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CN104897895B - The magnetic immunochemiluminescence detection kit of a kind of gentamicin and application thereof - Google Patents

The magnetic immunochemiluminescence detection kit of a kind of gentamicin and application thereof Download PDF

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CN104897895B
CN104897895B CN201510175688.1A CN201510175688A CN104897895B CN 104897895 B CN104897895 B CN 104897895B CN 201510175688 A CN201510175688 A CN 201510175688A CN 104897895 B CN104897895 B CN 104897895B
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gentamicin
enzyme
magnetic
kit
liquid
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CN104897895A (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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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/553—Metal or metal coated

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Abstract

The present invention relates to detect gentamicin magnetic immunochemiluminescence kit, including reagent have: enzyme-labelled antigen, enzyme-labelled antigen dilution, magnetic labeling antibody, magnetic labeling antibody dilution, gentamicin serial standards solution, chemical luminous substrate A, B liquid, concentrates redissolution liquid, concentrated cleaning solution.Enzyme-labelled antigen is the haptenic label of gentamicin of horseradish peroxidase-labeled, 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 formic acid phenylhydrazine is carried out condensation reaction, obtaining the gentamicin haptens product with carboxyl, magnetic labeling antibody is that gentamicin monoclonal antibody obtains with magnetic bead coupling.The invention still further relates to a kind of use the method for gentamicin in described kit supporting chemiluminescence detector detection milk and milk powder, this method has higher sensitivity, specific and shorter detection time to gentamicin detection.

Description

The magnetic immunochemiluminescence detection kit of a kind of gentamicin and application thereof
Technical field
The present invention relates to a kind of chemiluminescence detection kit and detection method thereof.Particularly detect in the sample such as milk, milk powder and celebrate The magnetic immunochemiluminescence detection kit of big mycin residual quantity.Belong to field of immunological detection.
Background technology
Gentamicin (Gentamicin) is a kind of aminoglycoside antibiotics produced by small single-cell bacteria fermentation.Extensively Ying Yuzhi The infection that amount Gram-negative bacteria causes.Gentamicin antimicrobial spectrum is relatively wide, can effectively suppress growth and the breeding of bacterium, be mesh One of veterinary drug that front Chinese agriculture, animal husbandry and aquatic products industry are conventional.But be as being widely used of gentamicin, some bacteriums by Gradually to which creating drug resistance, and the infringement to cranial nerve, the sense of hearing and kidney is serious, along with a large amount of uses of this medicine, Its residue detection is valued by the people the most gradually.China and European Union remain strict demand to gentamicin, in milk Big residue limits is not above 100 μ g/kg.
The conventional detection method of gentamicin residue amount mainly have high performance liquid chromatography (HPLC), gas 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 expensive instrument and equipment and the loaded down with trivial details operating process of complexity, and the height to reviewer Skill set requirements so that above-mentioned instrument detection method is not suitable for the examination of a large amount of sample.Enzyme-Linked Immunospot and colloid gold immune layer Although analysis detection method belongs to Rapid Screening method, but owing to method sensitivity is relatively low, false negative rate and false positive rate are higher, gradually Substituted by chemical luminous immune detection method highly sensitive, that the degree of accuracy is high, the detection time is short, the immunity of magnetic immunochemiluminescence The residual of gentamicin in detection reagent supporting magnetic immunochemiluminescence detector detection food, it is achieved the full-automation of detection process, Reducing manual operation error, sensitivity can reach 0.1 μ g/L, and the degree of accuracy is high, and testing cost is low, it is adaptable to celebrates in batch samples The examination of big mycin residual quantity.
Summary of the invention
The technical problem to be solved is to provide a kind of gentamicin detection kit certainly, uses this kit to celebrate During the detection of big mycin, there is higher sensitivity, the specific and degree of accuracy.
Further object is that the detection method that a kind of gentamicin is provided, use this kit fiting chemical luminescence to examine Survey instrument carries out not only having higher sensitivity, the specific and degree of accuracy during detection of gentamicin, and achieves full-automation Detection, shortens the detection time, reduces manual operation error.
