CN105017347B - Gentamicin haptens and its preparation method and application - Google Patents
Gentamicin haptens and its preparation method and application Download PDFInfo
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- CN105017347B CN105017347B CN201410238929.8A CN201410238929A CN105017347B CN 105017347 B CN105017347 B CN 105017347B CN 201410238929 A CN201410238929 A CN 201410238929A CN 105017347 B CN105017347 B CN 105017347B
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Abstract
The invention discloses a kind of gentamicin haptens and its preparation method and application.Gentamicin haptens provided by the invention, structural formula is as shown in formula I：
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 gramnegative bacterium,
Because it has broad-spectrum antibacterial and bactericidal action, it is widely used in veterinary clinic and animal feed additive.But GM serum
Valid density is very close with toxic concentration, and safe range is smaller, and toxic side effect is also easy to produce in its application process, especially heavy dose of
Use lack of standardization, it is exceeded to be easily caused in animal food GM residuals, and health is formed and endangered, causes renal toxicity and ear poison
Property.Many countries forbid have strict MRL (MRL), the Ministry of Agriculture of China using GM or regulation in animal productiong
No. 235 bulletins provide that GM MRL is 100ng/g in animal food.
Gentamicin residue detection method mainly has gas chromatography, high performance liquid chromatography, liquid chromatography-tandem at present
Mass spectrography, high performance capillary electrophoresis and immunological detection.Instrumental method detection is expensive, is not suitable for promoting the use of in basic unit
With a large amount of screenings.Immunological assay method sensitivity is higher, and high specificity is easy to use.Therefore, Large-scale Screening detection in order to avoid
Epidemiology detection method is actual, with more application prospect.Enzyme linked immunosorbent assay (ELISA) with its high sensitivity, specificity it is good,
Easy to operate, low cost and other advantages are suitable for the quick screening of a large amount of samples.
The content of the invention
It is an object of the invention to provide a kind of gentamicin haptens and its preparation method and application.
Gentamicin hapten molecule structural formula provided by the invention is as shown in formula I：
The preparation method of gentamicin haptens provided by the invention, comprises the following steps：
0.5g is added in two mouthfuls of flasks of 100mL to bromo methyl acid and 1.05g gentamicins and 10mL ethanol, stirring
To dissolving, after being heated to 45 DEG C of reactions 6 hours, solvent is removed under reduced pressure.It is recrystallized to give in ethanol-water system big to the celebrating of carboxylic benzyl
Mycin, obtain haptens product.
Another object of the present invention is application of the above-mentioned gentamicin haptens in immune detection, is specifically included by institute
The gentamicin antigen that gentamicin haptens obtains with carrier protein couplet is stated, and by gained gentamicin antigen-immunized animal
The gentamicin antibody being prepared, the antibody are gentamicin monoclonal antibody.
Wherein described carrier protein can be bovine serum albumin(BSA), thyroprotein, ovalbumin, human albumin, mouse blood
Albumin, rabbit serum proteins, hemocyanin or fibrinogen.
The present invention is also provided by above-mentioned gentamicin haptens or gentamicin antigen in detection gentamicin or preparation inspection
The application surveyed in the product of gentamicin, and in particular to the enzyme-linked immunologic detecting kit of preparation is in animal derived food is detected
The application of gentamicin residue.
Gentamicin haptens provided by the invention, gentamicin antigen synthetic method are simple, pass through immune animal and produce
The specific antibody of gentamicin is directed to, potency, the specificity of antibody are all relatively good；With the Antibody preparation enzyme linked immunological of gained
Detection kit, testing cost is low, easy to use, detection method is accurate, it is quick, large batch of sample can be detected simultaneously, be suitable to
The on-site supervision of gentamicin residue and the examination of great amount of samples in animal derived food.The gentamicin haptens of the present invention exists
Played a significant role in the detection of gentamicin.
Brief description of the drawings
Fig. 1：Gentamicin hapten synthesis route map
Fig. 2：Gentamicin haptens nuclear magnetic resonance figures
Fig. 3：Gentamicin ELISA standard curves
Embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.
Experimental method, material, reagent used in following embodiments etc., unless otherwise specified, be conventional method and
Conventional material.
Embodiment 1：The synthesis and identification (synthetic route such as Fig. 1) of gentamicin haptens
0.5g is added in two mouthfuls of flasks of 100mL to bromo methyl acid and 1.05g gentamicins and 10mL ethanol, stirring
To dissolving, after being heated to 45 DEG C of reactions 6 hours, solvent is removed under reduced pressure.It is recrystallized to give in ethanol-water system big to the celebrating of carboxylic benzyl
Mycin, obtain haptens product.
