CN105017347A - Gentamicin hapten and preparation method and application thereof - Google Patents
Gentamicin hapten and preparation method and application thereof Download PDFInfo
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- CN105017347A CN105017347A CN201410238929.8A CN201410238929A CN105017347A CN 105017347 A CN105017347 A CN 105017347A CN 201410238929 A CN201410238929 A CN 201410238929A CN 105017347 A CN105017347 A CN 105017347A
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Abstract
The invention discloses a gentamicin hapten and a preparation method and application thereof. The structure of the gentamicin hapten is presented as the formula I (please see the formula in the specification). A quick detection kit product built on the basis of the gentamicin hapten is convenient to use and low in detection cost, a detection method is efficient, accurate and quick, a large number of samples can be detected at the same time, and the product is suitable for on-site monitoring of gentamicin residues in animal-origin food and quick screening of the samples.
Description
Technical field
The present invention relates to a kind of gentamicin haptens and its preparation method and application.
Background technology
Gentamicin (Gentamicin, GM) belongs to aminoglycosides antibiotics, mainly acts on gram negative bacterium, because it has broad-spectrum antibacterial and germicidal action, is widely used in veterinary clinic and animal feedstuff additive.But the serum effective concentration of GM and toxic concentration are very close, and safety range is less, in its application process, easily produce toxic side effect, especially heavy dose of use lack of standardization, easily cause GM in animal food to remain to exceed standard, harm is formed to HUMAN HEALTH, causes renal toxicity and ototoxicity.Many countries forbid to use GM or regulation to have strict maximum residue limit(MRL) (MRL) in animal productiong, and No. 235, the Ministry of Agriculture of China bulletin regulation, in animal food, the MRL of GM is 100ng/g.
Current gentamicin residue detection method mainly contains vapor-phase chromatography, high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry, high performance capillary electrophoresis and immunological detection.Instrumental method detects expensive, is not suitable for promoting the use of in basic unit and screening in a large number.Immunological assay method sensitivity is higher, and high specificity is easy to use.Therefore, Large-scale Screening detect actual with immunological detection method, have more application prospect.The rapid screening that enzyme-linked immunosorbent assay (ELISA) is highly sensitive with it, specificity good, easy and simple to handle, low cost and other advantages is suitable for a large amount of sample.
Summary of the invention
The object of this invention is to provide a kind of gentamicin haptens and its preparation method and application.
Gentamicin hapten molecule structural formula provided by the invention is such as formula shown in I:
The haptenic preparation method of gentamicin provided by the invention, comprises the steps:
In 100mL two mouthfuls of flasks, add 0.5g to bromo methyl acid and 1.05g gentamicin and 10mL ethanol, be stirred to dissolving, be heated to 45 DEG C of reactions after 6 hours, remove solvent under reduced pressure.In ethanol-water system, recrystallization obtains, to carboxylic benzyl gentamicin, obtaining haptens product.
Another object of the present invention is the application of above-mentioned gentamicin haptens in immunodetection, specifically comprise the gentamicin antigen obtained by described gentamicin haptens and carrier protein couplet, and the gentamicin antibody to be prepared by gained gentamicin antigen-immunized animal, described antibody is gentamicin monoclonal antibody.
Wherein said carrier proteins can be bovine serum albumin, thyroprotein, ovalbumin, human serum protein, mouse serum protein, rabbit serum proteins, hemocyanin or Fibrinogen.
The present invention also provides the application in the product detecting gentamicin or preparation detection gentamicin by above-mentioned gentamicin haptens or gentamicin antigen, and the enzyme-linked immunologic detecting kit being specifically related to prepare is in the application detecting gentamicin residue in animal derived food.
Gentamicin haptens provided by the invention, gentamicin antigen synthetic method are simple, create the specific antibody for gentamicin by immune animal, and the tiring of antibody, specificity are all relatively good; Prepare enzyme-linked immunologic detecting kit with the antibody of gained, testing cost is low, easy to use, and detection method accurately, fast, can detect large batch of sample, is suitable for the on-site supervision of gentamicin residue and the examination of great amount of samples in animal derived food simultaneously.Gentamicin haptens of the present invention plays a significant role in the detection of gentamicin.
