CN104897895A - Gentamycin magnetic immunochemiluminescence detection kit and application thereof - Google Patents

Gentamycin magnetic immunochemiluminescence detection kit and application thereof Download PDF

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CN104897895A
CN104897895A CN201510175688.1A CN201510175688A CN104897895A CN 104897895 A CN104897895 A CN 104897895A CN 201510175688 A CN201510175688 A CN 201510175688A CN 104897895 A CN104897895 A CN 104897895A
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gentamicin
gentamycin
magnetic
enzyme
solution
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CN104897895B (en
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聂雯莹
冯静
杜美红
罗晓琴
朱亮亮
万宇平
孙倩倩
王然
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Beijing Kwinbon Biotechnology Co Ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
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Abstract

The invention relates to a magnetic immunochemiluminescence kit for detecting gentamycin, which comprises the following reagents, enzyme-labelled antigen, an enzyme-labelled antigen weak solution, a magnetic antibody, a magnetic antibody weak solution, a gentamycin series standard substance solution, a chemiluminescence substrate solution A, a chemiluminescence substrate solution B, a concentrated compound solution, and a concentrated washing liquid. Enzyme-labelled antigen is a horseradish peroxidase-labelled gentamycin hapten marker, an alcoholic hydroxyl group in a gentamycin original drug is oxidized by pyridinium dichromate cas to obtain keto, monoketal gentamycin and hydrazinobenzene formate are subjected to a condensation reaction to obtain carboxyl gentamycin hapten products, the magnetic antibody is obtained by coupling the gentamycin monoclonal antibody and magnetic bead. The invention also relates to a method for detecting gentamycin in milk and milk powder by employing the kit and a matched chemiluminescence detector, and the method has advantages of high sensitivity, specificity and short detection time for detecting gentamycin.

Description

A kind of magnetic immunochemiluminescence detection kit of gentamicin and application thereof
Technical field
The present invention relates to a kind of chemiluminescence detection kit and detection method thereof.Particularly detect the magnetic immunochemiluminescence detection kit of gentamicin residue amount in the sample such as milk, milk powder.Belong to field of immunological detection.
Background technology
Gentamicin (Gentamicin) is a kind of aminoglycoside antibiotics produced by small single-cell bacteria fermentation.The infection that extensively should cause in quality Gram-negative bacteria.Gentamicin antimicrobial spectrum is comparatively wide, can the Growth and reproduction of effective anti-bacteria, is one of veterinary drug of commonly using of current Chinese agriculture, animal husbandry and aquatic products industry.But along with widely using of gentamicin, some bacteriums are gradually to which creating drug resistance, and serious to the infringement of cranial nerve, the sense of hearing and kidney, and along with a large amount of uses of this medicine, its residue detection is also valued by the people gradually.China and European Union remain strict demand to gentamicin, and the maximum residue limit in milk can not higher than 100 μ g/kg.
The detection method that gentamicin residue amount is commonly used mainly contains high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), high performance liquid chromatography mass spectrum series process (HPLC-MS/MS), LC-MS-MS (LC-MS), thin-layered chromatography (TLC), immunoassay etc.Due to the instrument and equipment of costliness and the loaded down with trivial details operating process of complexity, and the high professional qualification requirement to reviewer, make above-mentioned instrument detection method be not suitable for the examination of a large amount of sample.Although Enzyme-Linked Immunospot and colloidal gold immunochromatographimethod detection method belong to Rapid Screening method, but because method sensitivity is lower, false negative rate and false positive rate higher, highly sensitive gradually, accuracy is high, the chemical luminous immune detection method that detection time is short substitute, in magnetic immunochemiluminescence immunologic function test reagent supporting magnetic immunochemiluminescence detector detection food, gentamicin is residual, realize the full-automation of testing process, reduce manual operation error, sensitivity can reach 0.1 μ g/L, accuracy is high, testing cost is low, be applicable to the examination of gentamicin residue amount in batch samples.
Summary of the invention
Technical matters to be solved by this invention, from being to provide a kind of gentamicin detection kit, has higher sensitivity, specificity and accuracy when adopting this kit to carry out the detection of gentamicin.
