CN103323594B - A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products - Google Patents
A kind of enzyme linked immunological kit and application thereof detecting QNS in aquatic products Download PDFInfo
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Abstract
The invention discloses a kind of enzyme linked immunological kit detecting QNS.The enzyme linked immunological kit of detection QNS provided by the present invention, comprises the ELISA Plate, antibody working fluid, ELIAS secondary antibody, quinolones serial standards, concentrated liquid, concentrated cleaning solution, substrate nitrite ion, the stop buffer of redissolving that are coated with QNS and carrier protein couplet thing.The enzyme linked immunological kit of detection QNS of the present invention is multi-residue determination kit, can the total residue of 12 kinds of QNSs in Simultaneously test aquatic products (flesh of fish, shrimp) sample, the invention also discloses a kind of method applying above-mentioned enzyme linked immunological kit detection QNS, it comprises: first carry out sample pre-treatments, then detect with kit, ultimate analysis testing result.Kit disclosed by the invention is easy and simple to handle, low cost, highly sensitive, the whole running time only needs 45 minutes, can on-site supervision and be applicable to the examination of great amount of samples.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunologic detecting kit detecting QNS, for detecting QNS content in aquatic products (fish, shrimp etc.) or residual quantity.Belong to field of immunological detection.
Technical background
Comprecin (4-quinolones), also known as pyridonecarboxylic acids or pyridone acids, it is the synthetic antibacterial drug that a class is newer, the exploitation of such medicine can trace back to 1962, Lesher isolates a kind of secondary product-acidum nalidixicum from synthesis antimalarial chloroquine, quinolones goes through the development of more than 40 year, have developed altogether four generation QNS, amount to 50 multi-medicaments.
Comprecin with the DNA (deoxyribonucleic acid) of bacterium (DNA) for target position, chromosomal irreversible lesion is caused by optionally anti-bacteria DNA gyrase and topoisomerase I V, and bacterial cell is no longer divided, mainly act on the antibacterials of gram-negative bacteria, to the effect of gram positive bacteria more weak (some kind has good antibacterial use to staphylococcus aureus).
Quinolone antimicrobial is by inventing successively and the difference of anti-microbial property, be divided into one, two, three, four generations, the kind of first generation Comprecin has acidum nalidixicum (Nalidixicacid) and PA (Piromidicacid) 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 kind of forth generation Comprecin has gatifloxacin (Gatifloxacin) and moxifloxacin (Moxifloxacin) etc.
QNS can be used for the various infection for the treatment of respiratory tract infection, urogenital infections, digestive system infection and other classes, also can be used for antitumor and antivirus action, but such medicine also exists very large bad reaction to human body, as gastrointestinal reaction, reaction hub, insane carbuncle can be brought out, affect cartilage development, easily produce crystalluria and easily cause hepatic injury etc.
Quinolones medicament relict analysis generally includes: select suitable solvent extraction, utilize liquid-liquid extraction method further, solid phase extractions etc. purify, concentrated, finally use high performance liquid chromatography (Highperformanceliquidchromatography, HPLC), high performance capillary electrophoresis (Capillaryelectrophorctic, CE), liquid chromatography mass coupling technique (Liquidchromatography-massspectrometry, etc. LC-MS) method detects, sometimes vapor-phase chromatography (Gaschromatography is also used, GC), high performance thin layer chromatography (Highpefformancthinlayerchromatogram, HpTLC), microbial method (Microbiologicalassay, MA), immunoassay (Immunoassay, the mensuration such as IA).
Current detection method mainly instrumental method, and current enzyme-linked immune analytic method, can only detect one or several medicines, also can not reach far away detect many residual requirements simultaneously for the QNS that kind is more., because enzyme linked immunological kit is because simple to operate, save time, be the prefered method that every country detects residue of veterinary drug meanwhile.So set up the many residual enzyme-linked immunologic detection reagent kits of QNS have important meaning in the test stage maintaining strict control over food security.
