CN101566592A - Method for detecting gentamicin through clinical magnetic resonance imaging - Google Patents

Method for detecting gentamicin through clinical magnetic resonance imaging Download PDF

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CN101566592A
CN101566592A CNA2009100270673A CN200910027067A CN101566592A CN 101566592 A CN101566592 A CN 101566592A CN A2009100270673 A CNA2009100270673 A CN A2009100270673A CN 200910027067 A CN200910027067 A CN 200910027067A CN 101566592 A CN101566592 A CN 101566592A
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gentamicin
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antibody
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胥传来
马伟
陈伟
徐丽广
李灼坤
彭池方
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Jiangnan University
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Abstract

The invention relates to a method for detecting gentamicin through clinical magnetic resonance imaging, and belongs to the technical field of immunodetection. The method is to add standard gentamicin supernatant or gentamicin supernatant to be measured, gentamicin antibodies and goat-anti and rabbit-anti antibodies into gentamicin modified magnetic nano particles, and use the antibodies and the gentamicin modified magnetic nano particles to form immune polymers so as to generate magnetic resonance signals and perform magnetic resonance detection. The magnetic resonance signal detection method utilizes the antibodies and the gentamicin modified magnetic nano particles to form the immune polymers, and effectively shortens the transverse relaxation time T2 value of protons in water so as to generate signals. In general, when the concentration of the gentamicin in a sample is larger, the quantity of the gentamicin caught by the antibodies is more, the quantity of the antibodies which are combined with the gentamicin on the surface of the magnetic nano particles is fewer, and the measured T2 value is larger. The method does not need complex sample pretreatment, is simple and convenient to operate, only needs to add the sample to be measured for incubation and directly measure the T2 value, and can complete the process by one step. Moreover, the particle diameter of superparamagnetic nano particles for marking is approximately 10 nanometers, so that the water solubility is high and the dispersity and the stability are good.