Another object of the present invention is the detection method providing a kind of gentamicin, when adopting this kit fiting chemical luminometer to carry out the detection of gentamicin, not only there is higher sensitivity, specificity and accuracy, and achieve full-automatic detection, shorten detection time, reduce manual operation error.
For achieving the above object, the invention provides a kind of gentamicin detection kit, its main agents comprised has: enzyme-labelled antigen, enzyme-labelled antigen dilution, magnetic labeling antibody, magnetic labeling antibody dilution, gentamicin serial standards solution, chemical luminous substrate A, B liquid.
Described enzyme-labelled antigen is the haptenic label of gentamicin of horseradish peroxidase-labeled, gentamicin haptens is, with Pyridinium dichromate, the alcoholic extract hydroxyl group in former for gentamicin medicine is oxidized to ketone group, obtain single ketones base gentamicin, then single ketones base gentamicin with condensation reaction is carried out to formic acid phenylhydrazine, obtain the gentamicin haptens product with carboxyl.
Described magnetic labeling antibody is that gentamicin monoclonal antibody and magnetic bead coupling obtain.
Described magnetic bead surfaces contains-OH ,-COOH or-NH 2reactive group.
Described gentamicin monoclonal antibody is that the conjugate obtained by gentamicin haptens and bovine serum albumin(BSA) prepares as immunogen immune Balb/c mouse.
Described chemical luminous substrate A liquid is the solution of the trishydroxymethylaminomethane containing luminol and p-cresol.Chemical luminescence for liquid B liquid is for containing citric acid, anhydrous Na 2hPO 4with CO (NH2) 2H 2o 2aqueous solution.
Described kit also comprises standard solution, concentrated liquid and the concentrated cleaning solution of redissolving.
Described kit can carry out the detection of gentamicin residue amount in animal derived sample by supporting chemiluminescence detector.
The present invention also provides a kind of method utilizing the supporting Chemiluminescence Apparatus of kit to detect gentamicin, comprises the following steps:
1) enzyme-labelled antigen and enzyme-labelled antigen dilution are diluted according to the volume ratio of 1:10 ~ 1:20, load in chemiluminescence detector enzyme-labelled antigen working fluid container;
2) magnetic labeling antibody and magnetic labeling antibody dilution are diluted according to the volume ratio of 1:10 ~ 1:20, load in chemiluminescence detector magnetic labeling antibody working fluid container;
3) chemiluminescence detector is from dividing do not move absorption 30 μ L ~ 80 μ L enzyme-labelled antigen, 30 μ L ~ 80 μ L sample extracting solutions and 30 μ L ~ 80 μ L magnetic labeling antibodies, join reaction zone successively, at room temperature react 15min, distinguish from 4min at Magneto separate, with cleansing solution 300 μ L ~ 500 μ L, complex precipitate is cleaned 3 ~ 5 times after abandoning supernatant;
4) compound of separator well is put into measurement camera bellows, add chemical luminous substrate A liquid and each 30 μ L ~ 80 μ L of B liquid, detect the relative light intensity (RLU) sent, the content of gentamicin and RLU proportion relation in sample, can calculate the residual concentration of gentamicin by RLU typical curve.
The chemiluminescence detector that in the present invention, analysis test method is used comprises power circuit, reaction cup memory storage, sample storage device, sample arm, reagent storage means, reagent arm, campaign-styled cold storage plant, cleaning device, automatic injection pump, twilight detector, also be configured with the Windows control software design of computing machine and Chinese interface simultaneously, data typing can be carried out, result gathers, quality control, the functions such as result storage and result queries, the programming of multiple analytical model can be completed, quantitative or qualitatively to report the result, automatic generation also stores and more New function, 2 auto modification typical curves.
Enzyme-labelled antigen of the present invention is the haptenic label of gentamicin of horseradish peroxidase-labeled, and it is stored in containing pH7.2 ~ 7.6, containing tween 0.03% ~ 0.05% Tween-20, in the phosphate buffer of 0.02mol/L ~ 0.05mol/L.Described percentage composition is mass percentage.
Described enzyme-labelled antigen dilution is pH7.2 ~ pH7.6, Na 3pO 4the buffer solution of concentration to be 0.01mol/L, NaCl concentration be 0.25mol/L.
