CN110632296A - Quick detection method for ofloxacin - Google Patents
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- CN110632296A CN110632296A CN201910930658.5A CN201910930658A CN110632296A CN 110632296 A CN110632296 A CN 110632296A CN 201910930658 A CN201910930658 A CN 201910930658A CN 110632296 A CN110632296 A CN 110632296A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention provides a rapid detection method of ofloxacin, which adopts gold magnetic particles of a coupling anti-ofloxacin monoclonal antibody to carry out immunoassay to detect the content of ofloxacin in a sample to be detected; the chromatographic test strip base plate for the immunoassay is sequentially provided with filter paper, a sample pad, a nitrocellulose membrane and absorbent paper in a lap joint manner, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an ofloxacin artificial antigen, and the quality control line is coated with a second antibody. The mode of combining the nano-gold magnetic particles and the monoclonal antibody is any one of an electrostatic adsorption method or a covalent coupling method; the inner core of the nano gold magnetic particle is a magnetic core formed by ferroferric oxide, cobalt and nickel, and the shell is formed by a colloidal gold material. The result judgment of the quick ofloxacin detection method provided by the invention comprises two modes of qualitative detection and quantitative detection.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a rapid detection method of ofloxacin.
Background
Ofloxacin (OFLX), also known as Ofloxacin, belongs to the third generation broad-spectrum quinolone antibacterial drugs, and has strong antibacterial action on gram-positive bacteria and gram-negative bacteria. Due to the characteristics of broad spectrum, high efficiency, low price and the like, the antibacterial agent is widely applied to veterinary drugs and antibacterial agents in aquaculture. In addition, the medicine can be used as feed additive to increase animal weight.
Most veterinary drugs are carcinogenic, mutagenic, and teratogenic. At present, the residual level of most of oxyfluorfloxacin veterinary drugs in animal bodies generally cannot cause acute poisoning of organisms, but the acute poisoning reaction of the organisms can be caused by taking a large amount of animal food with over-standard residual quantity at one time. People with sensitive constitution may have allergic reaction to ofloxacin veterinary drug residue, and in severe cases, the people can endanger life. The long-term use of the antibiotic veterinary drug can greatly increase the drug resistance of pathogenic bacteria, have adverse effect on the treatment effect of human diseases and threaten the health of human beings. In addition, under the condition of rapid development of current environment, economy and trade, the veterinary drug residue has influence on the sustainable development of the environment and the balance of the ecological environment, and is extremely unfavorable for the animal husbandry and the economic trade development of China.
The colloidal gold immunochromatography technology taking a membrane as a solid phase carrier is a novel detection technology developed on the basis of a monoclonal antibody technology and a new material technology in the 80 th of the 20 th century. The method has the characteristics of rapidness, convenience, no need of special equipment, intuitive result judgment and capability of carrying out field detection, is more and more emphasized by people in recent years, has rapid technical development and is widely applied to the field of detection. However, for some samples with extremely low content of the detection target, the detection sensitivity of the colloidal gold test strip is greatly limited. The biological sample detected at the same time may contain color substances which seriously affect the detection result and sensitivity.
Disclosure of Invention
In order to solve the technical problems, the invention provides a rapid detection method of ofloxacin, which adopts gold magnetic particles of a coupling anti-ofloxacin monoclonal antibody to carry out immunoassay to detect the content of ofloxacin in a sample to be detected; the chromatographic test strip base plate for the immunoassay is sequentially provided with filter paper, a sample pad, a nitrocellulose membrane and absorbent paper in a lap joint manner, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an ofloxacin artificial antigen, and the quality control line is coated with a second antibody.
Wherein the mode of combining the nano-gold magnetic particles and the monoclonal antibody is any one of an electrostatic adsorption method or a covalent coupling method.
The inner core of the nano gold magnetic particle is a magnetic core formed by ferroferric oxide, cobalt and nickel, and the shell is formed by a colloidal gold material.
Wherein the size of the gold magnetic particles is 10-500 nm.
Preferably, the size of the gold magnetic particles is 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450 nm.