Description

A kind of method of detecting gentamicin through clinical magnetic resonance imaging
Technical field
The present invention relates to a kind of method of magnetic nano-particle labelled antibody detection by quantitative gentamicin, belong to technical field of immunoassay.
Background technology
Gentamicin is the polycomponent microbiotic that is produced by micromonospora, contains C 1, C 2, C 1aEtc. component, structure is seen accompanying drawing 1.Gentamicin sulphate is a white powder, and hydroscopicity is arranged, and is soluble in water, is insoluble in common organic solvents, and temperature and acid, alkali are all stablized, and antimicrobial spectrum and kanamycins are basic identical.Characteristics are that Pseudomonas aeruginosa, proteus and penicillin resistant gold grape are all had an inhibiting effect.With neomycin, kanamycins the crossing drug resistant phenomenon is arranged.Be mainly used in the treatment of the severe infections of above-mentioned sensitive bacteria such as septicemia, respiratory tract infection, pneumonia, enteric infection, infection of biliary tract, urinary tract infections, soft tissue infection etc.The toxicological effect of gentamicin is similar with the aminoglycoside other medicines, has very strong renal toxicity and hepatotoxicity wind agitation, poisons very big.
The method that current gentamicin residue commonly used in the world detects has: microbial method, liquid chromatography (LC), gas chromatography (GC), gas-matter coupling method (GC-MS), liquid-matter coupling method (LC-MS) etc.Microorganism detection is inaccurate and detection sensitivity is not high, and instrument analytical method not only needs expensive instrument and equipment, also than higher, needs through complicated sample pre-treatment just can carry out operating personnel's requirement.The external research of having carried out already the aminoglycoside antibiotics immune analysis method, method commonly used is enzyme-linked immunosorbent assay (ELISA) or is called immuno-enzymatic quantitative analysis method (IEMA), these class methods are with the coating antigen coated elisa plate, add medicine and anti-drug antibodies, add ELIAS secondary antibody again, promptly detect antibody, add the substrate colour developing at last, behind the certain hour, detect the absorbance of a certain specific wavelength with microplate reader, according to drug concentrations to be measured in the known standard product cubage sample, this complex operation, time-consuming.
Magnetic resonance imaging (magnetic resonance imaging, MRI) technology owing to can be used for fast detecting that biological internal organs and soft tissue be can't harm has become the most effectively one of methods for clinical diagnosis that the diagnosis soft tissue lesions especially detects tumour.For superparamagnetic nano particle, it mainly acts on is to shorten spin spinrelaxation (T 2), and to longitudinal relaxation time (T 1) influence not quite.Domesticly do not see have the magnetic resonance signal that utilizes superparamagnetic nanoparticle to reach the report that external object is detected so far as yet, the present invention has remedied this blank.
Summary of the invention
The purpose of this invention is to provide a kind of immunologic detection method, be used for the fast detecting of gentamicin residue with sensitivity, specificity, accuracy, the simple magnetic nano-particle labelled antibody of method of operating.
Technical scheme of the present invention: a kind of method of detecting gentamicin through clinical magnetic resonance imaging, at first with the gentamicin modified magnetic nano particles, add gentamicin standard or supernatant to be measured, gentamicin antibody, goat-anti rabbit two is anti-, antibody and gentamicin modified magnetic nano particles form immune polymkeric substance, produce magnetic resonance signal thus, carry out magnetic resonance and detect; Step is:
(1) lotus root of gentamicin and magnetic nano-particle connection:
1 μ L carbodiimide EDC and 2.4mg N-hydroxy thiosuccinimide sulfo-NHS join the Fe of 0.5mL, 1.3mg/mL 3O 4In the aqueous solution, reaction 15min activation, the Fe after the activation 3O 4Add 1mg gentamicin (GM medicine), room temperature reaction 4 hours, dialysis is 24 hours in the PBS of 0.01M, pH7.4, removes unreacted gentamicin micromolecule, makes the Fe that gentamicin is modified 3O 4Magnetic nano-particle;
(2) magnetic resonance detects, and draws relaxation time T 2～C gentamicin concentration typical curve:
The Fe that the gentamicin of 20 μ L, 150 μ g/mL is modified 3O 4Add the gentamicin standard items of 20 μ L variable concentrations in the magnetic nano-particle, add 1 μ L, 5mg/mL gentamicin antibody, add 1 μ L goat-anti rabbit two again and resist, after hatching 2 hours, being diluted to volume is 250 μ L, joins in the microwell plate, survey magnetic resonance, with spin spinrelaxation T 2Draw T 2～C gentamicin concentration typical curve;
Control group is provided with: gentamicin antibody replaces with dexamethasone antibody, and other operation is the same;
Solutions employed is all the PBS damping fluid of 0.01M, pH7.4; Gentamicin standard items concentration is respectively 0,8,12,16,32,48,72,100,120,144ng/mL;
Survey magnetic resonance with Siemens's clinical magnetic resonance imaging equipment (MAGNETOM Avanto 1.5T MRI), echo time, (echo time) TE was 20-120ms, repetition time (repetitiontime) TR is 2000ms, and used coil is a head coil;
(3) magnetic resonance detection by quantitative:
