CN103323594A - Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application - Google Patents
Enzyme-linked immunoassay kit for detecting quinolone drugs in aquatic products and its application Download PDFInfo
- Publication number
- CN103323594A CN103323594A CN2012100774200A CN201210077420A CN103323594A CN 103323594 A CN103323594 A CN 103323594A CN 2012100774200 A CN2012100774200 A CN 2012100774200A CN 201210077420 A CN201210077420 A CN 201210077420A CN 103323594 A CN103323594 A CN 103323594A
- Authority
- CN
- China
- Prior art keywords
- solution
- enzyme
- liquid
- kit
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an enzyme-linked immunoassay kit for detecting quinolone drugs. The enzyme-linked immunoassay kit for detecting quinolone drugs provided in the invention includes an enzyme-labelled plate coated with a quinolone drug-carrier protein conjugate, an antibody working solution, an enzyme labeled secondary antibody, quinolone series standard substances, a concentrated complex solution, a concentrated washing solution, a substrate color-developing solution, and a stopping solution. The enzyme-linked immunoassay kit for detecting quinolone drugs is a multi-residue detection kit, and can simultaneously determine the total residue of 12 quinolone drugs in aquatic product (fish, shrimp) samples. The invention also discloses a method for detecting quinolone drugs by the enzyme-linked immunoassay kit. The method comprises: first conducting sample pretreatment, then carrying out detection with the kit, and finally analyzing the detection result. The kit disclosed in the invention has the advantages of simple operation, low cost, high sensitivity, and a total operation time of only 45 minutes, can perform on-site monitoring and is suitable for screening of a large number of samples.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunologic detecting kit that detects QNS, for detection of QNS content or the residual quantity in the aquatic products (fish, shrimp etc.).Belong to the immunology detection field.
Technical background
Comprecin (4-quinolones), claim again pyridonecarboxylic acids or pyridone acids, it is the newer synthetic antibacterial drug of a class, the exploitation of such medicine can be traced back to 1962, Lesher isolates a kind of secondary product-acidum nalidixicum from synthetic antimalarial chloroquine, quinolones is gone through the development in more than 40 year, developed altogether four generation QNS, amount to 50 multi-medicaments.
Comprecin is take the DNA (deoxyribonucleic acid) (DNA) of bacterium as target position, by optionally anti-bacteria dna gyrase and topoisomerase I V cause chromosomal irreversible lesion, and bacterial cell is no longer divided, Main Function is in the antibacterials of gram-negative bacteria, to a little less than the effect of gram positive bacteria (some kind has preferably antibacterial action to staphylococcus aureus).
Quinolone antimicrobial is by the difference of invention priority and anti-microbial property thereof, be divided into one, two, three, four generations, the kind of first generation Comprecin has acidum nalidixicum (Nalidixic acid) and PA (Piromidic acid) etc., the kind of second generation Comprecin has Nossacin (Cinoxacin) and methoxy oxolinic acid (Miloxacin) etc., the kind of third generation Comprecin has Norfloxacin (Norfloxaicin), Ofloxacin (Ofloxacin), Pefloxacin (Perfloxacin), Enoxacin (Enoxacin) and Ciprofloxacin (Ciprofloxacin) etc., the 4th generation Comprecin kind gatifloxacin (Gatifloxacin) and moxifloxacin (Moxifloxacin) etc. are arranged.
QNS can be used for treating the various infection of respiratory tract infection, urogenital infections, digestive system infection and other classes, also can be used for antitumor and antivirus action, but also there is very large bad reaction in such medicine to human body, such as gastrointestinal reaction, reaction hub, can bring out insane carbuncle, affect cartilage development, easily produce crystalluria and easily cause hepatic injury etc.
The quinolones medicament relict analysis generally includes: select suitable solvent extraction, further utilize liquid-liquid extraction method, solid phase extractions etc. purify, concentrated, use at last high performance liquid chromatography (High performance liquid chromatography, HPLC), high performance capillary electrophoresis (Capillary electrophorctic, CE), liquid chromatography mass coupling technique (Liquid chromatography-mas spectrometry, LC-MS) etc. method detects, sometimes also use vapor-phase chromatography (Gas chromatography, GC), high performance thin layer chromatography (High pefformancthinlay erchromatogram, HpTLC), microbial method (Microbiological assay, MA), the mensuration such as immunoassay (Immunoassay, IA).