Accompanying drawing explanation
Fig. 1: gentamicin hapten synthesis route map
Fig. 2: gentamicin haptens nucleus magnetic resonance figure
Fig. 3: gentamicin ELISA typical curve
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only for illustration of the present invention, and be not used for limiting the scope of the invention.
The experimental technique used in following embodiment, material, reagent etc., if no special instructions, be ordinary method and conventional material.
Embodiment 1: the haptenic synthesis of gentamicin and qualification (synthetic route is as Fig. 1)
In 100mL two mouthfuls of flasks, add 0.5g to bromo methyl acid and 1.05g gentamicin and 10mL ethanol, be stirred to dissolving, be heated to 45 DEG C of reactions after 6 hours, remove solvent under reduced pressure.In ethanol-water system, recrystallization obtains, to carboxylic benzyl gentamicin, obtaining haptens product.
Above-mentioned gentamicin hapten molecule structural formula is such as formula shown in I:
Get above-mentioned product nucleus magnetic resonance determination structure, as shown in Figure 2, the carboxyl fignal center of 11.0ppm, the aromatic ring fignal center of 7.44ppm and 8.09ppm, illustrate hapten synthesis success.
Embodiment 2: the preparation of gentamicin antigen
Gentamicin haptens and carrier protein couplet are obtained gentamicin antigen, and molecular structural formula is such as formula shown in II:
One, immunogen preparation
Get 14mg gentamicin haptens 1mL N, dinethylformamide (DMF) dissolves, respectively getting after 30mg carbodiimide (EDC) and N-hydroxy-succinamide (NHS) 0.2mL water fully dissolve adds in above-mentioned solution, stirred at ambient temperature 24h, can obtain reaction solution A; Take bovine serum albumin (BSA) 50mg, make it fully to be dissolved in 3.8mL PBS (pH7.2) solution, reaction solution A is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h; With 0.01mol/LPBS solution at 4 DEG C of dialysis 3d, change 3 dialyzates every day, to remove unreacted small-molecule substance; Packing, saves backup in-20 DEG C.
Two, coating antigen preparation
BSA is changed to ovalbumin (OVA) and prepares coating antigen as stated above.
Embodiment 3: the preparation of gentamicin monoclonal antibody
Animal immune: the immunogen obtained in embodiment 2 be injected in Balb/c Mice Body, immunizing dose is 150 μ g/, makes it produce antiserum(antisera).
Cytogamy and cloning: after mice serum measurement result is higher, get its splenocyte, in 8:1 ratio and SP2/0 myeloma cell fusion, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
Cell cryopreservation and recovery: the monoclonal hybridoma strain frozen storing liquid of gentamicin is made cell suspension, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation frozen storing liquid, move into and cultivate culture in glassware.
The production of monoclonal antibody and purifying: only Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5mL/, the monoclonal hybridoma strain of 7 days pneumoretroperitoneum injection gentamicins, gathered ascites after 7 days.Carry out ascites by sad-saturated ammonium sulphate method to purify ,-20 DEG C of preservations.This monoclonal antibody specificity is good, and detection sensitivity can reach 0.1 μ g/L.
The specificity of monoclonal antibody
Antibodies specific refers to the ability that its homospecificity antigen combines and comparing with such antigen-analogues ability, and conventional cross reacting rate is as judgement criteria.Cross reaction is less, and the specificity of antibody is then higher.
Gentamicin, Streptomycin sulphate, Vibriomycin, Liu Suanyan NEOMYCIN SULPHATE are done serial dilution, production standard curve, analyze and obtain IC50, be then calculated as follows cross reacting rate, the results are shown in Table 1.
Table 1 antibodies specific
Embodiment 4: by the enzyme-linked immunologic detecting kit of gentamicin monoclonal antibody preparation
One, the composition of enzyme-linked immunologic detecting kit
(1) bag is by the enzyme plate of gentamicin coupled antigen;
(2) ELIAS secondary antibody or ELIAS secondary antibody concentrated solution;
(3) antibody working fluid;
(4) standard solution: concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, and high standard product strength of solution is 1mg/L;
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl biphenyl amine aqueous solution;
(6) stop buffer is the sulphuric acid soln of 2mol/L;
(7) concentrated cleaning solution is the phosphate buffered saline buffer of 0.1 ~ 0.2mol/L pH7.2 ~ 7.4 of the tween 20 containing 0.5% ~ 1.0%, and described per-cent is percent weight in volume;
(8) concentrated redissolution liquid is the phosphate buffered saline buffer of pH7.2 ~ 7.6,0.05 ~ 0.1mol/L, and described per-cent is percent weight in volume.