The Fe that the gentamicin of 20 μ L, 150 μ g/mL is modified 3O 4Add the supernatant to be measured of 20 μ L in the magnetic nano-particle, add 1 μ L, 5mg/mL gentamicin antibody, it is anti-to add 1 μ L goat-anti rabbit two again, hatch 2 hours after, being diluted to volume is 250 μ L, joins in the microwell plate, surveys magnetic resonance, with spin spinrelaxation T 2With the T that draws 2～C gentamicin concentration typical curve contrasts, and obtains the concentration of gentamicin in the sample.
The magnetic nano-particle that described gentamicin is modified is the surface that gentamicin is linked to the magnetic nano-particle of carboxyl modified with lotus root linked method lotus root; Magnetic nano-particle is the magnetic nano-particle of two carboxyl modified, and its core is Fe 3O 4
Described antibody is the polyclonal antibody of anti-gentamicin, through the specific antibody of ammonium sulfate precipitation method purifying.
The magnetic nano-particle that described antibody and gentamicin are modified forms immune polymkeric substance: add gentamicin antibody in the magnetic nano-particle that gentamicin is modified after, gentamicin free in the gentamicin of magnetic nano particle sub-surface and the solution is competed antibody, combines with the specificity of antibody by antigen and forms Ag-Ab immunity polymkeric substance.
It is to utilize antibody and gentamicin modified magnetic nano particles to form immune polymkeric substance that described magnetic resonance signal detects, and effectively shortens proton spin spinrelaxation T in the water 2Be worth and the generation signal.Usually gentamicin concentration is big more in the sample, and many more by the gentamicin amount of antibody capture, then the antibody that combines with the gentamicin of magnetic nano particle sub-surface is few more, the T that then records 2Numerical value is big more.
Beneficial effect of the present invention:
(1) this law does not need the complicated sample pre-treatment, even for whole blood, and urine, tissue fluid.
(2) this law is easy and simple to handle, only need add and directly measure T after testing sample is hatched 2Value only needs can finish once going on foot.
(3) this law is used for the mark superparamagnetic nanoparticle, and particle diameter is 10nm, water-soluble height, dispersed good stability.Saturation magnetization is 65emu g -1
Description of drawings
Fig. 1 gentamicin structural formula.
Fig. 2 magnetic resonance imaging figure (left side) and little coloured picture (right side).
Fig. 3 magnetic resonance examination criteria curve.
Embodiment
The lotus root connection of embodiment 1 gentamicin and magnetic nano-particle:
1 μ L carbodiimide EDC and 2.4mg N-hydroxy thiosuccinimide sulfo-NHS join the Fe of 0.5mL, 1.3mg/mL 3O 4In the aqueous solution, activate about reaction 15min.Fe after the activation 3O 4Add 1mg gentamicin (GM medicine), room temperature reaction 4 hours, dialysis is 24 hours in the PBS of 0.01M, pH 7.4, removes unreacted medicine micromolecule.
Embodiment 2 magnetic resonance:
The Fe that the gentamicin of 20 μ L, 150 μ g/mL is modified 3O 4In add the gentamicin standard items of 20 μ L variable concentrations, add 1 μ L gentamicin antibody (5mg/mL), it is anti-to add 1 μ L goat-anti rabbit two again, hatch 2 hours after, being diluted to volume is 250 μ L, joins in the microwell plate, surveys magnetic resonance.
Control group is provided with: gentamicin antibody replaces with dexamethasone antibody, and other operation is the same.
Solutions employed is all the PBS damping fluid of 0.01M, pH7.4.Gentamicin standard items concentration is respectively 0,8,12,16,32,48,72,100,120,144ng/mL.
Embodiment 3 magnetic resonance detection by quantitative
Magnetic resonance equipment is MAGNETOM Avanto 1.5T MRI, Siemens's clinical magnetic resonance imaging equipment.
This tests used sweep time parameter: the echo time, (echo time) TE was 20-120ms, and repetition time (repetition time) TR is 2000ms, and used coil is a head coil.
The principle that magnetic resonance detects object is that the formation by antibody and the immune polymkeric substance of drug modification magnetic nano-particle causes water proton T 2The size of value reaches and detects the object purpose.The gentamicin that adds in the water more at most polymkeric substance form few more, T 2Be worth then big more.By the T that sets up 2～C/ gentamicin concentration typical curve can contrast the concentration of obtaining gentamicin in the sample.Typical curve is seen accompanying drawing 3.
Embodiment 4 actual samples are measured
Actual sample is handled: get pork tissue sample 1g, in the 50mL centrifuge tube, the PB of 10mL, 0.2M (phosphate buffer) mixes with tissue sample, and mixed liquor is hatched 30min at 60 ℃, the centrifugal 15min of 4000r/min then, and it is to be measured to get supernatant.
The Fe that the gentamicin of 20 μ L, 150 μ g/mL is modified 3O 4The middle 20 μ L supernatant to be measured that adds, add 1 μ L gentamicin antibody (5mg/mL), it is anti-to add 1 μ L goat-anti rabbit two again, after hatching 2 hours, being diluted to volume is 250 μ L, joins in the microwell plate, survey magnetic resonance with Siemens's clinical magnetic resonance imaging equipment (MAGNETOMAvanto 1.5T MRI), echo time, (echo time) TE was 20-120ms, and repetition time (repetition time) TR is 2000ms, and survey is the T of hydrogen proton in the water 2So selecting TR is that 2000ms is proper, used coil is a head coil, with T 2～C/ gentamicin concentration typical curve contrasts, and obtains the concentration of gentamicin in the sample.
Table 1 actual sample adds recovery
Figure A20091002706700061
The drafting of typical curve is a horizontal ordinate with standard items concentration, with Δ T 2Ordinate, data see the following form, and accompanying drawing 3 is seen in mapping.
Table 2 MR data
A B C D E F G H I
Gentamicin concentration ng/ml 144 120 100 72 48 32 16 12 8
ΔT 2 47.5 39.8 34.4 21.9 15.1 7.8 3.5 2.1 1