Present detection method mainly is instrumental method, and present enzyme-linked immune analytic method can only detect one or several medicines, and the QNS more for kind also can not reach the simultaneously how residual requirement of detection far away.Simultaneously, because enzyme linked immunological kit saves time because simple to operate, be the prefered method that every country detects residue of veterinary drug.So set up the many residual enzyme-linked immunologic detection reagent kits of QNS in the test stage of maintaining strict control over food security important meaning being arranged.
Summary of the invention
The enzyme-linked immunologic detecting kit that the purpose of this invention is to provide a kind of QNS.
A kind of enzyme-linked immunologic detecting kit of QNS, comprise box body, be located at the ELISA Plate in the box body and be located at the interior reagent of box body, it is characterized in that, described reagent comprises that the sheep anti mouse of monoclonal antibody specific, horseradish peroxidase mark of QNS is two anti-, QNS series standard solution, substrate nitrite ion, stop buffer, concentrated liquid, the concentrated cleaning solution of redissolving.
Each hole of described ELISA Plate is coated with the envelope antigen made from QNS and ovalbumin coupling; The preferred 5.0 μ g/mL of wherein said envelope antigen concentration.
Described QNS series standard solution is respectively 0ng/mL, 0.2ng/mL, 0.6ng/mL, 1.8ng/mL, 5.4ng/mL and 16.2ng/mL.
Described enzyme mark sheep anti-mouse antibody is horseradish peroxidase-sheep anti-mouse igg stoste, and its working concentration is preferably 1: 2000.
Described QNS antibody is the monoclonal antibody that is made by the artificial immunogen immune animal that norfloxacin derivatives and bovine serum albumin coupling are made, and its working concentration is preferably 1: 64000.
Described QNS monoclonal antibody is to be that the fluoroquinolones monoclonal hybridoma strain D-3-1 secretion of CGMCC NO.5885 produces by deposit number.
Hybridoma cell strain D-3-1 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 03 12nd, 2012, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preservation registration number is CGMCCNO.5885.
Described nitrite ion comprises A liquid and B liquid, and A liquid is the hydrogen peroxide urea solution; B liquid is the tetramethyl biphenyl amine aqueous solution.
Described concentrated redissolution liquid is that the pH value is 7.4 phosphate buffer.
Described concentrated cleaning solution is that the pH value is 7.2 phosphate buffer.
Described coated damping fluid is that the pH value is sodium carbonate-sodium bicarbonate buffer solution of 9.6.
Described confining liquid is that the pH value is 9.2, contains the carbonate buffer solution of calf serum.
Kit maximum detection range of the present invention is 0ng/mL～16.2ng/mL.
Description of drawings
Fig. 1 is norfloxacin derivatives synthetic reaction formula.
Fig. 2 is the working curve of QNS antibody of the present invention.
Embodiment
Embodiment 1: the preparation of solution of the present invention
The QNS standard solution that relates in the kit of the present invention, enzyme mark sheep anti-mouse antibody solution, QNS antibody-solutions, substrate chromophoric solution and wash solution prescription 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, QNS standard solution: the QNS sterling is used the 0.05mmol/L that contains 10% methyl alcohol with conventional method, the PBS of pH=7.4 is mixed with concentration and is respectively 0ng/mL, 0.2ng/mL, 0.6ng/mL, 1.8ng/mL, 5.4ng/mL with the QNS standard solution of 16.2ng/mL, described number percent is percent by volume.
2, enzyme mark sheep anti-mouse antibody solution: enzyme mark sheep anti-mouse antibody is horseradish peroxidase-sheep anti-mouse igg stoste, is mixed with 1: 2000 working concentration during use with wash solution.
3, QNS antibody-solutions: QNS antibody is the monoclonal antibody that makes with artificial immunizing antigen immune animal, gained QNS antibody is diluted to 1: 64000 working concentration with wash solution.
4, substrate chromophoric solution: the A formula of liquid is to add urea peroxide 1g, citric acid 10.3g, Na in every 1000mL deionized water
2HPO
412 H
2O 35.8g, Tween-20 100 μ L transfer to pH=5; The B formula of liquid is to add tetramethyl benzidine 700mg in every 1000mL deionized water, and the 10.3g citric acid transfers to pH=2.6.
5, the concentrated liquid that redissolves: the pH value is 7.4, contains the phosphate buffer of 10% ovalbumin, 0.2mol/L, and described number percent is mass percent.