Above-mentioned gentamicin hapten molecule structural formula is as shown in formula I：
Take above-mentioned product to determine structure with nuclear magnetic resonance, as shown in Fig. 2 11.0ppm carboxyl signal peak, 7.44ppm and
8.09ppm aromatic ring signal peak, illustrate hapten synthesis success.
Embodiment 2：The preparation of gentamicin antigen
Gentamicin haptens and carrier protein couplet are obtained into gentamicin antigen, molecular structural formula is as shown in formula II：
First, prepared by immunogene
Take 14mg gentamicin haptens 1mL DMFs (DMF) to dissolve, respectively take 30mg carbonizations two sub-
Amine (EDC) and n-hydroxysuccinimide (NHS) are added in above-mentioned solution after fully being dissolved with 0.2mL water, are stirred at room temperature
24h, you can obtain reaction solution A；Bovine serum albumin(BSA) (BSA) 50mg is weighed, is allowed to be substantially dissolved in 3.8mL PBS (pH7.2)
In solution, reaction solution A is slowly dropped in protein solution dropwise, and stir 24h at room temperature；It is molten with 0.01mol/L PBS
Liquid 4 DEG C dialyse 3d, 3 dialyzates are changed daily, to remove unreacted small-molecule substance；Packing, is saved backup in -20 DEG C.
2nd, prepared by coating antigen
BSA is changed to ovalbumin (OVA) coating antigen is prepared as stated above.
Embodiment 3：The preparation of gentamicin monoclonal antibody
Animal immune：The immunogene obtained in embodiment 2 is injected into Balb/c Mice Bodies, immunizing dose is 150 μ g/
Only, it is made to produce antiserum.
Cell fusion and cloning：After mice serum measurement result is higher, its splenocyte is taken, by 8:1 ratio and SP2/0 bones
Myeloma cells merge, and determine cell supernatant using indirect competitive ELISA, screen positive hole.Using limiting dilution assay to the positive
Hole carries out cloning, until obtaining the hybridoma cell strain of secrete monoclonal antibody.
Cell cryopreservation and recovery：Cell suspension is made with frozen stock solution in the monoclonal hybridoma strain of gentamicin,
Preserved for a long time in liquid nitrogen.Cryopreservation tube is taken out during recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, after centrifugation removes frozen stock solution, is moved into
Cultivate culture in glassware.
The production and purifying of monoclonal antibody：Balb/c mouse peritoneals are injected into sterilizing paraffin oil 0.5mL/ only, abdomen after 7 days
Chamber injects the monoclonal hybridoma strain of gentamicin, and ascites is gathered after 7 days.Ascites is carried out with octanoic acid-saturated ammonium sulfate method
Purifying, -20 DEG C of preservations.The monoclonal antibody specificity is good, and detection sensitivity is up to 0.1 μ g/L.
The specificity of monoclonal antibody
Antibody specificity refers to the ability of its homospecificity antigen binding and the ratio with such antigen-analogues ability
Compared with conventional cross reacting rate is as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
Gentamicin, streptomysin, dihydrostreptomycin, neomycin are serially diluted, make standard curve, analysis obtains
IC50, cross reacting rate is then calculated as follows, the results are shown in Table 1.
The antibody specificity of table 1
Embodiment 4：The enzyme-linked immunologic detecting kit prepared by gentamicin monoclonal antibody
First, the composition of enzyme-linked immunologic detecting kit
(1) it is coated with the ELISA Plate of gentamicin coupled antigen；
(2) ELIAS secondary antibody or ELIAS secondary antibody concentrate；
(3) antibody working solution；
(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, high standard product solution concentration are 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) terminate liquid is 2mol/L sulfuric acid solution；
(7) concentrated cleaning solution is the phosphorus of 0.1~0.2mol/L pH7.2~7.4 containing 0.5%~1.0% Tween-20
Phthalate buffer, the percentage are percent weight in volume；
(8) it is pH7.2~7.6 that liquid is redissolved in concentration, and 0.05~0.1mol/L phosphate buffer, the percentage is attached most importance to
Measure percent by volume.
The main agents of this kit are provided in the form of working solution, and the method for inspection is convenient and easy, have specificity high, clever
The features such as sensitivity is high, accuracy is high, the degree of accuracy is high.