Summary of the invention
The object of this invention is to provide a kind of enzyme-linked immunologic detecting kit of QNS.
A kind of enzyme-linked immunologic detecting kit of QNS, 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, described reagent comprises the monoclonal antibody specific of QNS, sheep anti mouse two anti-, QNS series standard solution, substrate nitrite ion, the stop buffer of horseradish peroxidase mark, concentrated liquid, the concentrated cleaning solution of redissolving.
Each hole of described ELISA Plate is coated with the envelope antigen made with QNS and ovalbumin coupling; Wherein said envelope antigen concentration is 5.0 μ g/mL preferably.
Described QNS series standard solution is 0ng/mL, 0.2ng/mL, 0.6ng/mL, 1.8ng/mL, 5.4ng/mL and 16.2ng/mL respectively.
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 the artificial immunogen immune animal of being made up of norfloxacin derivatives and bovine serum albumin coupling obtains, and its working concentration is preferably 1: 64000.
Described QNS monoclonal antibody is that the fluoroquinolones monoclonal hybridoma strain D-3-1 being CGMCCNO.5885 by deposit number secretes generation.
Hybridoma cell strain D-3-1 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 03 12nd, 2012, 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 hydrogen peroxide urea solution; B liquid is tetramethyl biphenyl amine aqueous solution.
Described concentrated redissolution liquid to be pH value be 7.4 phosphate buffer.
Described concentrated cleaning solution to be pH value be 7.2 phosphate buffer.
Described bag is buffered liquid to be pH value be 9.6 sodium carbonate-bicarbonate buffer solution.
Described confining liquid is pH value is 9.2, the carbonate buffer solution containing calf serum.
Kit maximum detection range of the present invention is 0ng/mL ~ 16.2ng/mL.
Accompanying drawing explanation
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 sensitivity impact that the QNS standard solution related in kit of the present invention, enzyme mark sheep anti-mouse antibody solution, QNS antibody-solutions, substrate chromophoric solution and wash solution formula detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method thereof are:
1, QNS standard solution: in conventional manner by the 0.05mmol/L of QNS sterling containing 10% methyl alcohol, the PBS of pH=7.4 is mixed with concentration and is respectively 0ng/mL, 0.2ng/mL, 0.6ng/mL, 1.8ng/mL, the QNS standard solution of 5.4ng/mL and 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 the working concentration of 1: 2000 with wash solution during use.
3, QNS antibody-solutions: QNS antibody is by the obtained monoclonal antibody of artificial immunizing antigen immune animal, gained QNS antibody wash solution is diluted to the working concentration of 1: 64000.
4, substrate chromophoric solution: A formula of liquid is add urea peroxide 1g, citric acid 10.3g, Na in every 1000mL deionized water
2hPO
412H
2o35.8g, Tween-20 100 μ L, is adjusted to pH=5; B formula of liquid is add tetramethyl benzidine 700mg in every 1000mL deionized water, and 10.3g citric acid, is adjusted to pH=2.6.
5, the concentrated liquid that redissolves: pH value is 7.4, the phosphate buffer containing 10% ovalbumin, 0.2mol/L, and described number percent is mass percent.
6, concentrated cleaning solution: pH value is 7.2, be the phosphate buffer of 0.8% Tween-20,0.01 ‰ thiomersal preservative, 0.01mol/L containing percent by volume, described number percent is mass percent.
7, bag is buffered liquid: pH value is the sodium carbonate-bicarbonate buffer solution of 9.6,0.05mol/L.
8, confining liquid: pH value is 9.2, be the carbonate buffer solution of 0.2% Tween-20,0.2mol/L containing 5% calf serum, percent by volume, described number percent is percent by volume.
9, stop buffer: compound concentration is the sulfuric acid solution of 2mol/L in conventional manner.
Embodiment 2: the bag quilt of ELISA Plate of the present invention
In the present invention, coated elisa plate adopts and QNS-OVA conjugate is placed in the bag of setting by solution, with the concentration set, the time of setting, reacts bag quilt in 37 DEG C of constant temperature ovens.
The coating buffer that the present invention adopts is the sodium carbonate-bicarbonate buffer solution of pH=9.6.In the present invention, in microwell plate, the QNS-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 5.0 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.
Enzyme-linked immunologic detecting kit of the present invention has highly sensitive, easy quick, cheap, feature that accuracy is high, can detect the total residue of 12 kinds of QNSs simultaneously.Be expected at animal food, as played a significant role in the quinolones medicament relict detection in aquatic products.
Embodiment 3: the preparation of derivant, immunogene, coating antigen and monoclonal antibody
(1) norfloxacin derivatives synthesis
A, by Norfloxacin 1mmol, is dissolved in 55ml methenyl choloride, adds DCC2mmol, and DMAP catalyzer is appropriate, p-ethyl benzene 1.5mmol, and stirring at room temperature 5h, TLC monitor raw material and disappear, and filter, and liquid phase are washed, anhydrous Na
2sO
4drying, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/5).
B, is dissolved in methyl alcohol by above-mentioned product, adds NaOH0.76g, 60 DEG C are stirred 5h, TLC and monitor raw material disappearance, decompression desolventizing, the dope obtained is dissolved in 1mol/LNaOH solution, regulate pH3 ~ 5, extraction into ethyl acetate, dry, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/1), obtain quinolones haptens.
(2) immunogenic synthesis
Prepared by A, A liquid: get 15mg Norfloxacin haptens, be dissolved in 1mLDMF, get after 15mgEDC 0.2ml water fully dissolves and be dissolved with in haptenic DMF in adding, stirred at ambient temperature 24h, can obtain reactant liquor A.
B, BSA coupling: take BSA40mg, makes it fully to be dissolved in 3mLPBS (PH=7.2), is dropwise slowly added drop-wise in protein solution by reactant liquor A, and in stirred at ambient temperature 24h,
C, dialysis: change 3 dislysates every day, to remove unreacted small-molecule substance with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Take haptens 20mg and OVA30mg, by above-mentioned steps reaction, obtain envelope antigen, for bag by.
(3) preparation of QNS monoclonal antibody
A, animal immune: by the above-mentioned immunogene (QNS-BSA) prepared by 100 μ g/, mix with physiological saline solution immunogene 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 immunogene 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), and the fusion agent (PEG4000) then slowly adding preheating in 45 seconds merges, and suspends evenly with HAT nutrient culture media, add appropriate feeder cells again, be incubated at 96 well culture plates, in 37 DEG C, cultivate in 5%CO2 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, sheep anti-mouse igg-HRP is added and IgM-HRP, OPD carry out chromogenic reaction after cleaning.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first mixes the Norfloxacin equal-volume of cell conditioned medium with 100 μ g/mL, 37 DEG C of water-bath effect 30min, then joins bag by good ELISA Plate.Replace Norfloxacin with PBS to compare, all the other steps are the same simultaneously.If the OD450nm value after Norfloxacin 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, finally obtain stably excreting anti-fluoroquinolones monoclonal hybridoma strain D-3-1, this cell line has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 03 12nd, 2012, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preservation registration number is CGMCCNO.5885.
E, 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.
The foundation of embodiment 4:ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Longitudinally press 80.0 μ g/mL, 40.0 μ g/mL, 20.0 μ g/mL, 10.0 μ g/mL with often kind of envelope antigen, 5.0 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the dilution series coated elisa plate of 0.625 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 the antibody (1: 1000 to 1: 512000) of the 50 a series of dilutions in μ L/ hole, add the horseradish peroxidase-sheep anti-mouse igg antibody of 1: 2000 of 50 μ L/ holes again, room temperature (20 ~ 25 DEG C) hatches 30min, washes plate five times, pats dry for the last time; Add substrate nitrite ion A liquid 50 μ L/ hole respectively, B liquid 50 μ L/ hole, room temperature (20 ~ 25 DEG C) hatches 15min, measures light absorption value.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with 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, 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, bag quilt: the solution by solution, envelope antigen being made into 5.0 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: the QNS solution 50 μ L/ hole adding variable concentrations, add 50 μ L/ hole ELIAS secondary antibody solution, add 50 μ L/ hole antibody working fluids again in the above-mentioned reacting hole closed, room temperature (20 ~ 25 DEG C) lucifuge hatches 30min, then wash plate five times, pat dry for the last time.
D, colour developing: add substrate solution A liquid 50 μ L/ hole, then add substrate solution B liquid 50 μ L/ hole, mixing of vibrating gently, react 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
E, measures: add stop buffer 50 μ L/ hole, mixing of vibrating gently, and setting microplate reader, in 450nm place (suggestion dual wavelength 450/630nm detects, and please runs through data in 5min), measures every hole OD value.
F, testing result calculates with inhibiting rate:
Percentage absorptance (%)=B/B
0, B is the mean absorbance values of standard solution or sample solution, B
0it is the mean absorbance values of 0ppb standard solution.
The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
Embodiment 5: the enzyme linked immunological kit composition detecting QNS
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: the monoclonal antibody preparing gained with artificial immunizing antigen (QNS-BSA) immune animal, is diluted to 1: 64000 working concentration by gained QNS antibody wash solution.
D, ELIAS secondary antibody: enzyme mark sheep anti-mouse antibody is the working concentration that horseradish peroxidase-sheep anti-mouse igg wash solution is diluted to 1: 2000.
E, substrate nitrite ion: A liquid: add urea peroxide 1g in 1000mL deionized water, citric acid 10.3g, Na
2hPO
412H
2o35.8g, Tween-20 100 μ L, is adjusted to pH=5; B liquid: add tetramethyl benzidine 700mg in 1000mL deionized water, 10.3g citric acid, is adjusted to pH=2.6.
F, stop buffer: compound concentration is the sulfuric acid solution of 2mol/L in conventional manner.
G, the concentrated liquid that redissolves: pH value is 7.4, the phosphate buffer containing 10% Tween-20,0.01 ‰ thiomersal preservative, 0.2mol/L.
H, thickening and washing solution: described concentrated cleaning solution is pH value is 7.2, containing 0.8% Tween-20,0.01 ‰ thiomersal preservative, 0.01 phosphate buffer.
Embodiment 6: the application detecting the enzyme linked immunological kit of QNS
(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, redissolution working fluid: the concentrated phosphoric acid salt buffer provided in kit is spent ionized water and use by after 1: 4 times of dilution.
C, 0.1M sodium hydroxide solution: take 2.0g NaOH, adds the mixing of 500mL deionized water dissolving.
(2) aquatic products (fish, shrimp) sample pre-treatments:
---get 2.0 ± 0.05g homogeneous sample in 50mL polystyrene centrifuge tube, add 1mL0.1M sodium hydroxide solution, then add 7mL acetonitrile, fully to vibrate 5min with oscillator; More than 3000g, room temperature (20-25 DEG C) centrifugal 10min;
---get 2mL supernatant in 10mL glass test tube, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up;
---add 1mL normal hexane vortex instrument whirling motion 30s, then add 1mL redissolution working fluid, whirling motion 30s, more than 3000g, room temperature (20-25 DEG C) centrifugal 5min;
---removing upper organic phase, take off layer 50 μ L 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/ hole, then add antibody working fluid 50 μ L/ hole, mixing of vibrating gently, reacts 30min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;
B, washing: carefully open cover plate film, dries liquid in hole, with wash operating solution 250 μ L/ hole, washs 5 times, pat dry;
C, colour developing: add substrate solution A liquid 50 μ L/ hole, then add substrate solution B liquid 50 μ L/ hole, mixing of vibrating gently, react 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
D, measures: add stop buffer 50 μ L/ hole, mixing of vibrating gently, and setting microplate reader, in 450nm place (suggestion dual wavelength 450/630nm detects, and please runs through data in 5min), measures every hole OD value.
In the present invention Analysis of test results process for: with the absorbance values (B) of the standard solution of each concentration the obtained absorbance (B divided by first standard solution (0 standard)
0) be multiplied by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) × 100%
With the semilog value of the concentration of QNS standard solution (ng/mL) for X-axis, percentage absorbance is Y-axis, drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the QNS content of each sample corresponding then can read from typical curve.
In the present invention, the analysis of testing result also can adopt regression equation method, calculates sample solution concentration.
In the present invention, the analysis of testing result can also utilize computer professional software, and this method is more convenient for the express-analysis of a large amount of sample, and whole testing process at most only needs can complete for 45 minutes.
The enzyme linked immunological kit that the present invention detects QNS mainly adopts the content of QNS in the qualitative or quantitative detection sample of competitive ELISA method; Require low to the pre-treatment of sample, sample pretreatment process is simple, can detect batch samples fast simultaneously; 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, accuracy high.Enzyme linked immunological kit of the present invention, structure is simple, easy to use, low price, carrying convenience, detection method are efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.Kit of the present invention plays a significant role in the detection of QNS.
The determination test of embodiment 7 kit technical parameter
(1) standard items precision test
Respectively the ELISA Plate prepared from three different time periods respectively extract a collection of ELISA Plate out, often criticize each extraction 10 kits, 20 micropores extracted out by every plate, measure the absorbance of 0.6ng/mL standard solution, calculate the coefficient of variation.
Table 1 standard repeatability test (CV%)
Can be drawn by above-mentioned test findings, between 20 the enzyme mark hole coefficient of variation 3.4% ~ 11.6% often criticizing each 10 ELISA Plate of kit, interassay coefficient of variation is 7.7%, meets the regulation that precision is less than or equal to 25%.
(2) sample preci-sion and accuracy test
Carry out interpolation mensuration with the QNS of 1.0 μ g/kg and 2.0 μ g/kg concentration to the flesh of fish and shrimp sample, get each three of the kit of three different batches respectively, each concentration repeats 6 times, calculates the coefficient of variation, the results are shown in Table 2 ~ 3.
The test of sample preci-sion and accuracy oppressed by table 2
Table 3 shrimp sample preci-sion and accuracy is tested
Result shows, flesh of fish sample TIANZHU XINGNAO Capsul is between 68.0% ~ 99.5%, shrimp sample TIANZHU XINGNAO Capsul, between 66.0% ~ 99.0%, has met " Ministry of Agriculture's file " agriculture doctor's method [2005] No. 17 annex 2 kits and has put on record with reference to the 4th standard of accruacy in judgment criteria.In the plate of flesh of fish sample, the coefficient of variation is all between 2.9% ~ 11.3%, between variation within batch coefficient 6.6% ~ 11.0%, in the plate of shrimp sample, the coefficient of variation is all between 3.7% ~ 11.5%, variation within batch coefficient, between 4.5% ~ 11.1%, has met " Ministry of Agriculture's file " agriculture doctor's method [2005] No. 17 annex 2 kits and has put on record with reference to the 4th precision standard in judgment criteria.
(3) cross reacting rate test
Using Ciprofloxacin as standard, if the cross reacting rate of Ciprofloxacin is 100%, the medicine for 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.By kit procedure operation, but the competitor added is respectively different quinolones analogs, makes and suppresses curve, calculate each competitor 50% inhibition concentration (IC according to linear equation
50).Cross reacting rate (%CR) is the IC of antibody to Ciprofloxacin
50with the IC of antibody to fluoroquinolones competitor
50the percentage of ratio, calculate by following formula:
The results are shown in table 4:
Table 4 QNS kit specific test
| Competitor | 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) stabilization of kit test
Kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, QNS added the practical measurement recovery all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 15 days under 37 DEG C of preservation conditions by kit, carry out accelerated aging tests, result shows that this kit indices meets the requirements completely.Consider that freezing situation may occur kit, kit is put into-20 DEG C of refrigerator freezings 15 days, measurement result also shows that kit indices is completely normal.Can show that kit at least can be preserved more than 12 months at 2 ~ 8 DEG C from above result.