For achieving the above object, the present invention provides a kind of gentamicin detection kit, and its main agents comprised has: enzyme mark resists At the bottom of former, enzyme-labelled antigen dilution, magnetic labeling antibody, magnetic labeling antibody dilution, gentamicin serial standards solution, chemiluminescence Thing A, B liquid.
Described enzyme-labelled antigen is the haptenic label of gentamicin of horseradish peroxidase-labeled, and 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, the then celebrating of single ketones base Big mycin with formic acid phenylhydrazine is carried out condensation reaction, obtain the gentamicin haptens product with carboxyl.
Described magnetic labeling antibody is that gentamicin monoclonal antibody obtains with magnetic bead coupling.
Described magnetic bead surfaces contains-OH ,-COOH or-NH2Active group.
Described gentamicin monoclonal antibody is that the conjugate obtained by gentamicin haptens and bovine serum albumin(BSA) is as immunogene Balb/c mouse prepares in immunity.
Described chemical luminous substrate A liquid is containing luminol and the solution of the trishydroxymethylaminomethane of p-cresol.Chemiluminescence Liquid B liquid is containing citric acid, anhydrous Na2HPO4With CO (NH2) 2 H2O2The aqueous solution.
Described kit also includes standard solution, concentrates redissolution liquid and concentrated cleaning solution.
Described kit can carry out the detection of gentamicin residue amount in animal derived sample with supporting chemiluminescence detector.
The present invention also provides for a kind of method utilizing kit supporting Chemiluminescence Apparatus detection gentamicin, comprises the following steps:
1) enzyme-labelled antigen is diluted according to the volume ratio of 1:10～1:20 with enzyme-labelled antigen dilution, loads chemiluminescence detection In instrument enzyme-labelled antigen working fluid container；
2) magnetic labeling antibody is diluted according to the volume ratio of 1:10～1:20 with magnetic labeling antibody dilution, loads chemiluminescence detection In instrument magnetic labeling antibody working fluid container；
3) chemiluminescence detector from the most dynamic draw 30 μ L～80 μ L enzyme-labelled antigens, 30 μ L～80 μ L sample extracts and 30 μ L～80 μ L magnetic labeling antibodies, be added sequentially to reaction zone, at room temperature react 15min, separates 4min in Magneto separate district, abandons With cleaning solution 300 μ L～500 μ L, complex precipitate is cleaned 3～5 times after supernatant；
4) compound of separator well is put into measurement camera bellows, add chemical luminous substrate A liquid and B liquid each 30 μ L～80 μ L, inspection Surveying the relative light intensity (RLU) sent, in sample, the content of gentamicin and RLU proportion relation, can pass through RLU Calibration curve calculates the residual concentration of gentamicin.
In the present invention, the chemiluminescence detector used by analysis test method includes that power circuit, reaction cup storage device, sample are deposited Storage device, sample arm, reagent storage means, reagent arm, campaign-styled cold storage plant, cleaning device, automatic injection pump, low-light Detector, the Windows being simultaneously also configured with computer and Chinese interface controls software, data typing can be carried out, result collects, Quality control, result storage and the function such as result queries, can complete the programming of multiple analytical model, quantitative or qualitative report result, Automatically generate and store and more New function, 2 automatic correction calibration curves.
Enzyme-labelled antigen of the present invention is the haptenic label of gentamicin of horseradish peroxidase-labeled, and it is stored in and contains PH7.2～7.6, containing tween 0.03%～0.05% Tween-20, in the phosphate buffer of 0.02mol/L～0.05mol/L.Described hundred Dividing content is weight/mass percentage composition.
Described enzyme-labelled antigen dilution is pH7.2～pH7.6, Na3PO4Concentration is 0.01mol/L, NaCl concentration is 0.25mol/L Cushioning liquid.
Described magnetic labeling antibody is that gentamicin monoclonal antibody obtains with magnetic bead coupling.The content of magnetic bead surfaces group is 0.1eq/g ～0.3eq/g, described magnetic labeling antibody is saved in pH7.2～7.6, containing tween 0.1%～0.3% Tween-20,0.02mol/L～0.05mol/L Phosphate buffer in.Described percentage composition is weight/mass percentage composition.