The main agents of this test kit provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, tolerance range is high, accuracy high.
Two, enzyme-linked immunologic detecting kit detects the application of actual sample
(1) Sample pretreatment method one
1, (chicken, chicken gizzard) Sample pretreatment method is organized
Take 2.0g ± 0.05g and remove the tissue samples of fat in 50mL polystyrene centrifuge tube, add 6mL0.1mol/L PBS solution (take 13.4g disodium hydrogen phosphate dodecahydrate, 20.0g sodium-chlor, 0.32g sodium hydroxide, 0.5g bis-hypophosphite monohydrate potassium dihydrogen, 0.5g Repone K add the mixing of 500mL deionized water dissolving), mixing 10min; Add 60min in 60 DEG C of water bath, take out and be cooled to room temperature and shake up; More than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugal 10min; Pipette supernatant liquor (with deionized water, the 2 × concentrated liquid that redissolves to be undertaken diluting (1 part 2 × concentrated liquid+1 part of deionized water that redissolves) redissolution for sample by 1:1 volume ratio with redissolution working fluid, the working fluid that redissolves can preserve one month at 4 DEG C of (39.2 ℉) environment) carry out diluting (50 μ L supernatant liquor+450 μ L redissolution working fluid) with the volume ratio of 1:9, get 50 μ L for analyzing.
2, fresh milk Sample pretreatment method
Get 20 μ L fresh milk samples in 2mL polystyrene centrifuge tube, adding 780 μ L redissolution working fluids, mixing, getting 50 μ L for analyzing.
3, milk powder Sample pretreatment method
Take 1.0g ± 0.05g milk powder sample in 10mL polystyrene centrifuge tube, add 5mL deionized water, fully vibrate to milk powder with vibrator and all dissolve; Taking out 50 μ L sample liquid and add 950 μ L redissolution working fluids, mixing, getting 50 μ L for analyzing.
(2) Sample pretreatment method two
1, chicken Sample pretreatment method
With homogenizer homogeneous sample; Take the equal pledge of 2.0g ± 0.05g in 50mL polystyrene centrifuge tube, add 4mL0.07mol/L carbonate buffer solution (take 3.26g anhydrous sodium carbonate and 0.35g sodium bicarbonate and add the mixing of 500mL deionized water dissolving) and 2mL methyl alcohol, with vortex instrument whirling motion 5min, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugal 5min; Pipette 100 μ L supernatant liquors in 2mL polystyrene centrifuge tube, add 900 μ L redissolution working fluids, with vortex instrument whirling motion 1min; Get 20 μ L for analyzing.
2, chicken gizzard Sample pretreatment method
With homogenizer homogeneous sample; Take the equal pledge of 2.0g ± 0.05g in 50mL polystyrene centrifuge tube, add 3mL0.1mol/L carbonate buffer solution (take 4.66g anhydrous sodium carbonate and 0.5g sodium bicarbonate and add the mixing of 500mL deionized water dissolving) and 3mL methyl alcohol, with vortex instrument whirling motion 5min, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugal 5min; Pipette 100 μ L supernatant liquors in 2mL polystyrene centrifuge tube, add 900 μ L redissolution working fluids, with vortex instrument whirling motion 1min; Get 20 μ L for analyzing.
3, milk Sample pretreatment method
Pipette 1mL milk sample in 2mL polystyrene centrifuge tube, add 1mL1% trichoroacetic acid(TCA) (take 5.0g trichoroacetic acid(TCA) and add the mixing of 500mL deionized water dissolving), with vortex instrument whirling motion 3min, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugal 5min; Pipette 100 μ L supernatant liquors in 2mL polystyrene centrifuge tube, add 900 μ L redissolution working fluids, with vortex instrument whirling motion 1min; Get 20 μ L for analyzing.