2nd, the application of enzyme-linked immunologic detecting kit detection actual sample
(1) Sample pretreatment method one
1st, (chicken, chicken gizzard) Sample pretreatment method is organized
Weigh 2.0g ± 0.05g and remove fatty tissue samples into 50mL polystyrene centrifuge tubes, add
6mL0.1mol/L PBS solutions (weigh 13.4g disodium hydrogen phosphate dodecahydrates, 20.0g sodium chloride, 0.32g sodium hydroxides,
The hypophosphite monohydrate potassium dihydrogens of 0.5g bis-, 0.5g potassium chloride add 500mL deionized water dissolvings and mixed), mix 10min；Add 60 DEG C
60min in water bath, taking-up are cooled to room temperature and shaken up；More than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugation
10min；Pipette supernatant and (2 × concentration is redissolved into liquid by 1 with deionized water with redissolving working solution:1 volume ratio is diluted (1 part
+ 1 part of deionized water of liquid is redissolved in 2 × concentration) it is used for the redissolution of sample, one can be preserved in 4 DEG C of (39.2 ℉) environment by redissolving working solution
Individual month) with 1:9 volume ratio is diluted (the μ L of 50 μ L of supernatant liquid+450 redissolve working solution), takes 50 μ L to be used to analyze.
2nd, fresh milk Sample pretreatment method
20 μ L fresh milks samples are taken into 2mL polystyrene centrifuge tubes, 780 μ L is added and redissolves working solution, mix, take 50 μ L
For analyzing.
3rd, milk powder Sample pretreatment method
1.0g ± 0.05g milk powder sample is weighed into 10mL polystyrene centrifuge tubes, 5mL deionized waters are added, with vibration
Device fully vibrates to milk powder and all dissolved；Take out 50 μ L samples liquid add 950 μ L redissolve working solution, mix, take 50 μ L be used for point
Analysis.
(2) Sample pretreatment method two
1st, chicken Sample pretreatment method
With homogenizer homogeneous sample；2.0g ± 0.05g homogeneous thing is weighed into 50mL polystyrene centrifuge tubes, is added
4mL0.07mol/L carbonate buffer solutions (weigh 3.26g natrium carbonicum calcinatums and 0.35g sodium acid carbonates add 500mL deionized waters
Dissolving mixes) and 2mL methanol, with vortex instrument whirling motion 5min, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugation 5min；
100 μ L of supernatant liquid are pipetted into 2mL polystyrene centrifuge tubes, 900 μ L is added and redissolves working solution, with vortex instrument whirling motion 1min；Take
20 μ L are used to analyze.
2nd, chicken gizzard Sample pretreatment method
With homogenizer homogeneous sample；2.0g ± 0.05g homogeneous thing is weighed into 50mL polystyrene centrifuge tubes, is added
3mL0.1mol/L carbonate buffer solutions (weigh 4.66g natrium carbonicum calcinatums and 0.5g sodium acid carbonates addition 500mL deionizations are water-soluble
Solution mixes) and 3mL methanol, with vortex instrument whirling motion 5min, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugation 5min；Move
100 μ L of supernatant liquid are taken into 2mL polystyrene centrifuge tubes, 900 μ L is added and redissolves working solution, with vortex instrument whirling motion 1min；Take 20 μ
L is used to analyze.
3rd, milk Sample pretreatment method
1mL milk sample is pipetted into 2mL polystyrene centrifuge tubes, 1mL1% trichloroacetic acids is added and (weighs 5.0g trichlorines
Acetic acid adds 500mL deionized water dissolvings and mixed), with vortex instrument whirling motion 3min, more than 3000g, (20-25 DEG C/68-77 of room temperature
℉) centrifuge 5min；100 μ L of supernatant liquid are pipetted into 2mL polystyrene centrifuge tubes, 900 μ L is added and redissolves working solution, use vortex instrument
Whirling motion 1min；20 μ L are taken to be used to analyze.
4th, milk powder Sample pretreatment method
1.0g ± 0.05g milk powder is weighed into 10mL polystyrene centrifuge tubes, 5mL deionized waters are added, with vortex instrument whirlpool
Dynamic 3min, makes milk powder fully dissolve, more than 3000g, room temperature (20-25 DEG C/68-77 ℉) centrifugation 5min；Pipette 100 μ L of supernatant liquid
Into 2mL polystyrene centrifuge tubes, add 900 μ L and redissolve working solution, with vortex instrument whirling motion 1min；20 μ L are taken to be used to analyze.