Wherein, the concentration of the anti-ofloxacin monoclonal antibody is 1-1000 mug/mL.
Preferably, the concentration of the anti-ofloxacin monoclonal antibody is 100 mu g/mL, 200 mu g/mL, 300 mu g/mL, 400 mu g/mL, 500 mu g/mL, 600 mu g/mL, 700 mu g/mL, 800 mu g/mL and 900 mu g/mL.
Wherein, the film spraying concentration of the ofloxacin artificial antigen is 0.1-10mg/mL, and the spraying amount is 0.1-10 muL/cm.
Preferably, the first and second electrodes are formed of a metal,
the film spraying concentration of the ofloxacin artificial antigen is 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL and 9 mg/mL;
the spraying amount of the ofloxacin artificial antigen is 0.5 mu L/cm, 1 mu L/cm, 2 mu L/cm, 3 mu L/cm, 4 mu L/cm, 5 mu L/cm, 6 mu L/cm, 7 mu L/cm, 8 mu L/cm and 9 mu L/cm.
Wherein the second antibody comprises a goat anti-mouse antibody and a donkey anti-mouse antibody, the membrane spraying concentration of the second antibody is 0.1-10mg/mL, and the spraying amount is 0.1-10 muL/cm.
Preferably, the first and second electrodes are formed of a metal,
the membrane spraying concentration of the secondary antibody is 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL and 9 mg/mL;
the spraying amount of the secondary antibody is 0.5 muL/cm, 1 muL/cm, 2 muL/cm, 3 muL/cm, 4 muL/cm, 5 muL/cm, 6 muL/cm, 7 muL/cm, 8 muL/cm and 9 muL/cm.
The determination of the result of the rapid ofloxacin detection comprises two qualitative and quantitative modes, and comprises the following specific steps: dripping the nano gold magnetic particles coupled with the monoclonal antibody into a sample adding hole of a chromatographic test strip, reading the nano gold magnetic particles by using an immunochromatography analyzer after a period of time, and recording the absorbance of a T line, the absorbance of a C line and the T/C value;
1) and (3) qualitative analysis: and qualitatively analyzing by using a visual observation result, wherein the content of the ofloxacin in the sample is higher than the lowest detection lower limit of 1pg/mL if the T line does not develop color, and the content of the ofloxacin in the sample is not contained or is lower than the lowest detection lower limit of 1pg/mL if the T line develops color.
2) Quantitative analysis: and measuring the absorbance of the T line and the C line by using an immunochromatography analyzer, recording the ratio of the absorbance of the T line to the absorbance of the C line as a T/C value, drawing a standard curve by taking the concentration of different ofloxacin as a horizontal coordinate and the T/C value as a vertical coordinate, and determining the ofloxacin content in the common sample by referring to the prepared standard curve graph.
The invention provides a rapid detection method of ofloxacin, which utilizes the magnetic property, the characteristic color and the characteristic of easy coupling antibody of nano gold magnetic particles to mark the anti-ofloxacin antibody by the nano gold magnetic particles, then adds the marker into a sample, simultaneously coats a total antigen of ofloxacin on a nitrocellulose membrane to form a detection line, coats a goat anti-mouse secondary antibody on the nitrocellulose membrane as a quality control line, then sequentially pastes a treated sample pad, the nitrocellulose membrane, absorbent paper and filter paper on a support plate, detects whether the sample contains ofloxacin and quantitatively detects the ofloxacin by utilizing a competition method.
When a sample to be detected contains ofloxacin with a certain concentration, the ofloxacin is firstly combined with a gold magnetic particle labeled antibody, the mixture is dripped into a sample adding hole of a test strip, no probe enrichment or a small amount of enrichment exists on the detection line because an antigen (ofloxacin) -nano gold magnetic particle antibody compound formed by a chromatographic reaction can not react with an ofloxacin holoantigen on a detection line, redundant nano gold magnetic particle markers continuously move to a control band position, because the secondary antibody and the nano gold magnetic particle labeled antibody have an immunoreaction and are enriched on the control band, the test strip is taken out after 1-10 minutes, results are observed on the detection band and a quality control band by naked eyes, and the quantitative interpretation of the results can also be carried out by an immunochromatography analyzer. If no color or no obvious signal exists at the quality control line, the test strip has quality problems and the test is invalid.