6, concentrated cleaning solution: the pH value is 7.2, and containing percent by volume is the phosphate buffer of 0.8% Tween-20,0.01 ‰ thimerosal antiseptic, 0.01mol/L, and described number percent is mass percent.
7, coated damping fluid: the pH value is sodium carbonate-sodium bicarbonate buffer solution of 9.6,0.05mol/L.
8, confining liquid: the pH value is 9.2, contains 5% calf serum, percent by volume is the carbonate buffer solution of 0.2% Tween-20,0.2mol/L, and described number percent is percent by volume.
9, stop buffer: the sulfuric acid solution take the conventional method compound concentration as 2mol/L.
Embodiment 2: ELISA Plate of the present invention coated
Coated elisa plate adopts the QNS-OVA conjugate is placed the coated solution of setting among the present invention, and with the concentration of setting, the time of setting, reaction is coated in 37 ℃ of constant temperature ovens.
The coating buffer that the present invention adopts is sodium carbonate-sodium bicarbonate buffer solution of pH=9.6.The QNS-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 5.0 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.
Enzyme-linked immunologic detecting kit of the present invention has highly sensitive, easy quick, cheap, characteristics that accuracy is high, can detect simultaneously the total residue of 12 kinds of QNSs.Be expected at animal food, play a significant role in detecting such as the quinolones medicament relict in the aquatic products.
Embodiment 3: the preparation of derivant, immunogene, coating antigen and monoclonal antibody
(1) norfloxacin derivatives is synthetic
A with Norfloxacin 1mmol, is dissolved in the 55ml methenyl choloride, adds DCC 2mmol, and the DMAP catalyzer is an amount of, p-ethyl benzene 1.5mmol, and stirring at room 5h, TLC monitoring raw material disappears, and filters, with liquid phase washing, anhydrous Na S
2O
4Drying, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/5).
B is dissolved in above-mentioned product in the methyl alcohol, adds NaOH0.76g, 60 ℃ of stirring at room 5h, TLC monitoring raw material disappears, the decompression desolventizing, the dope that obtains is dissolved in 1mol/L NaOH solution, regulate pH3～5, ethyl acetate extraction, drying, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/1), get the quinolones haptens.
(2) immunogenic synthetic
A, A liquid preparation: get 15mg Norfloxacin haptens, be dissolved among the 1mL DMF, get 15mg EDC and fully dissolve afterwards with 0.2ml water and be dissolved with among the haptenic DMF in adding, stir 24h under the room temperature, can obtain reactant liquor A.
B, the BSA coupling: take by weighing BSA40mg, make it fully to be dissolved among the 3mL PBS (PH=7.2), reactant liquor A dropwise slowly is added drop-wise in the protein solution, and under room temperature, stirs 24h,
C, dialysis: 3d changes dislysate 3 times every day with 4 ℃ of dialysis of 0.01mol/L PBS, to remove unreacted small-molecule substance.Packing saves backup in-20 ℃.
Take by weighing haptens 20mg and OVA 30mg, by the above-mentioned steps reaction, obtain envelope antigen, for coated.
(3) preparation of QNS monoclonal antibody
A, animal immune: with the above-mentioned immunogene of preparing (QNS-BSA) by 100 μ g/ only, with physiological saline solution immunogene 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 immunogene 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), and the fusion agent (PEG4000) that then slowly added preheating in 45 seconds merges, and suspends evenly with the HAT nutrient culture media, add again an amount of feeder cells, be incubated at 96 well culture plates, in 37 ℃, cultivate in the 5%CO2 incubator, partly change liquid with the HT nutrient culture media afterwards in 5 days, and entirely changed 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, OPD carries out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening that filters out mixes the Norfloxacin equal-volume of cell conditioned medium with 100 μ g/mL first, and 37 ℃ of water-bath effect 30min join in the coated good ELISA Plate again.Replace Norfloxacin with PBS simultaneously and compare, all the other steps are the same.If the OD450nm value after Norfloxacin 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, obtain at last the anti-fluoroquinolones monoclonal hybridoma of stably excreting strain D-3-1, this cell line has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 03 12nd, 2012, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preservation registration number is CGMCC NO.5885.
E, 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.
