阅读说明：本技术 水产品中氯霉素时间分辨荧光免疫层析定量检测方法 (Time-resolved fluorescence immunochromatographic assay quantitative detection method for chloramphenicol in aquatic products ) 是由 李云 马立才 赵义良 刘河冰 刘薇 于 2019-10-12 设计创作，主要内容包括：本发明公开了水产品中氯霉素时间分辨荧光免疫层析定量检测方法。本发明提供了检测试剂卡,包括底板和放置在其上的样品垫、含有铕微球标记的抗氯霉素抗体的释放垫、含有氯霉素检测线T以及质控线C的硝酸纤维素膜,和吸水垫。本发明采用时间分辨荧光免疫分析技术建立了一种水产品中氯霉素残留量的快速检测方法,方法的检测灵敏度(定量限0.1μg/kg)优于传统的胶体金荧光免疫层析技术,达到了酶联免疫吸附方法和仪器方法的灵敏度水平。(The invention discloses a method for quantitatively detecting chloramphenicol in an aquatic product by time-resolved fluorescence immunochromatography. The invention provides a detection reagent card, which comprises a bottom plate, a sample pad, a release pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad is arranged on the bottom plate, the release pad contains an europium microsphere labeled anti-chloramphenicol antibody, the nitrocellulose membrane contains a chloramphenicol detection line T and a quality control line C, and the water absorption pad is arranged on the nitrocellulose membrane. The invention adopts the time-resolved fluorescence immunoassay technology to establish a rapid detection method for the residual quantity of chloramphenicol in aquatic products, the detection sensitivity (the limit of quantitation is 0.1 mug/kg) of the method is superior to the traditional colloidal gold fluorescence immunochromatography technology, and the sensitivity level of an enzyme-linked immunosorbent assay method and an instrument method is reached.)

1. A chloramphenicol time-resolved fluorescence immunoassay reagent card comprises a bottom plate, a sample pad arranged on the bottom plate, a release pad containing an europium microsphere-labeled anti-chloramphenicol antibody, a nitrocellulose membrane containing a chloramphenicol detection line T and a quality control line C, and a water absorption pad.

2. The reagent card of claim 1, wherein: the anti-chloramphenicol antibody is an anti-chloramphenicol monoclonal antibody;

the amino acid sequence of the heavy chain variable region of the chloramphenicol-resistant monoclonal antibody is 1;

the amino acid sequence of the light chain variable region of the chloramphenicol-resistant monoclonal antibody is 2.

3. The reagent card of claim 2, wherein:

the europium microsphere-labeled anti-chloramphenicol antibody-containing release pad is formed by spraying an europium microsphere-labeled anti-chloramphenicol antibody solution on the release pad;

the coating concentration of the europium microsphere-labeled anti-chloramphenicol antibody solution is 0.02 mg/mL.

4. The reagent card of claim 3, wherein: in the europium microsphere-labeled anti-chloramphenicol antibody solution, the mass ratio of the anti-chloramphenicol antibody to the europium microsphere is 1: 25;

or the europium microspheres are 200nm carboxylated europium microspheres.

5. The reagent card of any one of claims 1-4, wherein:

the chloramphenicol detection line T is formed by a chloramphenicol antigen or a solution thereof;

or the coating concentration of the chloramphenicol antigen solution is specifically 0.3 mg/ml;

or, the quality control line C is formed from an IgG antibody or an IgG antibody solution;

or the coating concentration of the IgG antibody solution is specifically 0.875 mg/mL;

or, the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C is a nitrocellulose membrane coated with the chloramphenicol antigen solution to form the detection line T, and the IgG antibody solution is coated with the nitrocellulose membrane to form the quality control line C, so that the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C is obtained.

6. A method of making a reagent card according to any one of claims 1 to 5, comprising the steps of: firstly, preparing a release pad containing an europium microsphere-labeled anti-chloramphenicol antibody and preparing a nitrocellulose membrane containing a chloramphenicol detection line T and a quality control line C; assembling a sample pad, the release pad containing the europium microsphere-labeled anti-chloramphenicol antibody, the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C, and a water absorption pad on a bottom plate to obtain a reagent card;

the europium microsphere-labeled anti-chloramphenicol antibody-containing release pad is formed by spraying an europium microsphere-labeled anti-chloramphenicol antibody solution on the release pad;

the concentration of the europium microsphere-labeled anti-chloramphenicol antibody solution is 0.02 mg/mL.

The anti-chloramphenicol antibody is an anti-chloramphenicol monoclonal antibody;

the amino acid sequence of the light chain variable region of the chloramphenicol-resistant monoclonal antibody is 1;

the amino acid sequence of the heavy chain variable region of the chloramphenicol-resistant monoclonal antibody is 2;

the chloramphenicol detection line T is formed by a chloramphenicol antigen or a solution thereof;

or the coating concentration of the chloramphenicol antigen solution is specifically 0.3 mg/ml;

the quality control line C is formed by IgG antibody or IgG antibody solution;

or the concentration of the IgG antibody solution is specifically 0.875 mg/mL;

the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C is prepared by coating a chloramphenicol antigen solution on a nitrocellulose membrane to form a detection line T, and coating an IgG antibody solution on a nitrocellulose membrane to form a quality control line C.

