CN103575878A - Chemiluminiscence enzyme-linked immunosorbent assay kit for chloramphenicol in aquatic products and detection method of chloramphenicol - Google Patents
Chemiluminiscence enzyme-linked immunosorbent assay kit for chloramphenicol in aquatic products and detection method of chloramphenicol Download PDFInfo
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Abstract
The invention discloses a chemiluminiscence enzyme-linked immunosorbent assay (ELISA) kit for chloramphenicol in aquatic products and a detection method of the chloramphenicol and belongs to the field of chemiluminiscence enzyme immunoassays. The chemiluminiscence enzyme-linked immunosorbent assay kit comprises a box body, an ELISA plate and a reagent arranged in the box body, wherein the ELISA plate is coated with a coating antigen; the reagent comprises an enzyme-labeled antibody, namely a horse radish peroxidase labeled chloramphenicol antibody, a chloramphenicol serial standard solution, a concentrated phosphate buffering solution, a concentrated scrubbing solution and a luminous solution. The principle of the kit is that the coating antigen is adsorbed on a micro-pore plate to form a solid-phase antigen and the chloramphenicol antibody is connected with horse radish peroxidase to form an enzyme-labeled object; a luminol system is used as a luminous base material to establish a chloramphenicol enzymatic chemiluminiscence immunoassay. According to the kit, the sensitivity is 0.0125ng/mL, the in-batch variable coefficient is less than 8%, the between-batch variable coefficient is less than 20%, the recycling rate of fish and shrimp samples is 87%-101% and the linear detection range is within 0.0125mg/mL-4.05ng/mL. The chemiluminiscence enzyme-linked immunosorbent assay kit has the characteristics of high sensitivity, convenience, rapidness and accuracy.
Description
Technical field
Chemiluminescent enzyme-linked immunosorbent immunoassay kits and a detection method thereof, belong to chemiluminescence immunoassay technology field, for the detection of aquatic products chloromycetin content.
Background technology
In the streptothricial nutrient culture media of the initial Shi You Venezuela of chloromycetin (Chloramphenicol, CAP), extracting and make, is a class broad-spectrum antibiotic.Widespread use in the animal husbandry of chloromycetin Zeng China, has played vital role to disease control and the treatment of livestock and poultry.Chloromycetin exists serious toxic and side effect, can cause people's alpastic anemia, granular white blood cells deficiency disease, neonate, premature's Synthetic Grey disease etc., low concentration medicament residue also can bring out the drug resistance of pathogenic bacteria, and the mankind's health is formed to huge potential threat.In the U.S., only allow chloromycetin to be applied to non-consumption animal, be defined in any animal food and must not detect chloromycetin.The European Community forbids that chloromycetin is applied to milking cow and laying hen, and the content in other animal foods also limits, and stipulates that high residue amount is 100ng/kg.The Ministry of Agriculture deletes chloromycetin to classify banning drugs as from version < < Chinese veterinary pharmacopoeia > > in 2002, has in recent years been defined in animal food and must not have detected.
As the Hubei aquatic products industry of national fresh water fishery the first province, within continuous 13 years, freshwater product total amount keeps the whole nation first.180 of the existing aquatic products processing of our province enterprises, wherein export processing enterprise is 19.As country, prohibite the veterinary drug of use, in the animal derived foods such as aquatic products, residual chloromycetin is listed in one of emphasis essential items for inspection always.
At present, widely used CAP detection technique has liquid phase chromatography (LC), mass spectroscopy (MS), high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA) etc.The large-scale instrument analytical approachs such as LC have higher requirements to equipment and operator, and sample treatment is complicated, and detection time is long.