The foundation of embodiment 4:ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Vertically press 80.0 μ g/mL, 40.0 μ g/mL, 20.0 μ g/mL, 10.0 μ g/mL with every kind of envelope antigen, 5.0 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the serial dilution degree coated elisa plate of 0.625 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; The antibody (1: 1000 to 1: 512000) that adds the 50 a series of dilutions in μ L/ hole adds 1: 2000 the horseradish peroxidase-sheep anti-mouse igg antibody in 50 μ L/ holes again, and room temperature (20～25 ℃) is hatched 30min, washes plate five times, pats dry for the last time; Add respectively substrate nitrite ion A liquid 50 μ L/ holes, B liquid 50 μ L/ holes, room temperature (20～25 ℃) is hatched 15min, measures light absorption value.There are the envelope antigen concentration of obvious graded and antibody dilution to carry out specific assay as optium concentration take absorbance 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: 64000, and envelope antigen concentration is the mensuration that 5.0 μ g/mL carry out the sensitivity of antibody:
A, coated: as envelope antigen to be made into the solution of 5.0 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: add the QNS solution 50 μ L/ holes of variable concentrations, add 50 μ L/ hole ELIAS secondary antibody solution, add again 50 μ L/ hole antibody working fluids in the above-mentioned reacting hole that has sealed, room temperature (20～25 ℃) lucifuge is hatched 30min, then washes plate five times, pats dry for the last time.
D, colour developing: add substrate solution A liquid 50 μ L/ holes, add substrate solution B liquid 50 μ L/ holes again, the mixing that vibrates gently is with reacting 15min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.
E, measure: add stop buffer 50 μ L/ holes, the mixing that vibrates is gently set microplate reader in the 450nm place (suggestion detects with dual wavelength 450/630nm, please runs through data in 5min), measures every hole OD value.
F, testing result is calculated with inhibiting rate:
Percentage absorptance (%)=B/B
0, B is the mean light absorbency value of standard solution or sample solution, B
0It is the mean light absorbency value of 0ppb standard solution.
The concentration of medicine is the sensitivity of this antibody when calculating 50% inhibiting rate.
Embodiment 5: the enzyme linked immunological kit that detects QNS forms
A is coated with the solid phase carrier (ELISA Plate) of coating antigen (QNS-OVA);
B, standard items: 0ng/mL, 0.2ng/mL, 0.6ng/mL, 1.8ng/mL, 5.4ng/mL, 16.2ng/mL.
C, antibody working fluid: prepare the monoclonal antibody of gained with artificial immunizing antigen (QNS-BSA) immune animal, gained QNS antibody is diluted to 1: 64000 working concentration with wash solution.
D, ELIAS secondary antibody: enzyme mark sheep anti-mouse antibody is the working concentration that horseradish peroxidase-sheep anti-mouse igg was diluted to wash solution 1: 2000.
E, substrate nitrite ion: A liquid: add urea peroxide 1g, citric acid 10.3g, Na in the 1000mL deionized water
2HPO
412H
2O 35.8g, Tween-20 100 μ L transfer to pH=5; B liquid: add tetramethyl benzidine 700mg in the 1000mL deionized water, the 10.3g citric acid transfers to pH=2.6.
F, stop buffer: the sulfuric acid solution take the conventional method compound concentration as 2mol/L.
G, the concentrated liquid that redissolves: the pH value is 7.4, contains the phosphate buffer of 10% Tween-20,0.01 ‰ thimerosal antiseptic, 0.2mol/L.
H, thickening and washing solution: described concentrated cleaning solution is that the pH value is 7.2, contains 0.8% Tween-20,0.01 ‰ thimerosal antiseptic, 0.01 phosphate buffer.
Embodiment 6: detect the application of the enzyme linked immunological kit of QNS
(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 redissolves working fluid: the concentrated phosphoric acid salt buffer that provides in the kit is spent ionized water use after by 1: 4 times of dilution.
C, 0.1M sodium hydroxide solution: take by weighing 2.0g NaOH, add 500mL deionized water dissolving mixing.
(2) aquatic products (fish, shrimp) sample pre-treatments:
---get 2.0 ± 0.05g homogeneous sample to 50mL polystyrene centrifuge tube, add 1mL 0.1M sodium hydroxide solution, add again the 7mL acetonitrile, with the oscillator 5min that fully vibrates; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃);
---get the 2mL supernatant in the 10mL glass test tube, flow down in 50～60 ℃ of water-bath nitrogen and dry up;
---add the 1mL normal hexane with vortex instrument whirling motion 30s, add again 1mL redissolution working fluid, whirling motion 30s, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃);
---remove upper organic phase, take off layer 50 μ L and be used for analyzing.