Described magnetic labeling antibody is that gentamicin monoclonal antibody and magnetic bead coupling obtain.The content of magnetic bead surfaces group is 0.1eq/g ~ 0.3eq/g, and described magnetic labeling antibody is kept at pH7.2 ~ 7.6, containing tween 0.1% ~ 0.3% Tween-20, in the phosphate buffer of 0.02mol/L ~ 0.05mol/L.Described percentage composition is mass percentage.
Described magnetic labeling antibody dilution is pH7.2 ~ pH7.6, Na 3pO 4the buffer solution of concentration to be 0.02mol/L, NaCl concentration be 0.3mol/L.
The conjugate that described gentamicin monoclonal antibody is obtained by gentamicin haptens and bovine serum albumin(BSA) obtains as the titration of the screening of immunogen immune Balb/c mouse, Fusion of Cells, hybridoma, subclone and mouse ascites.
Described chemical luminous substrate A liquid is luminol content is 0.01 μ g/L ~ 0.03 μ g/L, p-cresol content be 0.001 μ g/L ~ 0.005 μ g/L, pH is the tris solution of 8.0 ~ 9.0, B liquid is that every 100mL aqueous solution contains citric acid 1.7g ~ 2.3g, anhydrous Na 2hPO 42.2g ~ 3.0g and volumn concentration are CO (NH2) 2H of 0.75% 2o 20.5mL ~ 0.8mL.
Described gentamicin standard solution concentration is respectively: 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, and standard dilutions is pH7.4, containing 0.05% Tween-20, and the phosphate buffer of 0.05mol/L.Described percentage composition is mass percentage.
Described concentrated redissolution liquid is specially concentrated phosphoric acid salt buffer, is often liter of NaH containing 4.0g ~ 7.0g 2pO 42H 2o, 30.0g ~ 33.0g Na 2hPO 412H 2the aqueous solution of O.
Described thickening and washing solution is pH=7.4 ~ 7.6 containing volume fraction 0.02% ~ 0.06% Tween-20,0.2mol/L ~ 0.5mol/L phosphate buffer.
Beneficial effect of the present invention is as follows:
1) kit of the present invention has higher sensitivity and specificity, can reach 0.3 μ g/L to the detection sensitivity of gentamicin.
2) the supporting chemiluminescence detector of kit of the present invention detects gentamicin residue amount in sample, realize the full-automation of testing process, reduce manual operation error, detection time is short, only needs 20min to get final product the detection of gentamicin residue amount in paired samples.
Accompanying drawing explanation
Fig. 1 is gentamicin hapten synthesis reaction equation.
Fig. 2 is gentamicin haptens hydrogen nuclear magnetic resonance spectrogram.
Fig. 3 is magnetic immunochemiluminescence detection kit typical curve of the present invention.
Embodiment
Embodiment 1: the preparation of the concrete component of kit
1. gentamicin hapten synthesis
Gentamicin haptens is, with Pyridinium dichromate, the alcoholic extract hydroxyl group in former for gentamicin medicine is oxidized to ketone group, obtains single ketones base gentamicin, then single ketones base gentamicin with condensation reaction is carried out to formic acid phenylhydrazine, obtain the gentamicin haptens product with carboxyl.Get 0.6g ~ 1.2g gentamicin and join N, dissolve in dinethylformamide, then add 0.80g ~ 0.85g Pyridinium dichromate, stir, add 0.1mL ~ 0.5mL acetic acid, 60 DEG C of oil baths add thermal agitation 6h, and question response is complete, stop reaction and evaporate to dryness, add water and extraction into ethyl acetate, drying, ethyl alcohol recrystallization, obtains product 1.Get product 1 and add methyl alcohol dissolving, then add 0.1g ~ 0.6g K 2cO 3, stir, add 0.2g ~ 0.7g formic acid phenylhydrazine wherein, at room temperature stir 8h, sediment to be had separates out centrifuging, with ethanol washing, then adds re-crystallizing in ethyl acetate and obtains gentamicin haptens product 0.3g ~ 0.6g.Synthetic reaction formula as shown in Figure 1.