Described magnetic labeling antibody dilution is pH7.2～pH7.6, Na3PO4Concentration is 0.02mol/L, NaCl concentration is 0.3mol/L Cushioning liquid.
The conjugate that described gentamicin monoclonal antibody is obtained by gentamicin haptens and bovine serum albumin(BSA) is as immunogene The titration of immunity Balb/c mouse, cell fusion, the screening of hybridoma, subclone and mouse ascites obtains.
Described chemical luminous substrate A liquid is that luminol content is 0.01 μ g/L～0.03 μ g/L, p-cresol content are 0.001 μ g/L～the tris solution that 0.005 μ g/L, pH is 8.0～9.0, B liquid is that every 100mL aqueous solution is containing lemon Lemon acid 1.7g～2.3g, anhydrous Na2HPO42.2g～3.0g and CO (NH2) 2 H that volumn concentration is 0.75%2O20.5mL ～0.8mL.
Described gentamicin standard solution concentration is respectively as follows: 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, Standard dilutions is pH7.4, containing 0.05% Tween-20, the phosphate buffer of 0.05mol/L.Described percentage composition is quality Percentage composition.
The described redissolution liquid that concentrates is specially concentrated phosphoric acid salt buffer, is every liter of NaH containing 4.0g～7.0g2PO4·2H2O、30.0 G～33.0g Na2HPO4·12H2The aqueous solution of O.
Described thickening and washing solution is the pH=7.4～7.6 containing volume fraction 0.02%～0.06% Tween-20,0.2mol/L～0.5 Mol/L phosphate buffer.
Beneficial effects of the present invention is as follows:
1) kit of the present invention has higher sensitivity with specific, can reach the detection sensitivity of gentamicin 0.3μg/L。
2) gentamicin residue amount in sample is detected by the supporting chemiluminescence detector of kit of the present invention, it is achieved detection process Full-automation, reduce manual operation error, detection the time short, it is only necessary to 20min has got final product gentamicin residue in paired samples The detection of amount.
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 calibration curve of the present invention.
Detailed description of the invention
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 the celebrating of single ketones base Big mycin, then single ketones base gentamicin with formic acid phenylhydrazine is carried out condensation reaction, obtain the gentamicin haptens with carboxyl Product.Take 0.6g～1.2g gentamicin and join dissolving in DMF, add 0.80g～0.85g dichromic acid Pyridine, stirring, 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 are evaporated, Add water and ethyl acetate extracts, be dried, ethyl alcohol recrystallization, obtain product 1.Take product 1 and add methyl alcohol dissolving, then add 0.1g～0.6g K2CO3, stirring, it is added thereto to 0.2g～0.7g formic acid phenylhydrazine, is stirred at room temperature 8h, sediment to be had separates out centrifugal point From, wash with ethanol, then add re-crystallizing in ethyl acetate and obtain gentamicin haptens product 0.3g～0.6g.Synthetic reaction formula such as figure Shown in 1.
Take above-mentioned gentamicin haptens product to measure through nucleus magnetic hydrogen spectrum, as in figure 2 it is shown, the position of chemical shift δ=11ppm is Carboxyl hydrogen resonance absorbing peak, chemical shift δ=7.8, the position of 6.8ppm are benzene ring hydrogen resonance absorbing peak, it was demonstrated that spacerarm Successful connection, the success of gentamicin hapten synthesis.
2. the preparation of enzyme-labelled antigen
Take 18mg～25mg gentamicin haptens, be dissolved in 0.8mL～1.2mL DMF (DMF)； Take 25mg～30mg dichloroethanes (EDC) and N-hydroxy-succinamide (NHS) fully dissolves with 0.1mL～0.3mL water In rear addition haptens lysate, stir 24h under room temperature, i.e. can get reactant liquor A；Weigh horseradish peroxidase (HRP) 40mg～55mg, is allowed to be substantially dissolved in the phosphate buffer of pH7.2,3.8mL, is dropwise slowly added dropwise by reactant liquor A In HRP solution, and stir 24h at room temperature；Dialyse 3 days in 4 DEG C with the phosphate buffer of 0.01mol/L, every day Change 3 dislysates, to remove unreacted small-molecule substance, obtain gentamicin enzyme-labelled antigen；Packing, standby in-20 DEG C of preservations With.