(3) detecting step
A, application of sample: add standard items/sample 50 μ L in the micropore of correspondence, directly add ELIAS secondary antibody 50 μ L/ holes, add antibody working fluid 50 μ L/ holes again, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate;
B, washing: carefully open the cover plate film, liquid in the hole is dried, with wash operating solution 250 μ L/ holes, wash 5 times, pat dry;
C, colour developing: add substrate solution A liquid 50 μ L/ holes, add substrate solution B liquid 50 μ L/ holes again, the mixing that vibrates gently is with reacting 15min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.
D, measure: add stop buffer 50 μ L/ holes, the mixing that vibrates is gently set microplate reader in the 450nm place (suggestion detects with dual wavelength 450/630nm, please runs through data in 5min), measures every hole OD value.
The Analysis of test results process is among the present invention: use the absorbance mean value (B) of standard solution of each concentration that obtains divided by the absorbance (B of first standard solution (0 standard)
0) multiply by again 100%, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
Take the semilog value of the concentration (ng/mL) of QNS standard solution as X-axis, the percentage absorbance is Y-axis, the drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the QNS content of corresponding each sample then can be read from typical curve.
The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.
The analysis of testing result can also utilize computer professional software among the present invention, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process at most only needed to finish in 45 minutes.
The enzyme linked immunological kit that the present invention detects QNS mainly adopts the content of QNS in the qualitative or quantitative test sample of competitive ELISA method; Low to the determination requirement, sample pretreatment process is simple, simultaneously the fast detecting batch samples; Adopt the QNS monoclonal antibody of high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, the accuracy high.Enzyme linked immunological kit of the present invention, simple in structure, easy to use, low price, carrying convenience, detection method be efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.Kit of the present invention will play a significant role in the detection of QNS.
Definite test of embodiment 7 kit technical parameters
(1) standard items precision test
Respectively extract a collection of ELISA Plate out respectively from the ELISA Plate of three different time period preparations, every batch is extracted 10 kits, and every plate is extracted 20 micropores out, measures the absorbance of 0.6ng/mL standard solution, calculates the coefficient of variation.
The repeatable test of table 1 standard (CV%)
Can draw by above-mentioned test findings, 20 enzyme mark hole coefficient of variation of every batch of each 10 ELISA Plate of kit are between 3.4%～11.6%, and interassay coefficient of variation is 7.7%, meets precision and is less than or equal to 25% regulation.
(2) sample preci-sion and accuracy test
With the QNS of 1.0 μ g/kg and 2.0 μ g/kg concentration the flesh of fish and shrimp sample are added mensuration, get respectively each three of the kits of three different batches, each concentration repeats 6 times, and the calculating coefficient of variation the results are shown in Table 2～3.
The test of table 2 flesh of fish sample preci-sion and accuracy
The test of table 3 shrimp sample preci-sion and accuracy
The result shows, flesh of fish sample adds the recovery between 68.0%～99.5%, the shrimp sample adds the recovery between 66.0%～99.0%, has met [2005] No. 17 annex 2 kits of " Ministry of Agriculture's file " agricultural doctor's method and has put on record with reference to the 4th standard of accruacy in the judgment criteria.The plate within variance coefficient of flesh of fish sample is all between 2.9%～11.3%, the variation within batch coefficient is between 6.6%～11.0%, the plate within variance coefficient of shrimp sample is all between 3.7%～11.5%, the variation within batch coefficient has met [2005] No. 17 annex 2 kits of " Ministry of Agriculture's file " agricultural doctor's method and has put on record with reference to the 4th precision standard in the judgment criteria between 4.5%～11.1%.