(3) detected with kit
Detected after sample is handled according to pre-treating method one with kit, it is micro- to the ELISA Plate for being coated with coating antigen
50 μ L standard items/sample is added in hole, adds 50 μ L antibody working solutions, gently vibration mixes, and covers cover plate film, 37 DEG C of constant temperature
Lucifuge reacts 30min in case.The liquid in hole is poured out, (20 × concentrated cleaning solution 1 is pressed with deionized water with wash operating solution:
19 volume ratios are diluted) 250 μ L/ holes fully wash 4-5 times, per minor tick 10s, patted dry with blotting paper to ensure to remove completely
Liquid in hole.The μ L of ELIAS secondary antibody 100 are added per hole, gently vibration mixes, with rearmounted 37 DEG C (99 ℉) lucifuges of cover plate membrane cover plate
30min is reacted in environment.The liquid in hole is poured out, is fully washed 4-5 times with the μ L/ holes of wash operating solution 250, per minor tick 10s,
Patted dry with blotting paper to ensure to remove the liquid in hole completely.The μ L of substrate solution A liquid 50 are added per hole, add the μ of substrate solution B liquid 50
L, gently vibration mix, with lucifuge colour developing 15min in 37 DEG C of insulating boxs after cover plate membrane cover plate.50 μ L terminate liquids are added, are gently shaken
Mixing is swung, setting ELIASA measures the absorbance per hole at 450nm.
Detected after sample is handled according to pre-treating method two with kit, it is micro- to the ELISA Plate for being coated with coating antigen
20 μ L standard items/sample is added in hole, adds the mixed liquor of 80 μ L antibody working solutions and ELIAS secondary antibody concentrate (by antibody work
Make liquid and ELIAS secondary antibody concentrate by 10:1 volume ratio is mixed and mixed), gently vibration mixes, and covers cover plate film, 25 DEG C of constant temperature
Lucifuge reacts 30min in case.The liquid in hole is poured out, is fully washed 4-5 times with the μ L/ holes of wash operating solution 250, per minor tick
10s, patted dry with blotting paper to ensure to remove the liquid in hole completely.The μ L of substrate solution A liquid 50 are added per hole, add substrate solution B
The μ L of liquid 50, gently vibration mix, with lucifuge colour developing 15min in 25 DEG C of insulating boxs after cover plate membrane cover plate.50 μ L terminate liquids are added, gently
Light vibration mixes, and setting ELIASA measures the absorbance per hole at 450nm.
(4) Analysis of test results
With the average value (diplopore) of the standard items or the absorbance of sample obtained divided by first standard (0 standard)
Absorbance, multiplied by the percentage absorptance for 100%, that is, obtaining standard items or sample.With gentamicin standard items percentage extinction
Rate is ordinate, using the logarithm of gentamicin standard concentration as abscissa, draws canonical plotting, as shown in Figure 3.By sample
Percentage absorptance substitute into standard curve, the concentration corresponding to sample is read from standard curve, is multiplied by corresponding to its and dilutes
Multiple is gentamicin actual concentrations in sample.
3rd, the determination of enzyme-linked immunologic detecting kit technical parameter
Minimum detection limit：Respectively to 20 portions of blank chicken, chicken gizzard, milk, milk powder sample, according to Sample pretreatment method one
After processing, detected with kit, the concentration corresponding to each percentage absorptance is found from standard curve, celebrated with 20 parts of samples
The average value of big mycin concentration represents test limit plus 3 times of standard deviations, as a result obtains test limit of this method to chicken, chicken gizzard sample
For 4 μ g/kg, the detection to milk sample is limited to 4 μ g/L, and the detection to milk powder sample is limited to 10 μ g/kg.
Respectively to 20 portions of blank chicken, chicken gizzard, milk, milk powder sample, after being handled according to Sample pretreatment method two, with examination
Agent box is detected, and the concentration corresponding to each percentage absorptance is found from standard curve, with 20 parts of sample gentamicin concentrations
Average value represent test limits plus 3 times of standard deviations, as a result detection of this method to chicken, chicken gizzard sample is limited to 4 μ g/kg,
Detection to milk sample is limited to 2 μ g/L, and the detection to milk powder sample is limited to 5 μ g/kg.
The degree of accuracy and precision：The degree of accuracy of ELISA measure represents that precision is represented with the coefficient of variation with the rate of recovery.
Blank chicken, chicken gizzard sample are taken respectively, with the addition concentration of 4,8,16 μ g/kg tri-, take blank milk sample, with 4,
8th, 16 μ g/L, tri- addition concentration, take blank milk powder sample, with the gentamicin medicine of 10,20,40 μ g/kg, tri- addition concentration
It is added, respectively according to a pair of chicken of Sample pretreatment method, chicken gizzard, milk, milk powder sample process after, use kit
Detected, it is 80%~100% to the rate of recovery of chicken, chicken gizzard sample as a result to obtain this method, milk, the milk powder sample rate of recovery
For 70%~110%.Variation within batch coefficient ＜ 15%, interassay coefficient of variation ＜ 25%.