Get above-mentioned gentamicin haptens product to measure through nucleus magnetic hydrogen spectrum, as shown in Figure 2, the position of chemical shift δ=11ppm is carboxyl hydrogen resonance absorbing peak, the position of chemical shift δ=7.8,6.8ppm is benzene ring hydrogen resonance absorbing peak, prove spacerarm successful connection, the success of gentamicin hapten synthesis.
2. the preparation of enzyme-labelled antigen
Get 18mg ~ 25mg gentamicin haptens, be dissolved in 0.8mL ~ 1.2mL DMF (DMF); Getting after 25mg ~ 30mg ethylene dichloride (EDC) and N-hydroxy-succinamide (NHS) 0.1mL ~ 0.3mL water fully dissolve adds in haptens lysate, and stirred at ambient temperature 24h, can obtain reactant liquor A; Take horseradish peroxidase (HRP) 40mg ~ 55mg, make it fully to be dissolved in pH7.2, in the phosphate buffer of 3.8mL, reactant liquor A is dropwise slowly added drop-wise in HRP solution, and in stirred at ambient temperature 24h; Dialyse 3 days in 4 DEG C with the phosphate buffer of 0.01mol/L, change 3 dislysates every day, to remove unreacted small-molecule substance, obtain gentamicin enzyme-labelled antigen; Packing, saves backup in-20 DEG C.
3. immunogenic preparation
HRP 30mg ~ 70mg is replaced with bovine serum albumin(BSA) (BSA) 40mg ~ 60mg, preparation method is the same, obtains immunogene.
4. the preparation of gentamicin monoclonal antibody
A) animal immune: by the above-mentioned immunogene (RAC-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 merge first 3 days, 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, add appropriate feeder cells again, 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) 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, gentamicin standard solution 50 μ L is added after cleaning, add cell conditioned medium liquid 50 μ L and sheep anti-mouse igg-HRP 50 μ L again, in 37 DEG C of reaction 30min, wash plate, add substrate solution nitrite ion 100 μ L again, lucifuge reaction 15min at 25 DEG C, then add stop buffer 50 μ L, measure OD 450nmvalue drops to less than 50% of control wells, is 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/, 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.
5. the preparation of magnetic labeling antibody
A) magnetic bead activation
Surface has the magnetic bead of-COOH group (to be purchased from DYNAL, particle diameter is 2.8 μm), its content is 0.1eq/g ~ 0.3eq/g, get 100 μ L magnetic beads, with containing pH5.0,0.05% the concentration of Tween-20 be that 2-(N-morpholine) ethyl sulfonic acid monohydrate (MES) 100mL of 25mmol/L washes twice, remove supernatant after Magneto separate; Before magnetic bead activation, prepare EDC and the NHS solution of 50mmol/L respectively with the above-mentioned MES solution of 4 DEG C of storages; Respectively to each 50 μ L of EDC and NHS solution adding new configuration in the centrifuge tube that magnetic bead is housed, vortex mixes, room temperature activation 30min; Centrifuge tube is placed on Magneto separate frame and carries out Magneto separate 4min, remove supernatant, then add wherein 100 μ L, pH5.0,25mmol/L MES cleaning 2 ~ 3 times after can obtain the magnetic bead that there is activated carboxylic on surface.Described percentage composition is mass percentage.
B) preparation of magnetic bead coupling gentamicin monoclonal antibody
6 μ g ~ 12 μ g gentamicin monoclonal antibodies are dissolved in the MES of 60 μ L, pH5.0,25mmol/L, add the magnetic bead of 3mg ~ 6mg activation wherein, and regulate cumulative volume to 100 μ L with above-mentioned concentration MES solution, gently mix magnetic bead and gentamicin monoclonal antibody; Coupling 30min ~ 40min or 4 DEG C of coupling 2h under room temperature condition, can utilize vortex instrument to make magnetic bead keep mixing state during this period; Centrifuge tube is placed on Magneto separate frame and carries out Magneto separate 3min ~ 5min, removes supernatant; In order to the unreacted-COOH of cancellation, 100 μ L can be added, the trishydroxymethylaminomethane (TRIS) of pH7.2 ~ pH7.6 reacts 15min or 100 μ L, pH8.0, phosphate buffer that ethanolamine concentration is 50mmol/L closes magnetic bead; The magnetic bead closed with the phosphate buffer cleaning of 100 μ L, 0.1% ~ 0.3%BSA, 0.1% Tween-20 3 ~ 5 times, redissolves magnetic bead in the BSA, 0.01% ~ 0.1% Tween-20, the 0.02%NaN that contain 0.1% ~ 0.5% 5phosphate buffer in, in 2 DEG C ~ 8 DEG C preservations.Described percentage composition is mass percentage.