(3) cross reacting rate test
With Ciprofloxacin as standard, if the cross reacting rate of Ciprofloxacin is 100%, the medicine that is used for the antibody cross reaction Journal of Sex Research is and Ciprofloxacin structure or intimate QNS: Ciprofloxacin, Enrofloxacin, Ofloxacin, Danofloxacin, Norfloxacin, Lomefloxacin, Pefloxacin, Enoxacin, oxolinic acid, flumequine, marbofloxacin, Amifloxacin, Difloxacin, sarafloxacin.Press the kit procedure operation, but the competition thing that adds is respectively different quinolones analogs, makes and suppress curve, calculate according to linear equation and respectively compete thing 50% inhibition concentration (IC
50).Cross reacting rate (%CR) is antibody to the IC of Ciprofloxacin
50With the IC of antibody to fluoroquinolones competition thing
50The percentage of ratio, calculate by following formula:
The results are shown in table 4:
Table 4 QNS kit specific test
| The competition thing | IC 50(ng/mL) | Cross reacting rate (%) |
| Ciprofloxacin | 0.713 | 100.0 |
| Enrofloxacin | 0.859 | 83.0 |
| Ofloxacin | 0.712 | 100.0 |
| Danofloxacin | 0.951 | 75.0 |
| Norfloxacin | 0.450 | 158.6 |
| Lomefloxacin | 0.990 | 72.0 |
| Pefloxacin | 0.422 | 169.0 |
| Enoxacin | 0.857 | 83.2 |
| Oxolinic acid | 0.784 | 91.0 |
| Flumequine | 0.977 | 73.0 |
| Marbofloxacin | 0.825 | 86.4 |
| Amifloxacin | 0.715 | 100.0 |
| Difloxacin | 891.250 | ＜0.1 |
| Sarafloxacin | 1782.500 | ＜0.1 |
(4) kit stability test
The kit preservation condition is 2～8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, QNS added the practical measurement recovery all within normal range.Consideration has improper preservation condition and occurs in transportation and use procedure, and kit was placed 15 days under 37 ℃ of preservation conditions, carries out the accelerated deterioration experiment, and the result shows that this kit indices meets the requirements fully.Consider that freezing situation may occur kit, kit was put into-20 ℃ of refrigerator freezings 15 days, measurement result shows that also the kit indices is fully normal.Can draw kit from above result can preserve more than 12 months at least at 2～8 ℃.
Claims (9)
1. enzyme linked immunological kit that detects QNS is characterized in that it contains: the ELISA Plate that is coated with coating antigen; ELIAS secondary antibody; The quinolones specific antibody.
2. enzyme linked immunological kit according to claim 1 is characterized in that: described kit also comprises QNS series standard product, substrate nitrite ion, concentrated liquid, concentrated cleaning solution, the stop buffer of redissolving.
3. enzyme linked immunological kit according to claim 1 and 2 is characterized in that: the mouse resource monoclonal antibody that the conjugate that described quinolones monoclonal antibody specific is norfloxacin derivatives and bovine serum albumin(BSA) makes as the immunogen immune mouse.
4. enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: described ELIAS secondary antibody is the sheep anti-mouse antibody of horseradish peroxidase-labeled.
5. enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: described coated damping fluid is that the pH value is sodium carbonate-sodium bicarbonate buffer solution of 9.6,0.05mol/L; Described confining liquid is that the pH value is 9.2, contains the carbonate buffer solution of 5% calf serum, 0.2% Tween-20,0.2mol/L, and described number percent is percent weight in volume.
6. enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: nitrite ion comprises A liquid and B liquid, the A formula of liquid is to add urea peroxide 1g, citric acid 10.3g, Na in every 1000mL deionized water
2HPO
412 H
2O 35.8g, Tween-20 100 μ L transfer to pH=5; The B formula of liquid is to add tetramethyl benzidine 700mg in every 1000mL deionized water, and the 10.3g citric acid transfers to pH=2.6.
7. enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: stop buffer is the sulfuric acid solution of 2mol/L.
8. enzyme linked immunological kit according to claim 1 and 2, it is characterized in that: described concentrated cleaning solution is that the pH value is 7.2, contains the phosphate buffer of 0.8% Tween-20,0.01 ‰ thimerosal antiseptic, 0.02mol/L; Described concentrated redissolution liquid is that the pH value is 7.4, contains the phosphate buffer of 10% ovalbumin, 0.2mol/L.