It is to adopt traditional enzyme linked immunosorbent assay to detect that the chloromycetin of having applied at present detects more in Patents, as: Southern Yangtze University's application number is 02137940, ELISA kit and detection method > > thereof in < < animal food by name; China Agricultural University's application number is 200410037254, the kit > > of < < chlorine detection mycin by name; China Agricultural University's application number is 200410037255, the kit > > of a < < chlorine detection mycin by name; China Agricultural University's application number is 200410037258, the enzyme linked immunological kit > > of < < chloromycetin by name; China Agricultural University's application number 200410037259, the enzyme linked immunological kit > > of a < < chlorine detection mycin by name; Beijing Wanger Bioisystech Co., Ltd's application number 200510086775, a < < enzyme linked immunological kit > > who detects chloromycetin medicine in animal food by name etc.In this method, conventional chromogenic substrate is tetramethyl benzidine (TMB) or o-phenylenediamine (OPD), and wherein OPD has carcinogenicity, and long-term use meeting works the mischief to human body; And the more unstable external interference that is subject to of this class substrate is affected final measurement result, as illumination or metallic ion can make substrate self colour developing.Enzyme linked immunosorbent assay is with substrate for enzymatic activity colour developing, conventionally needs colour developing 10---60 minutes, and make colour developing reach plateau, after colour developing, need by sour color development stopping measurement result.
Partial monopoly relates to colloid gold test paper method in addition, as: rich biotechnology (Hangzhou) company limited application number 200310109470, immune colloid gold test paper of < < chlorine detection mycin by name and preparation method thereof > >; Yunnan University's application number 200610019376, < < chloromycetin colloidal gold test > > by name etc.The method is suitable easy and simple to handle, and the used time is short in batch samples examination, but sensitivity is lower, and result is inaccurate, is prone to false positive or false negative.
Also have partial monopoly to have employing liquid chromatography-tandem mass spectrometry method for determining, as: Xuhui District, Shanghai central hospital application number 200410067401, the liquid chromatography-tandem mass spectrometry method for determining > > of a < < Chloramphenicol Residue in Honey residual quantity by name; The People's Republic of China Jiangsu Entry-Exit Inspection and Quarantine Bureau application number 200810195887, the method > > that < < multi-kind pesticide residues in bee products by name detects simultaneously, this class methods measurement result is accurate, but sample pretreatment process is loaded down with trivial details, need to use a large amount of organic solvents, detect and need extensive instrument.
In addition also has Tianjin Shengji Group Co.Ltd's application number 200810053150, the method for quick > > of forbidden drug in < < feed by name or veterinary drug, this patent adopts thin-layered chromatography to detect forbidden drug, this method is easy and simple to handle, but in sample preparation process, conventionally use a large amount of organic solvents, and testing result can only qualitatively be sentenced accurately detection of drugs content.Southern Yangtze University's application number 200910027458, the immuomagnetic bead chromatographic test strip of a < < fast detecting chloromycetin by name and preparation method thereof > >, adopts immuomagnetic bead chromatographic test strip detection the method and colloid gold test paper method similar.Jiangsu Inst of Atomic Medical Sciences application number 200910182284, the light-induced chemiluminescent immunoassay kit > > of a < < chloromycetin by name, adopt light induced chemiluminescence immunoassay method, the method need break through repeatedly mark, being about to CAP-OVA is coated in luminous particle, synthesizing biotinylated two is anti-, and Streptavidin is coated on photosensitive particulate.The present invention combines the chemical luminescent detecting technology with height sensitivity with the immune response of high specific, easy and simple to handle, the used time is short, without long-time colour developing and termination, and highly sensitive, specificity is good.
Summary of the invention
The object of the present invention is to provide a kind of chemical luminescence ELISA detection kit and detection method thereof of chlorine detection mycin, for the detection of aquatic products chloromycetin content.This kit has that detection sensitivity is high, applying flexible, feature easily.