Embodiment 2: the establishment of kit
Set up the magnetic immunochemiluminescence detection kit detecting gentamicin class medicine, make it contain following component:
The haptenic label of gentamicin of horseradish peroxidase-labeled
Enzyme-labelled antigen dilution
The conjugate of gentamicin monoclonal antibody and magnetic bead
Magnetic labeling antibody dilution
Gentamicin standard solution, concentration is respectively: 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, and standard dilutions is pH7.4, containing 0.05% Tween-20, the phosphate buffer of 0.05mol/L.Described percentage composition is mass percentage.
The concentrated liquid that redissolves is concentrated phosphoric acid salt buffer, is often liter of NaH containing 5.0g ~ 8.0g 2pO 42H 2o, 30.0g ~ 35.0g Na 2hPO 412H 2the aqueous solution of O.
Thickening and washing solution is pH=7.4 ~ 7.6 containing volume fraction 0.03% ~ 0.08% Tween-20,0.1mol/L ~ 0.7mol/L phosphate buffer.
Embodiment 3: the detection of gentamicin residue amount in sample
1. sample-pretreating method
(1) milk
Get 50 μ L fresh milk samples and add 950 μ L sample diluting liquids (being diluted by 1:1 volume ratio by concentrated redissolution liquid with deionized water), whirling motion mixes, and gets this solution for sample analysis.
(2) milk powder
Take 1.0g ± 0.05g powdered milk sample, add 5mL sample diluting liquid, whirling motion mixes, and therefrom takes out 200 μ L and is added in 600 μ L sample diluting liquids, and whirling motion mixes, and gets this solution for sample analysis.
2. detect and interpretation of result with kit
Enzyme-labelled antigen and enzyme-labelled antigen dilution are diluted according to the volume ratio of 1:10 ~ 1:20, loads in chemiluminescence detector enzyme-labelled antigen working fluid container, magnetic labeling antibody and magnetic labeling antibody dilution are diluted according to the volume ratio of 1:10 ~ 1:20, loads in chemiluminescence detector enzyme-labelled antigen working fluid container, each sample/standard items are arranged to the position on specimen holder, the test event title that input sample message and needs detect, sample hose/standard QC is put on the specimen holder set, chemiluminescence detector draws 50 μ L enzyme-labelled antigens successively, 50 μ L testing sample/standard items and 50 μ L magnetic labeling antibodies join in reaction cup, mixing, and at room temperature react 15min, Magneto separate 4min is carried out again by cleaning device, be pH7.2 ~ pH7.6 with cleansing solution again, the phosphate buffer of 300 μ L ~ 500 μ L cleans 3 times ~ 5 times, add chemical luminous substrate A liquid and each 50 μ L of B liquid again, detect its relative light intensity sent (RLU), in sample, the content of gentamicin becomes negative correlativing relation with RLU, the concentration of gentamicin can be calculated by RLU combined standard curve method.
The present invention adopts 6 gentamicin standard items (0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L) to carry out plotting curves.By the RLU value (RLU of the mean value of obtained standard items and sample RLU value divided by first standard items 0value) be multiplied by 100 again, with relative luminous intensity (%)=RLU/RLU 0for ordinate, the logarithm of gentamicin concentration is that horizontal ordinate does typical curve, and the concentration of each sample can read from typical curve.Typical curve as shown in Figure 3.
Embodiment 4: the mensuration of kit quality
1. the detectability of kit
Kit detectability is defined as: measure 20 negative samples, and the mean value of mensuration adds 3 times of standard deviations.The detection of this kit is limited to: milk 2 μ g/L, milk powder 5 μ g/kg.
2. the accuracy of kit and precision
Accuracy refers to the matching degree between measured value and true value, and kit accuracy is commonly used the recovery and represented.Precision is also known as repeatability, and the conventional coefficient of variation represents.
