirling motion 30s, then add 1mL phosphate buffer, proceed in 2mL centrifuge tube with vortex instrument whirling motion 1min, more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 15min;
---removing upper organic phase, take off layer 50 μ L for analyzing.
B, milk:
---get milk sample, more than 3000g, 10 DEG C of centrifugal 10min, absorb upper-layer fat;
---get 5mL and remove fatty milk sample in 10mL polystyrene centrifuge tube, add 250 μ L0.36M bis-hydration sodium nitroprusside solution and 250 μ L1M Zinc vitriol solution fully mix, more than 3000g, 4 ~ 12 DEG C of centrifugal 10min;
---pipette 2.2mL upper liquid in another 10mL polystyrene centrifuge tube, add 4mL ethyl acetate and to vibrate up and down 10min;
---more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 10min;
---pipette 2mL ethyl acetate upper layer organic phase, in the clean glass test tube of 10mL, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up;
---add 0.5mL phosphate buffer, whirling motion 2min dissolves dry residue;
---removing upper organic phase, get 50 μ L for analyzing.
(3) detecting step
A, application of sample: add chloromycetin series standard strength solution or sample solution 50 μ L in ELISA Plate micropore, then adds enzyme mark chloramphenicol antibody solution 50 μ L, room temperature (20 ~ 25 DEG C) constant-temperature incubation 15min;
B, washing: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 5 times, pat dry;
C, adds luminescent solution: every hole adds luminescent solution 100 μ L, reaction 3min;
D, detects: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument.
(4) result judges
Divided by first standard, (luminous value of (0 standard) is multiplied by 100 to the mean value of the standard items obtained and sample luminous value again, take inhibiting rate as ordinate, the logarithm of chloramphenicol concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution measure, RLU
0it is the luminous intensity values of blank (concentration is the standard solution of 0).
Embodiment 5: kit specific test
Medicine for antibody cross reaction Journal of Sex Research is and chloromycetin structure or intimate medicine: chloromycetin-glucosiduronate, chloramphenicol palmitate, thiamphenicol, Florfenicol, caproyl chloride mycin.By kit procedure operation, but the competitor added is respectively different chloromycetin analogs, makes and suppresses curve, calculate each competitor 50% inhibition concentration (IC50) according to linear equation.Cross reacting rate (%CR) is the IC of antibody to chloromycetin
50with the IC of antibody to competitor
50the percentage of ratio, calculate by following formula:
The results are shown in table 1:
Table 1 chloromycetin kit specific test
| Competitor | 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 |
| Caproyl chloride mycin | 299555.000 | ＜0.1 |
Embodiment 6: kit preci-sion and accuracy is tested
Add pork, chicken, pork liver, chicken gizzard, the flesh of fish, shrimp and milk sample with the chloromycetin of variable concentrations respectively and carry out interpolation recovery test, calculate different pharmaceutical and obtain the recovery in different sample, thus determine the accuracy of kit, each sample adds two concentration, each concentration adds 6 samples, extracts 3 batches of kits and tests.
Carry out the quantitative calculating of the recovery according to the linear equation of the typical curve formulated, the results are shown in following table 2.
Table 2 chloromycetin pharmaceutical kit accuracy test
From 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, between 75 ~ 110%, shows that this kit has good accuracy.
Claims (8)
1. a preparation method for chloromycetin chemical luminescence ELISA detection kit, comprises preparation and is located at the ELISA Plate in box body and prepares the reagent be located in box body; It is characterized in that, described kit also comprises box body, and each hole of described ELISA Plate is coated with the envelope antigen made by glutaraldehyde method coupling with chloromycetin haptens and ovalbumin; 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;
Wherein, described chloromycetin monoclonal antibody is that the conjugate be made up by glutaraldehyde method coupling of chloromycetin haptens and bovine serum albumin is prepared as immunogen immune Balb/c mouse;
Described chloromycetin haptens is dissolved in methyl alcohol by chloromycetin, adds the Pd/C of 5%, pass into hydrogen, keeps certain pressure, room temperature reaction 2h, and cross and filter Pd/C, solvent evaporated obtains; The mol ratio of described Pd/C and chloromycetin is 1: 10.
2. the preparation method of chloromycetin chemical luminescence ELISA detection kit according to claim 1, is characterized in that: described ELISA Plate is milky opaque polystyrene 96 hole chemiluminescence ELISA Plate.
3. the preparation method of chloromycetin chemical luminescence ELISA detection kit according to claim 1, is characterized in that: described envelope antigen concentration is 10 μ g/mL.
4. the preparation method of chloromycetin chemical luminescence ELISA detection kit according to claim 1, is characterized in that: the working concentration of described chloromycetin monoclonal antibody is 1: 16000.
5. the preparation method of chloromycetin chemical luminescence ELISA detection kit 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.
6. the preparation method of chloromycetin chemical luminescence ELISA detection kit according to claim 1, is characterized in that: described concentrated phosphoric acid salt buffer be often liter containing NaH
2pO
42H
2o5.74g, Na
2hPO
412H
2the aqueous solution of O32.6g.
7. the preparation method of chloromycetin chemical luminescence ELISA detection kit according to claim 1, is characterized in that: described concentrated cleaning solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
8. the preparation method of chloromycetin chemical luminescence ELISA detection kit according to claim 1, it is characterized in that: described chemical luminescence for liquid comprises A liquid and B liquid, A liquid is luminol content be 0.01M, three (methylol) aminomethane solution that p-cresol content is 0.001MpH=8.8; B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the hydrogen peroxide/urea 0.64mL of 0.75%, described number percent is mass percent.
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