Claims (1)

1, a kind of method of detecting gentamicin through clinical magnetic resonance imaging, it is characterized in that at first with the gentamicin modified magnetic nano particles, add gentamicin standard or supernatant to be measured, gentamicin antibody, goat-anti rabbit two is anti-, antibody and gentamicin modified magnetic nano particles form immune polymkeric substance, produce magnetic resonance signal thus, carry out magnetic resonance and detect; Step is:
(1) lotus root of gentamicin and magnetic nano-particle connection:
1 μ L carbon, two inferior gang EDC and 2.4mg N-hydroxy thiosuccinimide sulfo-NHS join the Fe of 0.5mL, 1.3mg/mL 3O 4In the aqueous solution, reaction 15min activation, the Fe after the activation 3O 4Add the 1mg gentamicin, room temperature reaction 4 hours, dialysis is 24 hours in the PBS of 0.01M, pH 7.4, removes unreacted gentamicin micromolecule, makes the Fe that gentamicin is modified 3O 4Magnetic nano-particle;
(2) magnetic resonance detects, and draws relaxation time T 2～C gentamicin concentration typical curve:
The Fe that the gentamicin of 20 μ L, 150 μ g/mL is modified 3O 4Add the gentamicin standard items of 20 μ L variable concentrations in the magnetic nano-particle, add 1 μ L, 5mg/mL gentamicin antibody, add 1 μ L goat-anti rabbit two again and resist, after hatching 2 hours, being diluted to volume is 250 μ L, joins in the microwell plate, survey magnetic resonance, with spin spinrelaxation T 2Draw T 2～C gentamicin concentration typical curve;
Control group is provided with: gentamicin antibody replaces with dexamethasone antibody, and other operation is the same;
Solutions employed is all the PBS damping fluid of 0.01M, pH7.4; Gentamicin standard items concentration is respectively 0,8,12,16,32,48,72,100,120,144ng/mL;
Survey magnetic resonance with the clinical magnetic resonance imaging equipment MAGNETOM Avanto 1.5T MRI of Siemens, sweep time, parameter was: echo time TE is 20-120ms, totally 6 echoes, and repetition time TR is 2000ms, used coil is a head coil;
(3) magnetic resonance detection by quantitative:
The Fe that the gentamicin of 20 μ L, 150 μ g/mL is modified 3O 4Add the supernatant to be measured of 20 μ L in the magnetic nano-particle, add 1 μ L, 5mg/mL gentamicin antibody, it is anti-to add 1 μ L goat-anti rabbit two again, hatch 2 hours after, being diluted to volume is 250 μ L, joins in the microwell plate, surveys magnetic resonance, with spin spinrelaxation T 2With the T that draws 2～C gentamicin concentration typical curve contrasts, and obtains the concentration of gentamicin in the sample.
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CN102243229A (en) * 2010-05-12 2011-11-16 上海中医药大学 Radioimmunoassay kit for measuring rat serum total IgE, and detection method thereof
CN103197070A (en) * 2013-03-17 2013-07-10 南昌大学 Quick detection method of NMR (nuclear magnetic resonance) food-borne pathogenic bacteria based on gamma-Fe2O3 nano-particle indirect inrichment
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CN103197070A (en) * 2013-03-17 2013-07-10 南昌大学 Quick detection method of NMR (nuclear magnetic resonance) food-borne pathogenic bacteria based on gamma-Fe2O3 nano-particle indirect inrichment
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CN104122285A (en) * 2014-07-09 2014-10-29 张祥林 Magnetic-bead-based low field NMR (nuclear magnetic resonance) rare cell detection method
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