According to the sample-pretreating method of embodiment 3, with the gentamicin of 2 μ g/L, 4 μ g/L and 8 μ g/L concentration, milk sample is added, with the gentamicin of 5 μ g/kg, 10 μ g/kg and 20 μ g/kg concentration, powdered milk sample is added, often kind of each concentration determination of sample 4 is parallel, measure with three batches of kits, the recovery of calculation sample and precision.Experimental result is in table 1.
The mensuration of table 1 accuracy and precision
As known from Table 1, in milk sample, the TIANZHU XINGNAO Capsul scope of gentamicin is 72.5% ~ 108.8%, and in powdered milk sample, the TIANZHU XINGNAO Capsul scope of gentamicin is 74.0% ~ 106.0%.Within-run and between-run analysis coefficient is all less than 15%.
3. specificity
Using gentamicin as standard, if the cross reacting rate of gentamicin is 100%, the medicine for antibody cross reaction Journal of Sex Research is and gentamicin structure or intimate competition medicine: gentamicin, streptomysin, dihydrostreptomycin, neomycin.By the operation of kit step, make and suppress curve, calculate 50% inhibition concentration (IC of each medicine according to linear equation 50).Cross reacting rate (%CR) is the IC of antibody to gentamicin 50with the IC of antibody to gentamicin competitor 50the percentage of ratio, calculate by following formula:
The results are shown in table 2:
Table 2 kit specific test
Competitor IC 50(μg/L) Cross reacting rate (%)
Gentamicin 0.24 100.0
Streptomysin 68.94 <1.0
Dihydrostreptomycin 85.39 <1.0
Neomycin 112.58 <1.0
As can be seen from Table 2, kit has higher specificity to gentamicin, to gentamicin structure or the equal no cross reaction of intimate competition medicine.

Claims (9)

1. a magnetic immunochemiluminescence detection kit for gentamicin, is characterized in that the reagent comprised has: enzyme-labelled antigen, enzyme-labelled antigen dilution, magnetic labeling antibody, magnetic labeling antibody dilution, gentamicin serial standards solution, chemical luminous substrate A, B liquid, the concentrated liquid that redissolves, concentrated cleaning solution.
2. kit according to claim 1, it is characterized in that: described enzyme-labelled antigen is the haptenic label of gentamicin of horseradish peroxidase-labeled, gentamicin haptens is, with Pyridinium dichromate, the alcoholic extract hydroxyl group in former for gentamicin medicine is oxidized to ketone group, obtain single ketones base gentamicin, then single ketones base gentamicin with condensation reaction is carried out to formic acid phenylhydrazine, obtain the gentamicin haptens product with carboxyl.
3. kit according to claim 1, is characterized in that: described magnetic labeling antibody is that gentamicin monoclonal antibody and magnetic bead coupling obtain.
4. kit according to claim 3, is characterized in that: described magnetic bead surfaces contains-OH ,-COOH or-NH 2reactive group.
5. kit according to claim 3, is characterized in that: described gentamicin monoclonal antibody is that the conjugate obtained by gentamicin haptens and bovine serum albumin(BSA) prepares as immunogen immune Balb/c mouse.
6. kit according to claim 1, is characterized in that: described chemical luminous substrate A liquid is the solution of the trishydroxymethylaminomethane containing luminol and p-cresol.Chemical luminescence for liquid B liquid is for containing citric acid, anhydrous Na 2hPO 4with CO (NH2) 2H 2o 2aqueous solution.
7. kit according to claim 1, is characterized in that: described kit also comprises standard solution, concentrated liquid and the concentrated cleaning solution of redissolving.
8. kit according to claim 1, is characterized in that supporting chemiluminescence detector to detect the residual quantity of gentamicin.