7. Use of the reagent card of any one of claims 1 to 6 in the preparation of a product for the detection or the auxiliary detection of chloramphenicol;

or the use of the reagent card according to any one of claims 1 to 6 for the preparation of a test or for the auxiliary detection of a chloramphenicol-containing product in a test sample;

or the use of the reagent card according to any one of claims 1 to 6 for the preparation of a product for the detection or the auxiliary detection of the chloramphenicol content in a sample to be tested;

or the use of said reagent card according to any one of claims 1 to 6 for the detection or for the auxiliary detection of chloramphenicol;

or the use of the reagent card according to any of claims 1 to 6 for the detection or assisted detection of the presence of chloramphenicol in a sample to be tested;

or the use of the reagent card according to any of claims 1 to 6 for the detection or assisted detection of the chloramphenicol content in a sample to be tested;

or the europium microsphere-labeled anti-chloramphenicol antibody of any one of claims 1 to 6, for use in detecting or aiding in detecting the presence or absence of chloramphenicol in a test sample or the chloramphenicol content of a test sample.

8. A method for detecting or assisting in detecting whether a sample to be detected contains chloramphenicol comprises the following steps: extracting a sample to be detected by using an organic solvent, and collecting an extracting solution; adding the extracting solution to a sample pad of the reagent card of any one of claims 1 to 6, reacting, and detecting the reaction product by using a fluorescence immunoassay analyzer;

the organic solvent comprises ethyl acetate and n-hexane;

if the fluorescence signal value of the detection line T of the reagent card is less than or equal to the fluorescence signal value of the quality control line C, the sample to be detected contains or is candidate to contain chloramphenicol;

and if the fluorescence signal value of the detection line T of the reagent card is greater than the fluorescence signal value of the quality control line C, the sample to be detected does not contain or candidate does not contain chloramphenicol.

9. A method for detecting or assisting in detecting the content of chloramphenicol in a sample to be detected comprises the following steps:

1) preparing a standard curve;

taking a sample which has the same source as the sample to be detected and has the chloramphenicol content of less than 0.1 mug/kg, extracting the sample by using an organic solvent, and collecting an extracting solution; the organic solvent comprises ethyl acetate and n-hexane;

adding chloramphenicol with different concentrations into the extracting solution to obtain standard solutions with different concentrations;

adding the standard substance solutions with different concentrations to the sample pad of the reagent card in any one of claims 1 to 6, reacting, and detecting the reaction product by using a fluorescence immunoassay analyzer to obtain the fluorescence signal values of the detection line T and the quality control line C of the standard substance solutions with different concentrations;

taking different concentrations of the added chloramphenicol as an X axis, and taking the ratio of the fluorescence signal value of the detection line T corresponding to the different concentrations to the fluorescence signal value of the quality control line C as a Y axis to make a standard curve;

2) extracting a sample to be detected by using an organic solvent, and collecting a sample extracting solution to be detected; the organic solvent comprises ethyl acetate and n-hexane;

adding the extracting solution of the sample to be detected on the sample pad of the reagent card of any one of claims 1 to 6, reacting, and detecting the reaction product by using a fluorescence immunoassay analyzer to obtain the fluorescence signal value of the detection line T and the fluorescence signal value of the quality control line C of the sample to be detected;

and substituting the fluorescence signal value of the detection line T and the fluorescence signal value of the quality control line C of the sample to be detected into the standard curve obtained in the step 1) to obtain the content of chloramphenicol in the sample to be detected.

10. The use according to claim 7 or the method according to claim 8 or 9, characterized in that:

the sample to be detected is a food animal or a tissue sample of the food animal;

or, the food animal is specifically a marine animal; the food animal tissue sample is specifically aquatic animal muscle;

or, the aquatic animal is specifically a fish, shrimp, or crab; the fish, shrimp or crab tissue sample is specifically fish muscle, shrimp muscle or crab muscle.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for quantitatively detecting chloramphenicol in an aquatic product by time-resolved fluorescence immunochromatography.

Background

Chloramphenicol (CAP) belongs to the class of amidol antibacterial drugs and was originally isolated from the culture broth of Streptomyces venezuelae. Chloramphenicol is usually in the form of needle-like crystalline powder or long-sheet crystalline powder, white, readily soluble in acetone and alcohols, and slightly soluble in water. The chloramphenicol can act on 50S subunit of 70S ribosome of bacteria to destroy the activity of amidotransferase, and inhibit protein synthesis, thereby inhibiting bacteria; it is a broad-spectrum antibacterial medicine, and can produce good inhibition action for several gram-negative and gram-positive bacteria, chlamydia and rickettsia, etc. Therefore, the chloramphenicol has great application in the prevention and treatment of bacterial diseases of food animals (especially aquatic animals). The use of the drug in animal breeding inevitably involves the risk of drug residues in animal food, thus endangering consumer health. Chloramphenicol can inhibit mitochondrial protein synthesis in mammalian bone marrow cells, and can cause non-dose indolent aplastic anemia, hemolysis, purpura and other symptoms in human beings. In order to protect consumer health, many countries in the world (such as the united states, the european union and japan) have banned the use of chloramphenicol in animal foods; the Ministry of agriculture in China stipulates that chloramphenicol is a forbidden drug in No. 235 bulletin 'the maximum residue limit of veterinary drugs in animal food' in 2002, 12 months and no chloramphenicol can be detected in animal food.