Blank chicken, chicken gizzard sample are taken respectively, with the addition concentration of 4,8,16 μ g/kg tri-, take blank milk sample, with 2,
4th, 8 μ g/L, tri- addition concentration, take blank milk powder sample, with the gentamicin medicine pair of 5,10,20 μ g/kg, tri- addition concentration
It is added, respectively according to Sample pretreatment method two to chicken, chicken gizzard, milk, milk powder sample process after, entered with kit
Row detection, it is 85%~115% to the rate of recovery of chicken, chicken gizzard, milk, milk powder sample as a result to obtain this method.Variation within batch system
Number ＜ 15%, interassay coefficient of variation ＜ 25%.
Kit of the present invention can at least preserve 12 months at 2~8 DEG C after measured.
Claims (2)
- A kind of 1. enzyme-linked immunologic detecting kit prepared by gentamicin monoclonal antibody, it is characterised in that its composition bag Include：(1) it is coated with the ELISA Plate of gentamicin coupled antigen；(2) ELIAS secondary antibody or ELIAS secondary antibody concentrate；(3) antibody working solution；(4) standard solution：Concentration is respectively 0 μ g/L, 0.1 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, high standard product solution concentration are 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) terminate liquid is 2mol/L sulfuric acid solution；(7) concentrated cleaning solution is the phosphate of 0.1~0.2mol/L pH7.2~7.4 containing 0.5%~1.0% Tween-20 Buffer solution, the percentage are percent weight in volume；(8) it is pH7.2~7.6 that liquid is redissolved in concentration, 0.05~0.1mol/L phosphate buffer；Wherein, the antibody working solution is the gentamicin monoclonal antibody that is prepared by gentamicin antigen, the gentamicin Antigen is coupled to obtain by gentamicin haptens with bovine serum albumin(BSA)；The preparation method of the gentamicin haptens is included such as Lower step：0.5g is added in two mouthfuls of flasks of 100mL to bromo methyl acid and 1.05g gentamicins and 10mL ethanol, stirring To dissolving, after being heated to 45 DEG C of reactions 6 hours, remove solvent under reduced pressure, be recrystallized to give in ethanol-water system big to the celebrating of carboxylic benzyl Mycin, as gentamicin haptens, its molecular structural formula are：
- 2. enzyme-linked immunologic detecting kit as claimed in claim 1, it is characterised in that the preparation side of the gentamicin antigen Method comprises the following steps：Take 14mg gentamicin haptens 1mL DMF to dissolve, respectively take 30mg EDC and NHS to be filled with 0.2mL water Added after dividing dissolving in above-mentioned solution, stir 24h at room temperature, you can obtain reaction solution A；Bovine serum albumin(BSA) 50mg is weighed, is made Be substantially dissolved in 3.8mL pH7.2 PBS solution, reaction solution A is slowly dropped in protein solution dropwise, and in room temperature Lower stirring 24h；With 0.01mol/L PBS solutions 4 DEG C dialyse 3d, 3 dialyzates are changed daily, to remove unreacted small molecule Material；Packing, is saved backup in -20 DEG C.
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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 |
| 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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| US4410634A (en) * | 1979-10-26 | 1983-10-18 | Dynasciences Corporation | Method of passively adsorbing immuno-reactive haptens to solid phases |
| CN103509068A (en) * | 2012-06-19 | 2014-01-15 | 北京勤邦生物技术有限公司 | Amikacin semi-antigen, and preparation method and application thereof |
| CN103713133A (en) * | 2012-09-29 | 2014-04-09 | 北京勤邦生物技术有限公司 | Test strip for detecting spiramycin, streptomycin, gentamycin and neomycin, and method |
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| US4410634A (en) * | 1979-10-26 | 1983-10-18 | Dynasciences Corporation | Method of passively adsorbing immuno-reactive haptens to solid phases |
| CN103509068A (en) * | 2012-06-19 | 2014-01-15 | 北京勤邦生物技术有限公司 | Amikacin semi-antigen, and preparation method and application thereof |
| CN103713133A (en) * | 2012-09-29 | 2014-04-09 | 北京勤邦生物技术有限公司 | Test strip for detecting spiramycin, streptomycin, gentamycin and neomycin, and method |
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