The most immunogenic preparation
HRP 30mg～70mg replaces with bovine serum albumin(BSA) (BSA) 40mg～60mg, and preparation method ibid, is exempted from Epidemic focus.
4. the preparation of gentamicin monoclonal antibody
A) animal immune: by the above-mentioned immunogene (RAC-BSA) prepared by 100 μ g/ only, with physiological saline solution immunogene Mix with Freund's complete adjuvant equal-volume, neck dorsal sc injection the immunity 6～8 female mouse of week old Balb/c, after initial immunity the 7th, 14, within 28 days, mixing with incomplete Freund's adjuvant equal-volume with immunogene, each supplementary immunization once, merges first 3 days and is combined with immunity Thing 100 μ g/ only, is not added with Freund's adjuvant supplementary immunization more once.
B) cell merges: carries out according to a conventional method, takes the splenocyte of immune mouse and be in the mouse myeloma of exponential phase Cell (SP2/0) mixes, and the fusion agent (PEG4000) being then slowly added to preheating in 45 seconds merges, and uses HAT culture medium Suspend uniformly, add appropriate feeder cells, be incubated at 96 well culture plates, in 37 DEG C, 5%CO2Incubator is cultivated, 5 Partly change liquid with HT culture medium after it, when 9 days, entirely change liquid.
C) screening of hybridoma: after cell merges, when cell grows to the 1/4 of culture hole area, uses substep screening method Screening hybridoma.Primary election uses indirect ELISA method, and with envelope antigen, (with square formation method conventional titration, it most preferably wraps in advance By concentration and positive serum dilution factor) coated elisa plate, add measured hole culture supernatant, hatch, after cleaning, add gentamicin mark Quasi-product solution 50 μ L, adds cell supernatant 50 μ L and sheep anti-mouse igg-HRP 50 μ L, reacts 30min in 37 DEG C, washes plate, Adding substrate solution nitrite ion 100 μ L, at 25 DEG C, lucifuge reaction 15min, adds stop buffer 50 μ L, measures OD450nm Value drops to less than the 50% of control wells, is judged to the positive, is all positive hole through 2～3 detections, enters with limiting dilution assay immediately Row subcloning.
D) prepared by monoclonal antibody: 2～3 subclones are built the hybridoma after strain and expands cultivation, collect between supernatant Meet ELISA and measure titer, frozen；And only take 8～10 week old Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, 7～10 days Pneumoretroperitoneum injection hybridoma 1～2 × 106/ only, extract mouse ascites, centrifuging and taking supernatant after 7～10 days, measure titer, and freeze Deposit standby.
5. the preparation of magnetic labeling antibody
A) magnetic bead activation
Surface has-magnetic bead (being purchased from DYNAL, particle diameter is 2.8 μm) of COOH group, and its content is 0.1eq/g～0.3eq/g, Take 100 μ L magnetic beads, with containing pH5.0, the 2-that concentration is 25mmol/L (N-morpholine) ethyl sulfonic acid of the Tween-20 of 0.05% Monohydrate (MES) 100mL washes twice, and removes supernatant after Magneto separate；Before magnetic bead activation, with the above-mentioned MES of 4 DEG C of storages Solution prepares EDC and the NHS solution of 50mmol/L respectively；Respectively to equipped with the centrifuge tube of magnetic bead adds newly configured EDC 50 μ Ls each with NHS solution, vortex mixes, room temperature activation 30min；Centrifuge tube is placed on Magneto separate frame and carries out Magneto separate 4min, Remove supernatant, then the MES being added thereto to 100 μ L, pH5.0,25mmol/L clean 2～3 times after i.e. can get surface There is the magnetic bead of activated carboxylic.Described percentage composition is weight/mass percentage composition.