The invention has the beneficial effects that:
the method for rapidly detecting ofloxacin provided by the invention has the following advantages:
1. the nano gold magnetic particles adopted by the invention have the superparamagnetism of magnetic nano particles and the performance of high-efficiency coupling antibody on the surface of colloidal gold; the nano gold magnetic particles can physically adsorb protein substances such as antibodies and the like by virtue of non-covalent bond actions such as electrostatic action, hydrophobic action, Au-SH action and the like on the surfaces of the nano gold magnetic particles, so that the coupling rate is high, the coupling effect is stable, and the activity of the antibodies can be ensured not to be influenced in the coupling process.
2. According to the invention, immunomagnetic separation and immunochromatography are organically integrated, so that the step of eluting ofloxacin from immunomagnetic beads is omitted, and the capture efficiency is improved; the step of spraying colloidal gold on the bonding pad is omitted, the immunological reaction is more uniform, and the coefficient of variation during quantitative detection is small; the workload and the substrate interference are reduced.
3. Can simultaneously carry out qualitative and quantitative detection on the object ofloxacin.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it should be obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a structural diagram of a nanogold magnetic particle coupled monoclonal antibody in the rapid detection method for ofloxacin provided in the embodiment of the present invention;
fig. 2 is a schematic diagram of steps and results of immunomagnetic separation and detection of magnetic nanoparticles of nanogold in the rapid detection method for ofloxacin provided by the embodiment of the invention.
Detailed Description
The following is a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements are also considered to be within the scope of the present invention.
The invention provides a rapid detection method of ofloxacin, which adopts gold magnetic particles of a coupling anti-ofloxacin monoclonal antibody to carry out immunoassay to detect the content of ofloxacin in a sample to be detected; the chromatographic test strip base plate for the immunoassay is sequentially provided with filter paper, a sample pad, a nitrocellulose membrane and absorbent paper in a lap joint manner, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an ofloxacin artificial antigen, and the quality control line is coated with a second antibody. The mode of combining the nano gold magnetic particles with the monoclonal antibody is any one of an electrostatic adsorption method or a covalent coupling method, the inner core of the nano gold magnetic particles is a magnetic core formed by ferroferric oxide, cobalt and nickel, and the shell is formed by a colloidal gold material; the size of the gold magnetic particles is 10-500 nm; the concentration of the anti-ofloxacin monoclonal antibody is 1-1000 mug/mL; the film spraying concentration of the ofloxacin artificial antigen is 0.1-10mg/mL, and the spraying amount is 0.1-10 mu L/cm; the secondary antibody comprises a goat anti-mouse antibody and a donkey anti-mouse antibody, the membrane spraying concentration of the secondary antibody is 0.1-10mg/mL, and the spraying amount is 0.1-10 mu L/cm.
The determination of the result of the rapid ofloxacin detection comprises two modes of qualitative detection and quantitative detection, and comprises the following specific steps: dripping the nano gold magnetic particles coupled with the monoclonal antibody into a sample adding hole of a chromatographic test strip, reading the nano gold magnetic particles by using an immunochromatography analyzer after a period of time, and recording the absorbance of a T line, the absorbance of a C line and the T/C value;
1) and (3) qualitative analysis: and qualitatively analyzing by using a visual observation result, wherein the content of the ofloxacin in the sample is higher than the lowest detection lower limit of 1pg/mL if the T line does not develop color, and the content of the ofloxacin in the sample is not contained or is lower than the lowest detection lower limit of 1pg/mL if the T line develops color.
2) Quantitative analysis: and measuring the absorbance of the T line and the C line by using an immunochromatography analyzer, recording the ratio of the absorbance of the T line to the absorbance of the C line as a T/C value, drawing a standard curve by taking the concentration of different ofloxacin as a horizontal coordinate and the T/C value as a vertical coordinate, and determining the ofloxacin content in the common sample by referring to the prepared standard curve graph.