9. the method for a test sample quinolones medicament relict comprises step:
(1) sample pre-treatments;
(2) detect with the described kit of claim 1～8;
(3) analyzing and testing result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210077420.0A CN103323594B (en) | 2012-03-22 | 2012-03-22 | A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210077420.0A CN103323594B (en) | 2012-03-22 | 2012-03-22 | A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103323594A true CN103323594A (en) | 2013-09-25 |
| CN103323594B CN103323594B (en) | 2016-03-30 |
Family
ID=49192471
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210077420.0A Active CN103323594B (en) | 2012-03-22 | 2012-03-22 | A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103323594B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104977406A (en) * | 2014-04-03 | 2015-10-14 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting fluoroquinolone medicine and application of kit |
| CN106645728A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for fluoroquinolones drugs in foods |
| CN107255717A (en) * | 2017-07-07 | 2017-10-17 | 北京倍肯恒业科技发展股份有限公司 | Du-6859a analyte detection ELISA kit is developed and detection method |
| CN109633147A (en) * | 2018-12-07 | 2019-04-16 | 杭州康力食品有限公司 | The detection method of fluoquinolone constituents in a kind of fresh royal jelly |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766632A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting quinolones in animal derived food |
| JP2007063180A (en) * | 2005-08-31 | 2007-03-15 | Frontier Kenkyusho:Kk | Method of detecting new quinolone antibacterial agent |
| CN101315376A (en) * | 2008-06-26 | 2008-12-03 | 江南大学 | ELISA detection method for carbostyril antibiotic relict |
-
2012
- 2012-03-22 CN CN201210077420.0A patent/CN103323594B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007063180A (en) * | 2005-08-31 | 2007-03-15 | Frontier Kenkyusho:Kk | Method of detecting new quinolone antibacterial agent |
| CN1766632A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting quinolones in animal derived food |
| CN101315376A (en) * | 2008-06-26 | 2008-12-03 | 江南大学 | ELISA detection method for carbostyril antibiotic relict |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104977406A (en) * | 2014-04-03 | 2015-10-14 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting fluoroquinolone medicine and application of kit |
| CN104977406B (en) * | 2014-04-03 | 2018-01-30 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of FQNS |
| CN106645728A (en) * | 2016-11-09 | 2017-05-10 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for fluoroquinolones drugs in foods |
| CN107255717A (en) * | 2017-07-07 | 2017-10-17 | 北京倍肯恒业科技发展股份有限公司 | Du-6859a analyte detection ELISA kit is developed and detection method |
| CN109633147A (en) * | 2018-12-07 | 2019-04-16 | 杭州康力食品有限公司 | The detection method of fluoquinolone constituents in a kind of fresh royal jelly |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103323594B (en) | 2016-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101413943B (en) | Method for detecting melamine and specific enzyme-linked immunologic reagent kit | |
| CN103524427B (en) | Preparation method as well as application of trimethoprem hapten | |
| CN103018453B (en) | A kind of chemical luminescence ELISA detection kit of QNS | |
| CN101256188B (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN103323593B (en) | A kind of test paper and application thereof detecting fluoroquinolones | |
| CN103018454A (en) | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit | |
| CN103941000B (en) | A kind of detect sulfa drugs and the test strips of FQNS and method | |
| CN101451999B (en) | Directly competitive ELISA kit for detecting implicit malachite green | |
| CN101571539B (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN102827076B (en) | Universal hapten, artificial antigen and broad-spectrum monoclonal antibody for fluoroquinolone medicines as well as preparation method and application of universal hapten | |
| CN102618502B (en) | Hybridoma cell strain capable of secreting monoclonal antibodies to quinolones and application of monoclonal antibodies thereof | |
| CN103575890A (en) | Chemiluminescence assay kit of ractopamine (RAC) and application thereof | |
| CN103575889B (en) | A kind of test strips and method detecting vancomycin | |
| CN1811436B (en) | Method for detecting Ennoxacin and its special enzyme-linked immune reagent kit | |
| CN103323594B (en) | A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products | |
| CN104897896A (en) | Lincomycin magnetic immunochemiluminescence detection kit and application thereof | |
| CN101776685A (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN101403752A (en) | Chemical luminescence ELISA detection kit for gatifloxacin | |
| CN107677807A (en) | A kind of kitasamycin magnetic immunochemiluminescence detection kit | |
| CN106083815A (en) | A kind of lomefloxacin hapten preparation method and applications | |
| CN104950105B (en) | The preparation method and its application in chemiluminescence immunoassay kit of chloramphenicol haptens and antigen | |
| CN103304495B (en) | A kind of olaquindox metabolite hapten preparation method and applications | |
| CN103389373B (en) | A kind of test paper and application thereof detecting sulfa drugs | |
| CN103645322A (en) | Chemiluminescent enzyme-linked immunosorbent assay (ELISA) kit for streptomycin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |