CN103018453B - A kind of chemical luminescence ELISA detection kit of QNS - Google Patents
A kind of chemical luminescence ELISA detection kit of QNS Download PDFInfo
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- CN103018453B CN103018453B CN201110279143.7A CN201110279143A CN103018453B CN 103018453 B CN103018453 B CN 103018453B CN 201110279143 A CN201110279143 A CN 201110279143A CN 103018453 B CN103018453 B CN 103018453B
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Abstract
The invention discloses a kind of chemical luminescence ELISA detection 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, each hole of described ELISA Plate is coated with the conjugate of envelope antigen and norfloxacin derivatives and carrier protein; Described reagent comprises: antibody, QNS series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the chemical luminescence for liquid of the monoclonal antibody of QNS, the sheep anti mouse of horseradish peroxidase mark; Chemical luminescence ELISA detection kit of the present invention have high sensitivity, easy fast, accuracy is high, detection of drugs kind is many feature, compare with traditional colorimetric ELISA method, the running time significantly reduces.Can be used as detecting 12 kinds of quinolones medicament relicts in animal tissue's (pork, chicken, pork liver, chicken gizzard), aquatic products (flesh of fish, shrimp) and honey to detect.
Description
Technical field
The present invention relates to a kind of chemiluminescence immune detection reagent kit detecting QNS, for detecting QNS content in animal tissue's (pork, chicken, pork liver, chicken gizzard), aquatic products (fish, shrimp etc.), honey or residual quantity.Belong to field of immunological detection.
Background technology
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 action 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 (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 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 (High performance liquid chromatography, HPLC), high performance capillary electrophoresis (Capillary electrophorctic, CE), liquid chromatography mass coupling technique (Liquidchromatography-mas spectrometry, etc. LC-MS) method detects, sometimes vapor-phase chromatography (Gaschromatography is also used, GC), high performance thin layer chromatography (High pefformancthinlay erchromatogram, HPTLC), microbial method (Microbiological assay, MA), immunoassay (Immunoassay, the mensuration such as IA).
The method of current detection method mainly instrumental analysis, and 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.Enzyme linked immunological kit, because simple to operate, saves time, and is the prefered method that every country detects residue of veterinary drug.Chemical luminous immune detection method have high specificity, stable fast, high, the stable reagent of wide, the simple to operate automaticity of sensing range and the advantages such as the term of validity long (6 ~ 18 months), its detectability is than euzymelinked immunosorbent assay (ELISA) and the high several order of magnitude of Physico-chemical tests method.Chemoluminescent substrate is the key reagents of chemiluminescence enzyme immunity detection method, makes low cost and functional, be applicable to that domestic equipment uses luminous substrate liquid, can reduce the use cost of chemiluminescence method, what be conducive in basic unit is universal.Setting up stable chemiluminescence enzyme immunoassay method, is also the basis of the development carrying out commercial chemistry luminescence reagent box.Set up QNS many chemical residue Luminescence Enzyme linked immune assay kit detection method and 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 chemical luminescence ELISA detection kit of QNS.This kit has that detection sensitivity is high, applying flexible, easily feature.
A chemical luminescence ELISA detection kit for QNS, comprises box body, the reagent being located at the ELISA Plate in box body and being located in box body; It is characterized in that, each hole of described ELISA Plate is coated with the envelope antigen made with QNS and ovalbumin coupling; Described reagent comprises: antibody, QNS series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, the chemical luminescence for liquid of the monoclonal antibody of QNS, the sheep anti mouse of horseradish peroxidase mark.
The preferred milky of described ELISA Plate opaque polystyrene 96 hole chemiluminescence ELISA Plate.
Each hole of described ELISA Plate is coated with the envelope antigen made with QNS and ovalbumin coupling; Wherein said envelope antigen concentration is 20.0 μ g/mL preferably.
Described QNS series standard solution dilutes and obtains from quinolones sterling, dilution is the 0.05m mol/L containing 10% methyl alcohol, the PBS of pH=7.4, quinolones standard concentration is 0ng/mL respectively, 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL and 8.1ng/mL, described number percent is percent by volume.
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: 32000.
Three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Described concentrated phosphoric acid salt buffer is often liter and contains NaH
2pO
42H
2o 5.74g, Na
2hPO
412H
2the aqueous solution of O 32.6g;
Described thickening and washing solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
Described bag is the solution (CB) containing 1.59g sodium carbonate and 2.53g sodium bicarbonate in every premium on currency by solution, pH=9.5.
Described lock solution is containing 10g ovalbumin (OVA, ovalbumin, also claim chicken ovalbumin or chicken ovalbumin, be made up of 385 amino acid residues, molecular weight is about 45kDa) and to add quality be 5 ‰ NaN in often liter of wash solution
3solution, described number percent is mass percent.
The preparation of solution of the present invention:
The sensitivity impact that the QNS standard solution related in kit of the present invention, enzyme mark sheep anti-mouse antibody solution, QNS antibody-solutions, chemiluminescent solution and wash solution and formula thereof detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method thereof are:
1, 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, 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, the QNS standard solution of 2.7ng/mL and 8.1ng/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: 32000.
4, chemiluminescent solution: three (methylol) aminomethane solution that A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.
5, concentrated phosphoric acid salt buffer: NaH
2pO
42H
2o 5.74g, Na
2hPO
412H
2o 32.6g is dissolved in the deionized water of 1L.
6, thickening and washing solution: by volume Tween-20 is added into pH=7.4 by mark 0.05%, in 0.1mol/L phosphate buffer.
7, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH=9.5.
8, lock solution preparation: 10g OVA is dissolved in 1L wash solution, then adds the NaN that weight ratio is 5 ‰
3.
The bag quilt of ELISA Plate of the present invention:
In the present invention, coated elisa plate adopts and QNS-OVA conjugate is placed in the bag of setting by solution, with the concentration set, reacts bag quilt in 37 DEG C of constant temperature ovens.
What the present invention adopted is the sodium carbonate-bicarbonate buffer solution of pH=9.5.In the present invention, in microwell plate, the 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 20.0 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.
The preparation of QNS antibody-solutions and enzyme mark sheep anti-mouse antibody solution:
In the present invention, QNS antibody-solutions, enzyme mark sheep anti-mouse antibody solution concentration are the key factors determining QNS enzyme linked immunological test kit measurement range and sensitivity in the present invention.
The QNS antibody-solutions related in the present invention can be diluted to the working concentration of 1: 32000 with wash solution.
The enzyme mark sheep anti-mouse antibody solution related in the present invention preferably with wash solution preparation concentration be 1: 2000.
The kit prepared according to above-mentioned QNS antibody-solutions concentration and enzyme mark sheep anti-mouse antibody solution concentration can reach the good range of linearity (standard lines scope can reach 0ng/mL ~ 8.1ng/mL) and good sensitivity (0.1ng/mL).The preparation of chemiluminescent solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly luminol-hydrogen peroxide system.
Three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Principle of the present invention is combined with enzymatic high sensitivity by the high degree of specificity that antibody-antigene reacts, and utilizes the chemiluminescence reaction of substrate for enzymatic activity to detect production concentration.
Chemical luminescence ELISA detection kit of the present invention has highly sensitive, easy feature fast and accurately, compares with traditional colorimetric ELISA method, and sensitivity can improve an order of magnitude.Play a significant role during the quinolones medicament relict be expected in animal food (as aquatic products, animal tissue, honey) detects.
Accompanying drawing explanation
Fig. 1 is norfloxacin derivatives synthetic reaction formula.
Fig. 2 is chemiluminescence reaction formula of the present invention.
Fig. 3 is the working curve of QNS antibody of the present invention.
Embodiment
Embodiment 1: the preparation of derivant, immunogene, coating antigen and monoclonal antibody
(1) norfloxacin derivatives synthesis
A, by Norfloxacin 1mmol, is dissolved in 15mL methenyl choloride, add DCC2mmol, DMAP catalytic amount, alanine tert-butyl ester 1.5mmol, stirring at room temperature 5h, TLC monitors raw material and disappears, and filters, liquid phase is washed, anhydrous Na S2O4 is dry, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/5).
B, is dissolved in glacial acetic acid by above-mentioned product, stirring at room temperature 2h, TLC monitors raw material and disappears, decompression desolventizing, is dissolved in 1mol/LNaOH solution by the dope obtained, and regulates pH3 ~ 5, extraction into ethyl acetate, drying, column chromatography purification (eluant, eluent, ethyl acetate/petroleum ether, 1/1) quinolones haptens, is obtained.
(2) immunogenic synthesis
Prepared by A, A liquid: get 15mg Norfloxacin haptens, be dissolved in 1mL DMF, get after 15EDC 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 3mL PBS (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/lPBS 4 degree dialysis 3d.Packing, saves backup in-20 DEG C.
Take haptens 20mg and OVA 30mg, by above-mentioned steps reaction, 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), then the fusion agent (PEG4000) slowly adding preheating in 45 seconds merges, suspend evenly with HAT nutrient culture media, then add appropriate feeder cells, be incubated at 96 well culture plates, in 37 DEG C, 5%CO
2cultivate in incubator, within 5 days, partly change liquid with HT nutrient culture media afterwards, when 9 days, entirely change liquid.
C, the screening of hybridoma: after Fusion of Cells, when cell grows to 1/4 of culture hole area, adopts a point step screening method screening hybridoma.Primary election adopts indirect ELISA method, with envelope antigen (wrapping by concentration and positive serum dilutability by its best of square formation method conventional titration in advance) coated elisa plate, add measured hole culture supernatant, hatch, 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 OD after Norfloxacin blocks
450nm value drops to less than 50% of control wells, be then judged to the positive, and detecting through 2 ~ 3 times is all positive hole, carries out subcloning immediately with limiting dilution assay.
D, monoclonal antibody preparation: 2 ~ 3 subclones are built the hybridoma after strain and expands cultivation, collects supernatant indirect ELISA mensuration and tires, frozen; And get 8 ~ 10 week age Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, after 7 ~ 10 days lumbar injection hybridoma l ~ 2 × 10
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 2: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 15min, washes plate five times, pats dry for the last time; Add the chemical luminescence for liquid in 100 μ L/ holes, measure luminous intensity values.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with luminous intensity values 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: 32000, and envelope antigen concentration is the mensuration that 20.0 μ g/mL carry out the sensitivity of antibody:
A, bag quilt: the solution by solution, envelope antigen being made into 20.0 μ g/mL with the carbonate bag of 0.05M pH=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, (enzyme-antibody-solutions of (1: 32000) and enzyme mark sheep anti-mouse antibody working fluid (1: 2000) be mixed with wash solution 10: 1 proportional arrangement is by volume in in the above-mentioned reacting hole closed with the quinolones monoclonal antibody of dilution to add 50 μ L/ holes again, room temperature (20 ~ 25 DEG C) lucifuge hatches 15min, then wash plate five times, pat dry for the last time.
D, luminous: the chemiluminescent solution 100 μ L/ hole adding Extemporaneous in each reacting hole, detect with chemical illumination immunity analysis instrument after reaction 3min.
E, testing result calculates with inhibiting rate:
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution measure, RLU
0it is the luminous intensity values of blank (concentration is the standard solution of 0).
The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
Embodiment 3: the chemiluminescence enzyme linked immunoassay reagent kit detecting QNS
(1) composition of the chemiluminescence enzyme linked immunoassay reagent kit of QNS is detected
A, is coated with the solid phase carrier (ELISA Plate) of coating antigen (QNS-OVA);
B, QNS standard solution: 0ng/mL, 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL and 8.1ng/mL.
C, enzyme mark sheep anti-mouse antibody solution: enzyme mark sheep anti-mouse antibody is horseradish peroxidase-sheep anti-mouse igg stoste, loads, is mixed with the working concentration of 1: 2000 during use with wash solution.
D, QNS antibody-solutions: the monoclonal antibody preparing gained with artificial immunizing antigen (QNS-BSA) immune animal, is diluted to 1: 32000 working concentration by gained QNS antibody wash solution.
E, luminescent solution: three (methylol) aminomethane solution that A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8, B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%.
F, concentrated phosphoric acid salt buffer is often liter and contains NaH
2pO
42H
2o 5.74g, Na
2hPO
412H
2the aqueous solution G of O 32.6g, thickening and washing solution: the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
(2) preparation of ELISA Plate
With coating buffer, envelope antigen is diluted to 20.0 μ g/mL, every hole adds 100 μ L, 2h placed by 37 DEG C of constant temperature ovens, and incline coating buffer, pats dry, then every hole adds confining liquid 150 μ L, 37 DEG C of constant temperature ovens place 2h, liquid in hole of inclining, and cleansing solution washing once, pat dry, preserve with masking foil vacuum seal.