The invention provides a rapid detection method of ofloxacin, which comprises the steps of preparing a coupling buffer solution, a cleaning buffer solution and a blocking agent.
The preparation method of the coupling buffer solution comprises the following steps: mixing 3mL of borax with the concentration of 19.07g/mL and 7mL of boric acid with the concentration of 12.37g/mL, and diluting by 10 times;
the preparation method of the cleaning buffer solution comprises the following steps: weighing 2- (N-morpholino) ethanesulfonic acid buffer solution 0.43g, dissolving in 200mL of sterile distilled water, and adjusting the pH to 5.5-6.0;
the preparation method of the sealant comprises the following steps: 50mg of skimmed milk powder was added to 1mL of phosphate buffer solution with pH 7.4 to prepare a blocking agent.
Example 1
The method for rapidly detecting the ofloxacin in the milk provided by the invention comprises the following steps:
1. preparing nano gold magnetic particles of the coupled monoclonal antibody:
1.1 processing nano gold magnetic particles: placing 300 μ L of the coupling buffer solution prepared by the above method into a 2mL centrifuge tube, mixing with 0.5mg of 50nm gold magnetic nanoparticles, magnetically separating for 3min, and discarding the supernatant;
1.2 coupling reaction: mixing 250 mu g of anti-ofloxacin monoclonal antibody with 0.75mg of nano gold magnetic particles prepared in the step 1.1, placing the mixture into 1mL of coupling buffer solution, placing the mixture on a rotator with the rotating speed of 15rpm at the temperature of 37 ℃, coupling for 45min, carrying out magnetic separation for 5min, discarding supernatant, and washing for 3 times by using 1mL of washing buffer solution prepared by the method;
1.3 sealing: after washing, 1mL of the blocking agent prepared as described above was mixed with magnetic beads and blocked for 1 hour.
2. Method for capturing ofloxacin in milk by using nano gold magnetic particles coupled with monoclonal antibody
Adding 25mL of sterilized milk into 225mL of phosphate buffer solution with the pH value of 7.4, and adding an ofloxacin standard product with a certain concentration, wherein the concentration of the ofloxacin standard product is adjusted to be 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL and 6.25 pg/mL;
and (3) taking 1mL of standard substances with various concentrations and 1mL of milk sample to be detected, adding 100 mu g of the sealed immune nano gold magnetic particles, mixing and incubating for 30min at 37 ℃ and 15rpm, carrying out magnetic separation for 5min after incubation, discarding the supernatant, washing with phosphate buffer solution with pH of 7.4, and redissolving in phosphate buffer solution with pH of 7.4.
3. Preparation of ofloxacin immunochromatography test strip
Spraying ofloxacin rabbit polyclonal antibody and rabbit anti-mouse secondary antibody onto a nitrocellulose membrane to be used as a detection line and a quality control line respectively, wherein the concentration of ofloxacin is 1.5mg/mL, the concentration of goat anti-mouse secondary antibody is 0.5mg/mL, the spraying amount is 0.5 muL/cm, vacuum drying is carried out at 37 ℃ overnight, a sample pad, the nitrocellulose membrane and absorbent paper are sequentially pasted on a PVC bottom plate, and the sample pad, the nitrocellulose membrane and the absorbent paper are cut into strips with the width of 4mm after being pasted, and then the strips are installed in a card; and placing the prepared test strip into a tin foil bag, adding a drying agent, sealing, and placing into a drying cylinder for storage.
