阅读说明：本技术 一种氧氟沙星快速检测方法 (Quick detection method for ofloxacin ) 是由 袁丽娟 向建军 张大文 扎西次旦 张莉 廖且根 于 2019-09-29 设计创作，主要内容包括：本发明提供了一种氧氟沙星快速检测方法,采用偶联抗氧氟沙星单克隆抗体的金磁微粒免疫检测待测样品中的氧氟沙星含量；所述免疫检测的层析试纸条底板上依次搭接设置有滤纸、样品垫、硝酸纤维素膜和吸水纸,所述硝酸纤维素膜上设置有检测线和质控线,所述检测线上包被有氧氟沙星人工抗原,所述质控线上包被有二抗。所述纳米金磁颗粒与所述单克隆抗体结合的方式为静电吸附法或共价偶联法中的任意一种；所述纳米金磁微粒的内核为四氧化三铁、钴和镍形成的磁核,外壳为胶体金材料形成。本发明提供的氧氟沙星快速检测方法结果判定包括定性和定量两种方式。(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.)

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.

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.