4, milk powder Sample pretreatment method
Take 1.0g ± 0.05g milk powder in 10mL polystyrene centrifuge tube, add 5mL deionized water, with vortex instrument whirling motion 3min, milk powder is fully dissolved, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugal 5min; Pipette 100 μ L supernatant liquors in 2mL polystyrene centrifuge tube, add 900 μ L redissolution working fluids, with vortex instrument whirling motion 1min; Get 20 μ L for analyzing.
(3) detect with test kit
Detect with test kit after sample is processed according to pre-treating process one, in the enzyme plate micropore being coated with coating antigen, add 50 μ L standard substance/samples, then add 50 μ L antibody working fluids, mixing of vibrating gently, cover cover plate film, lucifuge reaction 30min in 37 DEG C of thermostat containers.Pour out the liquid in hole, fully wash 4-5 time with wash operating solution (being diluted by 1:19 volume ratio by 20 × concentrated cleaning solution with deionized water) 250 μ L/ hole, every minor tick 10s, pats dry the liquid ensureing to remove completely in hole with thieving paper.Every hole adds ELIAS secondary antibody 100 μ L, mixing of vibrating gently, reacts 30min with in cover plate membrane cover plate rearmounted 37 DEG C (99 ℉) light protected environment.Pour out the liquid in hole, fully wash 4-5 time with wash operating solution 250 μ L/ hole, every minor tick 10s, pats dry the liquid ensureing to remove completely in hole with thieving paper.Every hole adds substrate solution A liquid 50 μ L, then adds substrate solution B liquid 50 μ L, mixing of vibrating gently, with lucifuge colour developing 15min in 37 DEG C of thermostat containers after cover plate membrane cover plate.Add 50 μ L stop buffers, mixing of vibrating gently, setting microplate reader measures the absorbance in every hole in 450nm place.
Detect with test kit after sample is processed according to pre-treating process two, 20 μ L standard substance/samples are added in the enzyme plate micropore being coated with coating antigen, add the mixed solution (antibody working fluid and ELIAS secondary antibody concentrated solution mixed by 10:1 volume ratio and mix) of 80 μ L antibody working fluids and ELIAS secondary antibody concentrated solution again, to vibrate gently mixing, cover cover plate film, lucifuge reaction 30min in 25 DEG C of thermostat containers.Pour out the liquid in hole, fully wash 4-5 time with wash operating solution 250 μ L/ hole, every minor tick 10s, pats dry the liquid ensureing to remove completely in hole with thieving paper.Every hole adds substrate solution A liquid 50 μ L, then adds substrate solution B liquid 50 μ L, mixing of vibrating gently, with lucifuge colour developing 15min in 25 DEG C of thermostat containers after cover plate membrane cover plate.Add 50 μ L stop buffers, mixing of vibrating gently, setting microplate reader measures the absorbance in every hole in 450nm place.
(4) Analysis of test results
With the mean value (diplopore) of the absorbance of obtained standard substance or the sample absorbance divided by first standard (0 standard), then be multiplied by 100%, namely obtain the percentage light absorption ratio of standard substance or sample.With gentamicin standard substance percentage light absorption ratio for ordinate zou, with the logarithm of gentamicin standard concentration for X-coordinate, drawing standard graphic representation, as shown in Figure 3.The percentage light absorption ratio of sample substituted in typical curve, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is gentamicin actual concentrations in sample.
Three, the determination of enzyme-linked immunologic detecting kit technical parameter
Lowest detectable limit: respectively to 20 portions of blank chicken, chicken gizzard, milk, milk powder sample, after processing according to Sample pretreatment method one, detect with test kit, the concentration corresponding to each percentage light absorption ratio is found from typical curve, add that 3 times of standard deviations represent detectability with the mean value of 20 these gentamicin concentrations of increment, result obtains the detection of the method to chicken, chicken gizzard sample and is limited to 4 μ g/kg, is limited to 4 μ g/L to the detection of milk sample, is limited to 10 μ g/kg to the detection of milk powder sample.