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 wherein Enter 3mg～6mg activation magnetic bead, and with above-mentioned concentration MES solution regulation cumulative volume to 100 μ L, gently mixing magnetic bead and Gentamicin monoclonal antibody；Coupling 30min～40min or 4 DEG C of coupling 2h under room temperature condition, the most available vortex instrument makes Magnetic bead keeps mixing state；Centrifuge tube is placed on Magneto separate frame and carries out Magneto separate 3min～5min, removes supernatant；For cancellation Unreacted-COOH, can add 100 μ L, pH7.2～pH7.6 trishydroxymethylaminomethane (TRIS) reaction 15min or 100 μ L, pH8.0, ethanolamine concentration are that the phosphate buffer of 50mmol/L closes magnetic bead；With 100 μ L, 0.1%～0.3%BSA, The phosphate buffer of 0.1% Tween-20 cleans the magnetic bead 3～5 times closed, magnetic bead is redissolved in the BSA containing 0.1%～0.5%, 0.01%～0.1% Tween-20,0.02%NaN5Phosphate buffer in, in 2 DEG C～8 DEG C of preservations.Described percentage composition is quality Percentage composition.
Embodiment 2: the establishment of kit
Set up the magnetic immunochemiluminescence detection kit of detection gentamicin class medicine so that it is containing following component:
The haptenic label of gentamicin of horseradish peroxidase-labeled
Enzyme-labelled antigen dilution
Gentamicin monoclonal antibody and the conjugate of magnetic bead
Magnetic labeling antibody dilution
Gentamicin standard solution, concentration is respectively as follows: 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, Standard dilutions is pH7.4, containing 0.05% Tween-20, the phosphate buffer of 0.05mol/L.Described percentage composition is quality Percentage composition.
Concentrating redissolution liquid is concentrated phosphoric acid salt buffer, is every liter of NaH containing 5.0g～8.0g2PO4·2H2O, 30.0g～35.0g Na2HPO4·12H2The aqueous solution of O.
Thickening and washing solution is the 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
Take 50 μ L fresh milk samples add 950 μ L sample dilutions (with deionized water by concentration redissolution liquid carry out by 1:1 volume ratio Dilution), whirling motion mixes, and takes this solution for sample analysis.
(2) milk powder
Weighing 1.0g ± 0.05g powdered milk sample, add 5mL sample diluting liquid, whirling motion mixes, and is taken out 200 μ L and adds extremely In 600 μ L sample dilutions, whirling motion mixes, and takes this solution for sample analysis.
2. with kit detection and interpretation of result
Enzyme-labelled antigen is diluted according to the volume ratio of 1:10～1:20 with enzyme-labelled antigen dilution, loads chemiluminescence detector enzyme In mark antigen working fluid container；Magnetic labeling antibody is diluted according to the volume ratio of 1:10～1:20 with magnetic labeling antibody dilution, loads In chemiluminescence detector enzyme-labelled antigen working fluid container；Each sample/standard items are arranged the position on specimen holder, inputs sample Information and the test event title of needs detection；Putting on the specimen holder set by sample cell/standard QC, chemiluminescence is examined Survey instrument is drawn 50 μ L enzyme-labelled antigens, 50 μ L testing samples/standard items and 50 μ L magnetic labeling antibodies successively and is joined in reaction cup, mixed Even, and at room temperature react 15min, then carry out Magneto separate 4min by cleaning device, then with cleaning solution be pH7.2～pH7.6, The phosphate buffer of 300 μ L～500 μ L cleans 3 times～5 times, adds chemical luminous substrate A liquid and each 50 μ L of B liquid, inspection Surveying its relative light intensity sent (RLU), in sample, the content of gentamicin becomes negative correlativing relation with RLU, can pass through RLU Combined standard curve method calculates the concentration of gentamicin.
The present invention uses 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) Carry out plotting curves.By the mean value of the standard items obtained and sample RLU value divided by the RLU value (RLU of first standard items0 Value) it is multiplied by 100 again, with relative luminous intensity (%)=RLU/RLU0For ordinate, the logarithm of gentamicin concentration is horizontal seat Mark does calibration curve, and the concentration of each sample can read from calibration curve.Calibration curve is as shown in Figure 3.
Embodiment 4: the mensuration of kit quality
1. the detection limit of kit
The definition of kit detection limit is: measuring 20 negative samples, the mean value of mensuration adds 3 times of standard deviations.This kit Detection be limited to: milk 2 μ g/L, milk powder 5 μ g/kg.