4. The result of visual inspection and instrumental determination of the sample by a competitive method
Diluting the gold magnetic particles capturing the ofloxacin to the concentration of 100 mu g/mL, dropwise adding 100 mu L into a sample adding hole of a test strip, reading by an immunochromatography analyzer after 10min, recording the values of the absorbance of the T line, the absorbance of the C line and the T/C, and drawing a standard curve by taking the concentration of the standard solution of the ofloxacin as a horizontal coordinate and the values of the absorbance of the T line and the T/C as a vertical coordinate respectively. And qualitatively analyzing by using a visual observation result, wherein the content of the ofloxacin in the milk sample is higher than 1pg/mL if the line T does not develop color, and the content of the ofloxacin in the milk sample is not contained or is lower than 1pg/mL if the line T develops color. Finally, the concentration of ofloxacin in the milk sample is determined by referring to the standard curve chart.
Example 2
The method for rapidly detecting the ofloxacin in the beef provided by the invention comprises the following steps:
1. preparing nano gold magnetic particles of the coupled monoclonal antibody:
1.1 processing nano gold magnetic particles: putting 250 μ L of the coupling buffer solution prepared by the method into a 2mL centrifuge tube, mixing 0.75mg of 50nm nano gold magnetic particles with the coupling buffer solution, carrying out magnetic separation for 4min, and then discarding the supernatant;
1.2 coupling reaction: and (3) mixing 200 mu g of the prepared anti-ofloxacin monoclonal antibody with 1.0mg of the nano gold magnetic particles prepared in the step 1.1, and placing the mixture in 1mL of coupling buffer solution. At 37 deg.C, placing on a rotator with rotation speed of 10rpm, coupling for 50min, magnetically separating for 5min, discarding supernatant, and washing with 1mL of washing buffer prepared by the above method for 3 times.
1.3 sealing: after washing, 1mL of the blocking agent prepared as described above was mixed with magnetic beads and blocked for 1 hour.
2. Method for capturing ofloxacin in beef by using nano gold magnetic particles coupled with monoclonal antibody
Adding 25g of sterilized beef into 225mL of phosphate buffer solution with pH value of 7.4, and adding an ofloxacin standard product with a certain concentration, wherein the concentration of the ofloxacin standard product is adjusted to be 100pg/mL, 50pg/mL, 25pg/mL, 12.5pg/mL and 6.25 pg/mL;
and taking 1mL of standard substances with various concentrations and 1g of beef sample to be detected, adding 150 mu g of the sealed immune nano gold magnetic particles, mixing and incubating for 60min at 37 ℃ and 15rpm, carrying out magnetic separation for 3min after incubation, discarding the supernatant, washing with phosphate buffer solution with pH of 7.4, and redissolving in phosphate buffer solution with pH of 7.4.
3. Preparation of ofloxacin immunochromatography test strip
Spraying ofloxacin rabbit polyclonal antibody and rabbit anti-mouse secondary antibody onto the nitrocellulose membrane as a detection line and a quality control line respectively, wherein the concentration of ofloxacin is 1.0mg/mL, the concentration of goat anti-mouse secondary antibody is 0.4mg/mL, the spraying amount is 1.0 muL/cm, vacuum drying is carried out at 37 ℃ overnight, the sample pad, the nitrocellulose membrane and the absorbent paper are sequentially pasted on a polyvinyl chloride bottom plate, and the sample pad, the nitrocellulose membrane and the absorbent paper are cut into strips with the width of 4mm after being pasted, and then the strips are installed in a card. And placing the prepared test strip into a tin foil bag, adding a drying agent, sealing, and placing into a drying cylinder for storage.
4. The result of visual inspection and instrumental determination of the sample by a competitive method
And (3) diluting the gold magnetic particles capturing the ofloxacin to the concentration of 50 mu g/mL, dropwise adding 100 mu L of the gold magnetic particles into a sample adding hole of a test strip, reading the gold magnetic particles after 10min by using an immunochromatography analyzer, recording the absorbance of the T line, the absorbance of the C line and the value of T/C, and drawing a standard curve by taking the concentrations of different ofloxacin as abscissa and the values of the T line and the T/C as ordinate. And qualitatively analyzing by using a visual observation result, wherein the content of the ofloxacin in the beef sample is higher than 1pg/mL if the line T does not develop color, and the content of the ofloxacin in the beef sample is not contained or is lower than 1pg/mL if the line T develops color. Finally, the concentration of ofloxacin in the beef sample is determined by referring to the standard curve chart.