Respectively to 20 portions of blank chicken, chicken gizzard, milk, milk powder sample, after the process of Sample pretreatment method two, detect with test kit, the concentration corresponding to each percentage light absorption ratio is found from typical curve, add that 3 times of standard deviations represent detectability with the mean value of 20 these gentamicin concentrations of increment, result obtains the detection of the method to chicken, chicken gizzard sample and is limited to 4 μ g/kg, is limited to 2 μ g/L to the detection of milk sample, is limited to 5 μ g/kg to the detection of milk powder sample.
The accuracy that accuracy and precision: ELISA measures represents with the rate of recovery, and precision represents with the variation coefficient.
Get blank chicken, chicken gizzard sample respectively, concentration is added with 4,8,16 μ g/kg tri-, get blank milk sample, concentration is added with 4,8,16 μ g/L tri-, get blank milk powder sample, the gentamicin medicine adding concentration with 10,20,40 μ g/kg tri-adds it, respectively according to after Sample pretreatment method a pair chicken, chicken gizzard, milk, milk powder sample process, detect with test kit, it is 80% ~ 100% that result obtains the rate of recovery of the method to chicken, chicken gizzard sample, and milk, the milk powder sample rate of recovery are 70% ~ 110%.Variation within batch coefficient < 15%, interassay coefficient of variation < 25%.
Get blank chicken, chicken gizzard sample respectively, concentration is added with 4,8,16 μ g/kg tri-, get blank milk sample, concentration is added with 2,4,8 μ g/L tri-, get blank milk powder sample, the gentamicin medicine adding concentration with 5,10,20 μ g/kg tri-adds it, respectively according to Sample pretreatment method two to after chicken, chicken gizzard, milk, milk powder sample process, detect with test kit, it is 85% ~ 115% that result obtains the rate of recovery of the method to chicken, chicken gizzard, milk, milk powder sample.Variation within batch coefficient < 15%, interassay coefficient of variation < 25%.
Test kit of the present invention at least can preserve 12 months at 2 ~ 8 DEG C after measured.
Claims (7)
1. a gentamicin haptens, is characterized in that molecular structural formula is such as formula shown in I:
2. the haptenic preparation method of gentamicin according to claim 1, is characterized in that comprising the steps:
In 100mL two mouthfuls of flasks, add 0.5g to bromo methyl acid and 1.05g gentamicin and 10mL ethanol, be stirred to dissolving, be heated to 45 DEG C of reactions after 6 hours, remove solvent under reduced pressure.In ethanol-water system, recrystallization obtains carboxylic benzyl gentamicin.
3. a gentamicin antigen, it is characterized in that being obtained by gentamicin haptens according to claim 1 and carrier protein couplet, molecular structural formula is such as formula shown in II:
4. a preparation method for gentamicin antigen according to claim 3, is characterized in that comprising the steps:
Get 14mg gentamicin haptens 1mL DMF to dissolve, respectively get after 30mg EDC and NHS 0.2mL water fully dissolve and add in above-mentioned solution, stirred at ambient temperature 24h, can obtain reaction solution A; Take carrier proteins 50mg, make it fully to be dissolved in 3.8mL PBS (pH7.2) solution, reaction solution A is dropwise slowly added drop-wise in protein solution, and in stirred at ambient temperature 24h; By 0.01mol/L PBS solution at 4 DEG C of dialysis 3d, change 3 dialyzates every day, to remove unreacted small-molecule substance; Packing, saves backup in-20 DEG C.
5. the antibody prepared by the arbitrary described gentamicin antigen of claim 3-4.
6. antibody as claimed in claim 5, is characterized in that described antibody is gentamicin monoclonal antibody.
7. in gentamicin haptens according to claim 1 or claim 3-4, arbitrary described gentamicin antigen detects application in the product of gentamicin detecting gentamicin or preparation.
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| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
| CN112028786A (en) * | 2020-08-12 | 2020-12-04 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106525835A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for gentamycin in food |
| CN112028786A (en) * | 2020-08-12 | 2020-12-04 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| CN112028786B (en) * | 2020-08-12 | 2022-02-11 | 华南农业大学 | Tyramine hapten, antigen and antibody, and preparation method and application thereof |
| 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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