9. utilize the kit described in any one of claim 1 ~ 8 to detect a method for gentamicin, comprise the following steps:
1) enzyme-labelled antigen and enzyme-labelled antigen dilution are diluted according to the volume ratio of 1:10 ~ 1:20, load in chemiluminescence detector enzyme-labelled antigen working fluid container;
2) magnetic labeling antibody and magnetic labeling antibody dilution are diluted according to the volume ratio of 1:10 ~ 1:20, load in chemiluminescence detector magnetic labeling antibody working fluid container;
3) chemiluminescence detector draws 30 μ L ~ 60 μ L enzyme-labelled antigens, 30 μ L ~ 60 μ L sample extracting solutions and 30 μ L ~ 60 μ L magnetic labeling antibodies respectively, join in reaction cup memory storage successively, at room temperature react 15min, carry out Magneto separate 2min ~ 4min by cleaning device again, after abandoning supernatant, with cleansing solution 300 μ L ~ 500 μ L, 3 times ~ 5 times are cleaned to complex precipitate;
4) compound of separator well is put into measurement camera bellows, add chemical luminous substrate A liquid and each 50 μ L of B liquid, detect the relative light intensity (RLU) sent, in sample, the content of gentamicin becomes negative correlativing relation with RLU, can be calculated the residual concentration of gentamicin by RLU typical curve.
CN201510175688.1A 2015-04-14 2015-04-14 The magnetic immunochemiluminescence detection kit of a kind of gentamicin and application thereof Active CN104897895B (en)

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CN107643407A (en) * 2017-09-16 2018-01-30 北京勤邦生物技术有限公司 A kind of magnetic immunochemiluminescence detection kit of Trenbolone and its application
CN107677807A (en) * 2016-09-30 2018-02-09 青岛大学 A kind of kitasamycin magnetic immunochemiluminescence detection kit
CN108362896A (en) * 2017-12-27 2018-08-03 北京勤邦生物技术有限公司 A kind of magnetic immunochemiluminescence detection kit of apramycin and its application
CN109799332A (en) * 2018-12-25 2019-05-24 北京农业质量标准与检测技术研究中心 The magnetic immunofluorescence quantitative detecting method of rod method phenol monomethyl ether
CN112904008A (en) * 2021-02-04 2021-06-04 浙江省食品药品检验研究院 Enzyme linked immunosorbent assay kit for detecting protein A and other impurities in biological products and application thereof

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CN1771437A (en) * 2003-03-31 2006-05-10 科学与工业研究委员会 Rapid heat-mediated method for enzyme-linked immunosorbent assay procedure
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CN105319356A (en) * 2015-12-16 2016-02-10 北京勤邦生物技术有限公司 Immunomagnetic bead for aflatoxin B1 enrichment purification and preparation method and application thereof
CN105301254A (en) * 2015-12-22 2016-02-03 北京勤邦生物技术有限公司 Immunomagnetic beads for gathering purification of zearalenone and preparing method and application of immunomagnetic beads
CN105911293A (en) * 2016-05-26 2016-08-31 安徽伊普诺康生物技术股份有限公司 Kit for determining immunoglobulin A and preparation method thereof
CN107677807A (en) * 2016-09-30 2018-02-09 青岛大学 A kind of kitasamycin magnetic immunochemiluminescence detection kit
CN107607721A (en) * 2017-09-16 2018-01-19 北京勤邦生物技术有限公司 A kind of magnetic immunochemiluminescence detection kit of chlorpromazine and its application
CN107643407A (en) * 2017-09-16 2018-01-30 北京勤邦生物技术有限公司 A kind of magnetic immunochemiluminescence detection kit of Trenbolone and its application
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CN107643407B (en) * 2017-09-16 2020-08-28 北京勤邦生物技术有限公司 Trenbolone magnetic immunochemiluminescence detection kit and application thereof
CN108362896A (en) * 2017-12-27 2018-08-03 北京勤邦生物技术有限公司 A kind of magnetic immunochemiluminescence detection kit of apramycin and its application
CN108362896B (en) * 2017-12-27 2020-08-28 北京勤邦生物技术有限公司 Magnetic immunochemiluminescence detection kit for apramycin and application thereof
CN109799332A (en) * 2018-12-25 2019-05-24 北京农业质量标准与检测技术研究中心 The magnetic immunofluorescence quantitative detecting method of rod method phenol monomethyl ether
CN112904008A (en) * 2021-02-04 2021-06-04 浙江省食品药品检验研究院 Enzyme linked immunosorbent assay kit for detecting protein A and other impurities in biological products and application thereof

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