Embodiment 4: the application detecting the chemiluminescence enzyme linked immunoassay reagent 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, phosphate buffer: the concentrated phosphoric acid salt buffer provided in kit is spent ionized water and use by after 1: 1 times of dilution.
C, chemiluminescent solution: before using, A liquid and B liquid are mixed by volume at 1: 1.
(2) sample pre-treatments
A, animal tissue's (pork, chicken):
---with homogenizer homogeneous structure sample;
---take the equal pledge of 2.0 ± 0.05g in 50mL polystyrene centrifuge tube;
---add 0.6mL 0.1M hydrochloric acid solution, then add in 5.4mL anhydrous acetonitrile and mix; Vibration 3min, more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 10min;
---get in the clean glass test tube of upper organic phase 1mL to 10mL, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up;
---add 1mL normal hexane, with vortex instrument whirling motion 30s, then add 1mL phosphate buffer whirling motion 20s, more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 5min;
---removing upper strata normal hexane, takes off layer 50 μ L for analyzing.
B, aquatic products (flesh of fish, shrimp):
---get 2.0 ± 0.05g homogeneous sample in 50mL polystyrene centrifuge tube, add 1mL 0.1M hydrochloric acid solution, then add 5mL acetonitrile, immediately with vibration 3min; More than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 5min;
---get 1mL 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 phosphate buffer, whirling motion 20s, more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 5min;
---removing upper organic phase, take off layer 50 μ L for analyzing.
C, liver (pork liver, chicken gizzard):
---get 2.0 ± 0.05g homogeneous sample in 50mL polystyrene centrifuge tube, add 6mL 3%NaCL, then add 2mL methyl alcohol, whirling motion 30s, more than 3000g, room temperature (20 ~ 25 DEG C) centrifugal 5min;
---get supernatant liquor 200 μ L and 600 μ L phosphate buffers mix;
---get 50 μ L for analyzing.
D, honey:
---take in honey sample 1.0 ± 0.05g to 10mL polystyrene centrifuge tube;
---add 1mL deionized water, whirling motion mixing 5min, makes it fully dissolve;
---get 100 μ L and 900 μ L phosphate buffers dilute, whirling motion mixing 1min;
---get 50 μ L for analyzing.
(3) detecting step
A, application of sample: add QNS series standard strength solution or sample solution 50 μ L in ELISA Plate micropore, then every hole adds enzyme mark sheep anti-mouse antibody solution 50 μ L, then adds QNS antibody-solutions 50 μ L, room temperature (20 ~ 25 DEG C) constant-temperature incubation 15min;
B, washing: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 5 times, pat dry;
C, adds luminescent solution: every hole adds luminescent solution 100 μ L, reaction 3min;
D, detects: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument.
(4) result judges
The mean value of the standard items obtained and sample luminous intensity values is multiplied by 100 again divided by the luminous intensity values of first standard (0 standard), take inhibiting rate as ordinate, the logarithm of Du-6859a substrate concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
Relative luminous intensity (%)=RLU/RLU
0, RLU is the luminous intensity values that standard items or sample solution measure, RLU
0it is the luminous intensity values of blank (concentration is the standard solution of 0).
Embodiment 5: kit specific test
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 1:
Table 1 QNS kit specific test
| Competitor | IC 50(ng/mL) | Cross reacting rate (%) |
| Ciprofloxacin | 0.222 | 100.0 |
| Enrofloxacin | 0.267 | 83.0 |
| Ofloxacin | 0.222 | 100.0 |
| Danofloxacin | 0.296 | 75.0 |
| Norfloxacin | 0.140 | 158.6 |
| Lomefloxacin | 0.308 | 72.0 |
| Pefloxacin | 0.131 | 169.0 |
| Enoxacin | 0.267 | 83.2 |
| Oxolinic acid | 0.244 | 91.0 |
| Flumequine | 0.304 | 73.0 |
| Marbofloxacin | 0.257 | 86.4 |
| Amifloxacin | 0.222 | 100.0 |
| Difloxacin | 222.000 | ＜0.1 |
| Sarafloxacin | 246.667 | ＜0.1 |
Embodiment 6: kit preci-sion and accuracy is tested
Add pork, chicken, pork liver, chicken gizzard, the flesh of fish, shrimp and honey sample with different QNSs respectively and carry out interpolation recovery test, calculate different pharmaceutical and obtain the recovery in different sample, thus determine the accuracy of kit, each sample adds 1 concentration, each concentration adds 6 samples, extracts 3 batches of kits and tests.