Claims (8)
1. detect an enzyme linked immunological kit for QNS, it is characterized in that it contains: the ELISA Plate being coated with coating antigen; ELIAS secondary antibody; Quinolones specific antibody; Described quinolones specific antibody is the mouse resource monoclonal antibody that the conjugate of norfloxacin derivatives and bovine serum albumin(BSA) obtains as immunogen immune mouse; Described norfloxacin derivatives is dissolved in 55mL methenyl choloride by Norfloxacin 1mmol, adds DCC2mmoL, and DMAP catalyzer is appropriate, p-ethyl benzene 1.5mmoL, and stirring at room temperature 5h, TLC monitor raw material and disappear, and filter, and liquid phase are washed, anhydrous Na
2sO
4drying, column chromatography purification, is dissolved in methyl alcohol by obtained product, adds NaOH0.76g, and 60 DEG C are stirred 5h, TLC monitors raw material and disappears, and decompression desolventizing, is dissolved in 1mol/LNaOH solution by the dope obtained, and regulates pH3 ~ 5, be extracted with ethyl acetate, dry, column chromatography purification, obtains.
2. enzyme linked immunological kit according to claim 1, is characterized in that: described kit also comprises bag and is buffered liquid, confining liquid, QNS serial standards, 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: described ELIAS secondary antibody is the sheep anti-mouse antibody of horseradish peroxidase-labeled.
4. enzyme linked immunological kit according to claim 2, is characterized in that: the sodium carbonate-bicarbonate buffer solution of described bag is buffered liquid to be pH value be 9.6,0.05mol/L; Described confining liquid is pH value is 9.2, containing percent by volume be 5% calf serum, percent by volume is the carbonate buffer solution of 0.2% Tween-20,0.2mol/L.
5. enzyme linked immunological kit according to claim 2, is characterized in that: nitrite ion comprises A liquid and B liquid, and A formula of liquid is add urea peroxide 1g, citric acid 10.3g, Na in every 1000mL deionized water
2hPO
412H
2o35.8g, Tween-20 100 μ L, is adjusted to pH=5; B formula of liquid is add tetramethyl benzidine 700mg in every 1000mL deionized water, and 10.3g citric acid, is adjusted to pH=2.6.
6. enzyme linked immunological kit according to claim 2, is characterized in that: described stop buffer is the sulfuric acid solution of 2mol/L.
7. enzyme linked immunological kit according to claim 2, it is characterized in that: described concentrated cleaning solution is pH value is 7.2, containing percent by volume be 0.8% Tween-20, mass percent is the phosphate buffer of 0.01 ‰ thiomersal preservative, 0.02mol/L; Described concentrated redissolution liquid is pH value is 7.4, is the phosphate buffer of 10% ovalbumin, 0.2mol/L containing mass percent.
8. detect a method for sample quinolones medicament relict, comprise step:
(1) sample pre-treatments;
(2) detect with the kit described in any one of claim 1 ~ 7;
(3) testing result is analyzed.
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| 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 |
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| 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 |
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| CN103323594B true CN103323594B (en) | 2016-03-30 |
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| 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 |
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| CN101315376A (en) * | 2008-06-26 | 2008-12-03 | 江南大学 | ELISA detection method for carbostyril antibiotic relict |
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| CN101315376A (en) * | 2008-06-26 | 2008-12-03 | 江南大学 | ELISA detection method for carbostyril antibiotic relict |
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