The present invention mainly adopts chemiluminescence enzyme immunoassay method chlorine detection mycin.Its technical scheme: a kind of chloromycetin chemiluminescence enzyme immunoassay kit, comprise box body, be located at the ELISA Plate in box body and be located at the reagent in box body; It is characterized in that, each hole of described ELISA Plate is coated with antigen, and wherein envelope antigen is the comlete antigen of being made by the coupling of EDC (carbodiimide) method by the little molecule of chloromycetin and carrier protein; Described reagent comprises: chloromycetin series standard solution, enzyme labelled antibody, concentrated phosphoric acid salt buffer, thickening and washing solution, luminous solution.
In the chemical luminescence ELISA detection kit of above-mentioned chloromycetin:
Described envelope antigen is the comlete antigen of being made by carbodlimide method coupling by the little molecule of chloromycetin and carrier protein; Described microwell plate is the detachable or non-dismountable enzyme mark orifice plate of 48 holes or 96 holes or other hole counts; Described enzyme labelled antibody is to be made by the coupling of sodium periodate method by horseradish peroxidase and chloramphenicol antibody; Described chloromycetin series standard solution is respectively to dilute and obtain from chloromycetin sterling, and in chloromycetin standard items, chloramphenicol concentration is respectively: 0.0125ng/mL, 0.025ng/mL, 0.05ng/mL, 0.15ng/mL, 0.45ng/mL, 1.35ng/mL, 4.05ng/mL; Described chemical luminescence for liquid is containing damping fluid, luminol, p-Coumaric Acid, sodium perborate etc.
By the method for described kit chlorine detection mycin, the basis of mensuration is antigen-antibody reaction.In white Chemiluminescent plate, add chloromycetin standard solution or the sample of handling well and enzyme labelled antibody in turn, after inculation washing, add luminescent solution to detect luminous value, to add the luminous value drawing standard curve of chloromycetin standard items, with the luminous value that adds sample, from typical curve, calculate the content of chloromycetin in sample.
Described kit test method, adds standard solution or the sample of handling well and enzyme mark thing to every hole in the ELISA Plate being coated with, and at 25-49 ℃, incubation is 15 minutes.After washing, every hole adds luminescent solution, after 1-5 minute, on chemiluminescence detector, detects.Standard items luminous value with gained, luminous value divided by first standard (0 standard) is multiplied by 100 again, and the rate that is inhibited (B/B0) be 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.
The method of described chlorine detection mycin, its sample preparation:
Fishes and shrimps sample: get 10g and remove fatty sample homogenization, weigh the sample and 6mL ethyl acetate mixing 10min of 3g homogenization, the centrifugal 5min of 4000g, transferase 12 mL ethyl acetate upper strata is in clean glass tube, 50 ℃ of gentle nitrogen flow evaporator ethyl acetate, fat residue is dissolved in 1mL normal hexane and mixes 1min, and adds 1mL sample diluting liquid.Vortex mixed 1min, 4000g centrifuging 5min.(noting: as lower floor's emulsification, test tube can be placed in to 80 ℃ of water-bath 5min left and right, and again carry out centrifugal), discards upper strata, draw 100μ L lower floor solution for detection of.Final detection result should be multiplied by extension rate 1.
The sensitivity of described kit curve is 0.0125ng/mL.
Described kit precision can reach variation within batch coefficient and be less than 8%, and interassay coefficient of variation is less than 20%.
Described kit fishes and shrimps sample recovery rate is 87.4---and 101%.
Accompanying drawing explanation
The selection in Fig. 1-reaction time
Fig. 2-chloromycetin chemiluminescent enzyme-linked immunosorbent immune response typical curve
The schematic diagram directly perceived of Fig. 3-kit
In figure: box body (1), ELISA Plate (2), enzyme labelled antibody working fluid (3), chloromycetin standard solution (4)---(11), concentrated phosphoric acid salt buffer (12), concentrated cleaning solution (13), luminescent solution (14)
The schematic diagram directly perceived of Fig. 4-ELISA Plate
Embodiment
The preparation of embodiment 1, envelope antigen, enzyme labelled antibody and dilution selection
1. the preparation of envelope antigen
Take carrier protein (bovine serum albumin(BSA) BSA) and dissolve with ultrapure water, being mixed with concentration is 5---the BSA solution of 30mg/mL.Take chloromycetin and dissolve with ultrapure water, being mixed with concentration is 1---the CAP solution of 5mg/mL.Take EDCHCl, add ultrapure water and dissolve, being mixed with concentration is 1---the EDC solution of 5mg/mL.In EDC solution, add 0.1---0.5mL CAP solution, mix 2min, again the CAP solution having activated is joined in 0.5mL BSA solution, after mixing up and down, room temperature lucifuge is mixed 2h, collects coupled product and is transferred to 30kDa super filter tube, the centrifugal 5min of 3000rpm at 4 ℃ of refrigerated centrifuges, add equal-volume glycerine and mix ,-20 ℃ frozen.
2. the preparation of enzyme labelled antibody
Take HRP and be dissolved in ultrapure water, being mixed with concentration is 1---the HRP solution of 10mg/mL, and 4 ℃ of hold over night, make it abundant dissolving;---1mL newly join 0.01---the 1M NaIO that gets above-mentioned HRP solution 0.5mL add wherein 0.025 next day
4aqueous solution, mixes rear placement refrigerator lucifuge and stirs 5---after 20 minutes, takes out.Take out reactant liquor and pack in bag filter, 4 ℃ of acetum dialysed overnight to pH4.0.Take out the potpourri after dialysis next day, regulates potpourri pH value, makes it approach 9.6; Immediately by 0.5---the chloromycetin monoclonal antibody of 5mg adds, and room temperature lucifuge stirs.---0.5mL concentration is 1---NaBH4 of 10mg/mL that adds 0.05 is got in mixed liquor in reaction after finishing; Then mixed liquor ultrafiltration being added to equivalent glycerine preserves.
3. envelope antigen and enzyme labelled antibody concentration screening
Longitudinally use envelope antigen according to 1: the serial dilution degree coated elisa plate of 20K, 1: 40K, 1: 80K, 1: 100K, 1: 200K, 1: 400K, every kind of coated 2 row of concentration, 100 μ L/ holes, 0---4 ℃ of placements are spent the night.Inferior daily cleansing solution is washed plate 5 times, pats dry at every turn; 50---300 μ L/ hole confining liquid sealings, 25---placing 0.5 for 40 ℃---2 hours, washes plate 5 times, pats dry at every turn.
Odd column adds 50 μ L/ hole PBS damping fluids, even column adds the chloromycetin standard solution that 50 μ L/ hole concentration are 4ppb, then laterally uses enzyme labelled antibody according to 1: the dilutability of 30K, 1: 50K, 1: 60K, 1: 80K adds, and every kind of concentration adds 2 row, every hole adds 50 μ L, room temperature places 15---and 60 minutes, wash plate 5 times, pat dry at every turn, add 100 μ L/ hole TMB developers, room temperature lucifuge places 5---45 minutes, add 50 μ L/ hole stop buffers, 450nm place test OD value.
Table 1-envelope antigen and enzyme labelled antibody concentration are selected
Different antigen concentrations, in conjunction with different antibodies concentration gained absorbance and the absorbance that adds competition to obtain after antigen, are selected antigen coated concentration 1: 400K as can be seen from Table 1, and enzyme labelled antibody working concentration selects 1: 80K, corresponding absorbance is suitable compared with other concentration.
4. the reaction time is selected
Envelope antigen is diluted to 3 μ g/mL with the carbonate buffer solution of 0.1M pH=9.5, and every hole adds 200 μ L to be placed in that 4 ℃ of refrigerators are coated to spend the night.Take out the ELISA Plate of envelope antigen with every hole after the PBST washing of 0.01M pH=7.4 5 times and add 300 μ L 1%BSA (PBST of 0.01M pH=7.4) confining liquids next day, is placed in 37 ℃ of water-baths sealings and washs 5 times after 1 hour again.
In the ELISA Plate being coated with, every hole adds standard solution 100 μ L, concentration of standard solution is 0,0.1,10 and 1000ng/mL (non-specific point) and enzyme mark thing 50 μ L, enzyme mark thing dilutability is 1: 10K, at 37 ℃, difference incubation is 5,15,30,60 minutes.Wash after 5 times, every hole adds TMB nitrite ion 100 μ L and 37 ℃ of incubations 15 minutes, last every hole added 50 μ L stop buffers, detects OD450nm value.
After 15 minutes reaction time, tend to balance as seen from Figure 1, each standard point OD450nm variation later in 30 minutes is not obvious, and in order to shorten detection time, selecting the reaction time is 15 minutes.
5. chloromycetin chemiluminescent enzyme-linked immunosorbent immune response typical curve is set up
By coupled antigen with the carbonate buffering of 0.1M pH=9.5 according to 1: 400K dilution, every hole adds 200 μ L to be placed in that 4 ℃ of refrigerators are coated to spend the night.Take out the ELISA Plate of envelope antigen with every hole after the PBST washing of 0.01M pH=7.4 5 times and add 300 μ L 1%BSA (PBST of 0.01M pH=7.4) confining liquids next day, is placed in 37 ℃ of water-baths sealings and washs 5 times after 1 hour again.
In the ELISA Plate being coated with, every hole adds standard solution 100 μ L, concentration of standard solution is 0,0.0125,0.025,0.05,0.15,0.45,1.35,4.05ng/mL and enzyme mark thing 50 μ L, enzyme mark thing dilutability is 1: 80K, at room temperature incubation is 15 minutes.Wash after 5 times, every hole adds luminol chemiluminescence liquid 180 μ L, after 5 minutes, on chemiluminescence detector, detects.
6. the assembling of the chemiluminescence enzyme linked immunoassay reagent kit of chlorine detection mycin
1) (comlete antigen that chloromycetin and carrier protein couplet are made, coated concentration is 1: solid phase carrier 400K) (NUNC white Chemiluminescent plate) to be coated with coupled antigen
2) (working concentration is 1 to horseradish peroxidase-labeled chloramphenicol antibody working fluid: 80K)
3) chloromycetin standard solution is 8 bottles: concentration is respectively 0,0.0125,0.025,0.05,0.15,0.45,1.35,4.05ng/mL
4) concentrated phosphoric acid salt buffer solution: every liter containing NaCl 80g, KH
2pO
42.0g, Na
2hPO
412H
2the aqueous solution of 029.0g, KCl 2.0g
5) concentrated cleaning solution: be that to add volume ratio in above-mentioned concentrated phosphoric acid salt buffer be 0.05% Tween-20
6) luminescent solution: the 2mmol/L luminol of 0.1M pH=8.5Tris-HCl damping fluid preparation, wherein reinforcing agent is that p-Coumaric Acid concentration is 0.25mmol/L, oxygenant is that sodium perborate concentration is 20mmol/L.
7) preparation of ELISA Plate
By coupled antigen with the carbonate buffer solution of 0.1M pH=9.5 according to 1: 400K dilution, every hole adds 200 μ L to be placed in that 4 ℃ of refrigerators are coated to spend the night.Take out the ELISA Plate of coated antibody with every hole after the PBST washing of 0.01M pH=7.4 5 times and add 300 μ L 1%BSA (PBST of 0.01M pH=7.4) confining liquids next day, being placed in 37 ℃ of water-bath sealings washs 5 times after 1 hour again, pat dry, with masking foil vacuum seal, at 4 ℃, preserve.
7. the mensuration program of the chemiluminescence enzyme linked immunoassay reagent kit of chloromycetin of the present invention
The points for attention that kit is used
1) use before all reagent is placed to equilibrium at room temperature 30 minutes to 1 hour, after use, put back to immediately 4 ℃ of Refrigerator stores.
2) in use must be able to not allow micropore dry.
3) in strict accordance with instructions, operate.
4) in use, avoid light direct projection, in the process of hatching, use masking foil lucifuge.
● the preparation of reagent
5) sample diluting liquid: the concentrated phosphoric acid salt buffer providing in kit is used afterwards with 10 times of distilled water dilutings.
6) cleansing solution: the concentrated cleaning solution providing in kit is used afterwards with 10 times of distilled water dilutings.
7) luminescent solution: the 2mmol/L luminol of 0.1M pH=8.5Tris-HCl damping fluid preparation, wherein reinforcing agent is that p-Coumaric Acid concentration is 0.25mmol/L, oxygenant is that sodium perborate concentration is 20mmol/L.
Sample pre-treatments
8) tissue sample (shrimp, fish and meat): get 10g and remove fatty sample homogenization, weigh the sample and 6mL ethyl acetate mixing 10min of 3g homogenization, the centrifugal 5min of 4000g, transferase 12 mL ethyl acetate upper strata is in clean glass tube, 50 ℃ of gentle nitrogen flow evaporator ethyl acetate, fat residue is dissolved in 1mL normal hexane and mixes 1min, and adds 1mL sample diluting liquid.Vortex mixed 1min, 4000g centrifuging 5min.(noting: as lower floor's emulsification, test tube can be placed in to 80 ℃ of water-bath 5min left and right, and again carry out centrifugal), discards upper strata, draw 100μ L lower floor solution for detection of.Final detection result should be multiplied by extension rate 1.
● detecting step
9) in the ELISA Plate being coated with, every hole adds standard solution 100 μ L, standard solution (or sample of handling well) concentration of standard solution is 0,0.0125,0.025,0.05,0.15,0.45,1.35,4.05ng/mL and enzyme mark thing 50 μ L, at room temperature incubation 15 minutes respectively.
10) washing, the middle liquid that portals that inclines, the cleansing solution to adding 300 μ L/ holes in ELISA Plate, pats dry after standing 2 minutes, repeats five times.
11) every hole adds luminol chemiluminescence liquid 180 μ L, after 5 minutes, on chemiluminescence detector, detects.
● result judgement
Standard items luminous value with gained, luminous value divided by first standard (0 standard) is multiplied by 100 again, and the rate that is inhibited (B/B0) be 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.
% inhibiting rate=% standard items (sample) luminous value/0 standard items luminous value.
8. the sensitivity of kit, accuracy
● the sensitivity of kit
Detect 10 0 standard models simultaneously, obtain
value, and this value substitution typical curve is tried to achieve to sensitivity is 0.0125ng/mL.
● with batch in error and batch between the accuracy of expression of error
Error in batch: each standard items concentration is done 5 times and repeated, error in representing to criticize with its variation within batch coefficient.
Error between batch: the measurement operation that repeats to have set up, make continuously error between representing to criticize with its interassay coefficient of variation 5 times.
Table 2-criticize interior, batch between error
The precision of embodiment seven, kit
Get 3 parts of 10g shrimps, add a certain amount of chloromycetin to be mixed with that concentration is 0.2,0.8, the shrimp sample of 2ng/mL, organize preprocess method to process, and try to achieve the recovery according to embodiment tri-, the results are shown in table 4, the recovery is 87.4---101%.
Table 3-shrimp sample recovery rate
Embodiment bis-, the specific test of kit
Judge that the specific index of kit is cross reacting rate, by chloromycetin and with chloromycetin, have the several drugs of similar structures and function to measure cross reacting rate.Typical curve by various medicines obtains its 50% inhibition concentration.With following formula, calculate the cross reacting rate of kit to other drug.Cross reacting rate is less, illustrates that this kit is better to the detection specificity of chloromycetin.Cross reacting rate (%)=(suppressing the chloromycetin of the concentration of 50% chloromycetin/suppress 50% like substrate concentration) * 100%
Table 4-element cross reacting rate
Claims (9)
1. a chemiluminescent enzyme-linked immunosorbent immunoassay kits for chloromycetin, is characterized in that being coated with by (1) microwell plate of envelope antigen, (2) chloromycetin standard items, (3) enzyme labelled antibody working fluid, (4) damping fluid, (5) cleansing solution, (6) luminescent solution forms.
2. kit according to claim 1, is characterized in that envelope antigen (1) is the comlete antigen of being made by the coupling of EDC (carbodiimide) method by the little molecule of chloromycetin and carrier protein (bovine serum albumin(BSA) BSA).
3. kit according to claim 1, is characterized in that this enzyme labelled antibody of enzyme mark chloramphenicol antibody (3) is that horseradish peroxidase is connected with chloramphenicol antibody by sodium periodate method, forms enzyme labelled antibody.
4. kit according to claim 1, is characterized in that being coated with the microwell plate (1) of envelope antigen, it is characterized by the detachable or non-dismountable microwell plate of 48 holes, 96 holes or other hole counts.
5. kit according to claim 1, is characterized in that being coated with the microwell plate of envelope antigen, its preparation: to be coated with damping fluid dilution, it is coated that every hole adds 50-300 μ L to be placed in 4 ℃ of refrigerators by envelope antigen.After next day the microwell plate of envelope antigen being taken out to washing, every hole adds 50-300 μ L confining liquid, is placed in 25-40 ℃ of water-bath sealing and washs after 0.5-2 hour again, pats dry, and with vacuum seal, at 4 ℃, preserves.
6. kit according to claim 1, is characterized in that luminescent solution (6), and this luminescent solution comprises T damping fluid, luminol, p-Coumaric Acid, sodium perborate.
7. the method with kit chlorine detection mycin claimed in claim 1, it is characterized in that adding chloromycetin standard solution or the sample of handling well and enzyme labelled antibody in microwell plate, after inculation washing, add luminescent solution to detect luminous value, to add the luminous value drawing standard curve of chloromycetin standard items, with the luminous value that adds sample, from typical curve, calculate the content of chloromycetin in sample.
8. the method for chlorine detection mycin according to claim 7, is characterized in that being operating as: to every hole in the ELISA Plate being coated with, add standard solution or the sample of handling well and enzyme mark thing, at 25-40 ℃, incubation is 15 minutes.After washing, every hole added after luminescent solution 1-5 minute to be detected on chemiluminescence detector.With the standard items luminous value of gained, divided by the luminous value of first standard (0 standard), be multiplied by again 100, the rate that is inhibited (B/B
0), take inhibiting rate as ordinate, the logarithm of chloramphenicol concentration is that horizontal ordinate is made typical curve, the concentration of each sample can be read from typical curve.
9. according to the method for the chlorine detection mycin described in claim 7,8, it is characterized in that sample preparation:
Fishes and shrimps sample: get 10g and remove fatty sample homogenization, weigh the sample and 6mL ethyl acetate mixing 10min of 3g homogenization, the centrifugal 5min of 4000g, transferase 12 mL ethyl acetate upper strata is in clean glass tube, 50 ℃ of gentle nitrogen flow evaporator ethyl acetate, fat residue is dissolved in 1mL normal hexane and mixes 1min, and adds 1mL sample diluting liquid.Vortex mixed 1min, 4000g centrifuging 5min.(noting: as lower floor's emulsification, test tube can be placed in to 80 ℃ of water-bath 5min left and right, and again carry out centrifugal), discards upper strata, draw 100μ L lower floor solution for detection of.Final detection result should be multiplied by extension rate 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105486823A (en) * | 2014-10-13 | 2016-04-13 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit for detecting pretilachlor and detection method thereof |
| 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 |
-
2012
- 2012-08-09 CN CN201210280415.XA patent/CN103575878A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105486823A (en) * | 2014-10-13 | 2016-04-13 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit for detecting pretilachlor and detection method thereof |
| 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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Application publication date: 20140212 |