2. the degree of accuracy of kit and precision
The degree of accuracy refers to that the matching degree between measured value and true value, the kit degree of accuracy are commonly used the rate of recovery and represented.Precision is also known as can Repeatability, 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 to milk sample Being added, be added powdered milk sample with the gentamicin of 5 μ g/kg, 10 μ g/kg and 20 μ g/kg concentration, every kind of sample is every Individual concentration mensuration 4 is parallel, is measured with three batches of kits, calculates the rate of recovery and the precision of sample.Experimental result is shown in Table 1。
Table 1 degree of accuracy and the mensuration of 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, celebrating is big The TIANZHU XINGNAO Capsul scope of mycin is 74.0%～106.0%.Within-run and between-run analysis coefficient is respectively less than 15%.
The most specific
Using gentamicin as standard, if the cross reacting rate of gentamicin is 100%, for the medicine of antibody cross reaction Journal of Sex Research Thing is and gentamicin structure or intimate competition medicine: gentamicin, streptomysin, dihydrostreptomycin, neomycin. By kit step operation, make suppression curve, calculate 50% inhibition concentration (IC of each medicine according to linear equation50).Intersect anti- Rate (%CR) antibody IC to gentamicin should be50With the antibody IC to gentamicin competitor50The percentage of ratio, press Formula calculates:
Result is listed in table 2:
Table 2 kit specific test
Competitor IC50(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
From table 2 it can be seen that gentamicin is had higher specific by kit, to gentamicin structure or function phase As compete the equal no cross reaction of medicine.

Claims (5)

1. a magnetic immunochemiluminescence detection kit for gentamicin, including reagent have: enzyme-labelled antigen, enzyme-labelled antigen is dilute Release liquid, magnetic labeling antibody, magnetic labeling antibody dilution, gentamicin serial standards solution, chemical luminous substrate A, B liquid is dense Contracting redissolution liquid, concentrated cleaning solution；It is characterized in that: described enzyme-labelled antigen is that the gentamicin half of horseradish peroxidase-labeled is anti- Former label, 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 formic acid phenylhydrazine is carried out condensation reaction, obtain the celebrating with carboxyl the most mould Element haptens product；Described magnetic labeling antibody is that gentamicin monoclonal antibody obtains with magnetic bead coupling.
Kit the most according to claim 1, it is characterised in that: described gentamicin monoclonal antibody is by gentamicin The conjugate that haptens and bovine serum albumin(BSA) obtain prepares as immunogen immune Balb/c mouse；Described magnetic bead surfaces contains There are-OH ,-COOH or-NH2Active group.
Kit the most according to claim 1, it is characterised in that: described chemical luminous substrate A liquid be containing luminol and The solution of the trishydroxymethylaminomethane of p-cresol, chemical luminous substrate B liquid is containing citric acid, anhydrous Na2HPO4With CO(NH2)2·H2O2The aqueous solution.
Kit the most according to claim 1, it is characterised in that: this kit can detect with supporting chemiluminescence detector The residual quantity of gentamicin.
5. the method utilizing the kit described in any one of claim 1～4 to detect gentamicin, comprises the following steps:
1) enzyme-labelled antigen is diluted according to the volume ratio of 1:10～1:20 with enzyme-labelled antigen dilution, loads chemiluminescence detection In instrument enzyme-labelled antigen working fluid container；
2) magnetic labeling antibody is diluted according to the volume ratio of 1:10～1:20 with magnetic labeling antibody dilution, loads chemiluminescence detection In instrument magnetic labeling antibody working fluid container；
3) chemiluminescence detector draw respectively 30 μ L～60 μ L enzyme-labelled antigens, 30 μ L～60 μ L sample extracts and 30 μ L～ 60 μ L magnetic labeling antibodies, are added sequentially in reaction cup storage device, at room temperature react 15min, then carry out by cleaning device Magneto separate 2min～4min, cleans 3 times～5 times complex precipitate with concentrated cleaning solution 300 μ L～500 μ L after abandoning supernatant；
4) compound of separator well being put into measurement camera bellows, add chemical luminous substrate A liquid and each 50 μ L of B liquid, detection sends Relative light intensity, in sample, the content of gentamicin becomes negative correlativing relation with relative light intensity, can pass through relative light intensity scale Directrix curve calculates the residual concentration of gentamicin.
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