Carry out the quantitative calculating of the recovery according to the linear equation of the typical curve formulated, the results are shown in following table 2.
Table 2 QNS kit accuracy test
From said determination result, the recovery of pork sample between 75.2 ~ 95.0%, the recovery of chicken sample between 80.3 ~ 89.9%, the recovery of pork liver sample between 80.0 ~ 99.7%, the recovery of chicken gizzard sample between 76.3 ~ 94.9%, the recovery of flesh of fish sample between 80.2 ~ 109.9%, the recovery of shrimp sample between 80.2 ~ 109.7%, the recovery of honey sample is between 70.8 ~ 89.5%.The overall recovery, between 70 ~ 110%, shows that this kit has good accuracy.
Claims (9)
1. a chemical luminescence ELISA detection kit for QNS, comprises box body, the reagent being located at the ELISA Plate in box body and being located in box body; It is characterized in that, each hole of described ELISA Plate is coated with the envelope antigen made with norfloxacin derivatives and ovalbumin coupling; Described reagent comprises: the sheep anti-mouse antibody of quinolones monoclonal antibody, horseradish peroxidase-labeled, quinolones series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, chemical luminescence for liquid, wherein, described norfloxacin derivatives is by Norfloxacin 1mmol, is dissolved in 15mL methenyl choloride, add DCC2mmol, DMAP catalytic amount, alanine tert-butyl ester 1.5mmol, stirring at room temperature 5h, TLC monitors raw material and disappears, filter, liquid phase is washed, anhydrous Na S
2o
4drying, column chromatography purification; Column chromatography purification products therefrom is dissolved in 1mol/L NaOH solution, regulates pH3 ~ 5, extraction into ethyl acetate, dry, column chromatography purification obtains.
2. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: described ELISA Plate is milky opaque polystyrene 96 hole chemiluminescence ELISA Plate.
3. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: described envelope antigen concentration is 20 μ g/mL.
4. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: the working concentration of described quinolones monoclonal antibody is 1: 32000.
5. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: the monoclonal antibody of described quinolones is that the conjugate be made up of norfloxacin derivatives and bovine serum albumin coupling prepares as immunogen immune Balb/c mouse.
6. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: described quinolones series standard solution concentration is respectively: 0ng/mL, 0.1ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL and 8.1ng/mL.
7. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: described concentrated phosphoric acid salt buffer be often liter containing NaH
2pO
42H
2o 5.74g, Na
2hPO
412H
2the aqueous solution of O 32.6g.
8. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: described thickening and washing solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
9. the chemical luminescence ELISA detection kit of quinolones according to claim 1, is characterized in that: three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH=8.8; B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%, described number percent is mass percent.
Priority Applications (1)
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| CN103630657B (en) * | 2013-11-07 | 2015-10-28 | 洛阳莱普生信息科技有限公司 | A kind of QNS rapid chemical luminescence detection kit and method of testing thereof |
| CN103645320B (en) * | 2013-11-19 | 2015-12-02 | 洛阳莱普生信息科技有限公司 | A kind of AMOZ chemiluminescent enzyme-linked immunosorbent immunity quick detection kit |
| CN104165991A (en) * | 2014-07-24 | 2014-11-26 | 广州万联生物科技有限公司 | Enrofloxacin chemiluminescent enzyme-linked immunosorbent assay kit and use method thereof |
| CN106771142A (en) * | 2016-11-30 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of Ofloxacin detection method and kit |
| CN108614114A (en) * | 2016-12-12 | 2018-10-02 | 丹阳亿太生物科技发展有限公司 | A kind of chemiluminescence enzyme-linked immunoassay of detection sarafloxacin |
| CN107247136A (en) * | 2017-08-14 | 2017-10-13 | 天津科技大学 | A kind of preparation method of Norfloxacin electrochemical immunosensor |
| CN109115749A (en) * | 2018-08-12 | 2019-01-01 | 河北英茂生物科技有限公司 | A kind of Broadspectrum specificity molecularly imprinted polymer, chemical luminescence reagent kit and the detection method and application of quinolone drugs |
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