The rapid detection method for ofloxacin provided by the invention has the advantage that the concentration range of quantitative detection of ofloxacin is 6.25-100 pg/mL.
Fig. 1 is a structural diagram of a nanogold magnetic particle coupled monoclonal antibody in the rapid ofloxacin detection method provided by the embodiment of the invention, and fig. 2 is a schematic diagram of the steps and results of immunomagnetic separation and detection of nanogold magnetic particles in the rapid ofloxacin detection method provided by the embodiment of the invention.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the spirit of the present invention, and these changes and modifications are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. A quick detection method of ofloxacin is characterized in that: adopting gold magnetic particles coupled with the anti-ofloxacin monoclonal antibody to carry out immunoassay to detect the ofloxacin content in a sample to be detected; the chromatographic test strip base plate for the immunoassay is sequentially provided with filter paper, a sample pad, a nitrocellulose membrane and absorbent paper in a lap joint manner, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with an ofloxacin artificial antigen, and the quality control line is coated with a second antibody.
2. The rapid detection method for ofloxacin according to claim 1, characterized in that: the mode of combining the nano-gold magnetic particles and the monoclonal antibody is any one of an electrostatic adsorption method or a covalent coupling method.
3. The rapid detection method for ofloxacin according to claim 1, characterized in that: the inner core of the nano gold magnetic particle is a magnetic core formed by ferroferric oxide, cobalt and nickel, and the shell is formed by a colloidal gold material.
4. The rapid detection method for ofloxacin according to claim 1, characterized in that: the size of the gold magnetic particles is 10-500 nm.
5. The rapid detection method for ofloxacin according to claim 1, characterized in that: the concentration of the anti-ofloxacin monoclonal antibody is 1-1000 mug/mL.
6. The method for rapidly detecting ofloxacin according to any one of claims 1-5, wherein: the film spraying concentration of the ofloxacin artificial antigen is 0.1-10mg/mL, and the spraying amount is 0.1-10 mu L/cm.
7. The method for rapidly detecting ofloxacin according to any one of claims 1-5, wherein: the secondary antibody comprises a goat anti-mouse antibody and a donkey anti-mouse antibody, the membrane spraying concentration of the secondary antibody is 0.1-10mg/mL, and the spraying amount is 0.1-10 mu L/cm.
8. The method for rapidly detecting ofloxacin according to any one of claims 1-5, wherein: the determination of the result of the rapid ofloxacin detection comprises two modes of qualitative detection and quantitative detection, and comprises the following specific steps: dripping the nano gold magnetic particles coupled with the monoclonal antibody into a sample adding hole of a chromatographic test strip, reading the nano gold magnetic particles by using an immunochromatography analyzer after a period of time, and recording the absorbance of a T line, the absorbance of a C line and the T/C value;
1) and (3) qualitative analysis: qualitative analysis is carried out by using a visual observation result, if the T line does not develop color, the content of the ofloxacin in the sample is higher than the lowest detection lower limit of 1pg/mL, and if the T line develops color, the content of the ofloxacin in the sample is not contained or is lower than the lowest detection lower limit of 1 pg/mL;
2) quantitative analysis: and measuring the absorbance of the T line and the C line by using an immunochromatography analyzer, recording the ratio of the absorbance of the T line to the absorbance of the C line as a T/C value, drawing a standard curve by taking the concentration of different ofloxacin as a horizontal coordinate and the T/C value as a vertical coordinate, and determining the ofloxacin content in the common sample by referring to the prepared standard curve graph.
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| CN103439497A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Salmonella enrichment and rapid detection method |
| CN105606812A (en) * | 2015-12-07 | 2016-05-25 | 福建省淡水水产研究所 | Colloidal gold immunochromatographic test strip for quinolone drugs |
| CN106749632A (en) * | 2016-12-28 | 2017-05-31 | 河南科技学院 | A kind of Ofloxacin hemocyanin coating antigen and preparation method thereof and Test paper card |
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