At present, the main methods for detecting chloramphenicol residues in aquatic products include immunoassay (including immunochromatography and enzyme-linked immunosorbent assay Elisa) and chromatographic methods, such as Gas Chromatography (GC) and High Performance Liquid Chromatography (HPLC). The colloidal gold immunochromatographic assay technology as a traditional quick detection technology has the defects of relatively low sensitivity and incapability of quantitative detection, so that the requirement of trace residue detection of chloramphenicol in aquatic products cannot be met. The sensitivity of the ELISA meets the requirement, but compared with the immunochromatography detection method, the ELISA detection method has the defects of relatively complex operation and long detection time. The chromatographic method generally needs expensive detection instruments and has the problems of complex pretreatment, complex operation, long detection time and high detection cost, so the chromatographic method is not suitable for detecting large-batch samples in a basic laboratory.

The Time-resolved fluoroimmunoassay (TRFIA) is a novel nonradioactive immunoassay technology established by combining the high specificity of comprehensive immune reaction and the high sensitivity of lanthanide labeling by taking lanthanide (including Eu, Tb, Sm, Dy, and the like) as a marker. The lanthanide has the advantages of large Stokes shift (>200nm) and long fluorescence lifetime (1-2ms), can avoid interference with excitation light and background fluorescence signals to the maximum extent, and greatly improves the detection sensitivity. In terms of sensitivity, the time-resolved fluoroimmunoassay technique is much higher than the Elisa method and the colloidal gold technique. In view of the above characteristics, TRFIA has been widely used in clinical diagnosis in human and veterinary medicine, food safety inspection, and other fields.

Disclosure of Invention

In order to detect chloramphenicol residues in aquatic products, the invention provides the following technical scheme:

the invention aims to provide a chloramphenicol time-resolved fluorescence immunoassay reagent card.

The detection reagent card provided by the invention comprises a bottom plate, a sample pad arranged on the bottom plate, a release pad containing an europium microsphere labeled anti-chloramphenicol antibody, a nitrocellulose membrane containing a chloramphenicol detection line T and a quality control line C, and a water absorption pad.

In the reagent card, the anti-chloramphenicol antibody is an anti-chloramphenicol monoclonal antibody;

the amino acid sequence of the heavy chain variable region of the chloramphenicol-resistant monoclonal antibody is sequence 1;

the amino acid sequence of the light chain variable region of the chloramphenicol-resistant monoclonal antibody is sequence 2.

In the reagent card, the release pad containing the europium microsphere-labeled anti-chloramphenicol antibody is formed by spraying an europium microsphere-labeled anti-chloramphenicol antibody solution on the release pad;

the coating concentration of the europium microsphere-labeled anti-chloramphenicol antibody solution is 0.02 mg/mL.

In the reagent card, in the europium microsphere-labeled anti-chloramphenicol antibody solution, the mass ratio of the anti-chloramphenicol antibody to the europium microsphere is 1: 25;

or the europium microspheres are 200nm carboxylated europium microspheres.

In the reagent card, the chloramphenicol detection line T is formed by a chloramphenicol antigen or a solution thereof;

or the coating concentration of the chloramphenicol antigen solution is specifically 0.3 mg/ml;

or, the quality control line C is formed from an IgG antibody or an IgG antibody solution;

or the coating concentration of the IgG antibody solution is specifically 0.875 mg/mL;

or, the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C is a nitrocellulose membrane coated with the chloramphenicol antigen solution to form the detection line T, and the IgG antibody solution is coated with the nitrocellulose membrane to form the quality control line C, so that the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C is obtained.

It is another object of the present invention to provide a method for preparing the first object reagent card.

The method provided by the invention comprises the following steps: firstly, preparing a release pad containing an europium microsphere-labeled anti-chloramphenicol antibody and preparing a nitrocellulose membrane containing a chloramphenicol detection line T and a quality control line C; assembling a sample pad, the release pad containing the europium microsphere-labeled anti-chloramphenicol antibody, the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C, and a water absorption pad on a bottom plate to obtain a reagent card;

the europium microsphere-labeled anti-chloramphenicol antibody-containing release pad is formed by spraying an europium microsphere-labeled anti-chloramphenicol antibody solution on the release pad;

the concentration of the europium microsphere-labeled anti-chloramphenicol antibody solution is 0.02 mg/mL.

The anti-chloramphenicol antibody is an anti-chloramphenicol monoclonal antibody;

the amino acid sequence of the heavy chain variable region of the chloramphenicol-resistant monoclonal antibody is sequence 1;

the amino acid sequence of the light chain variable region of the chloramphenicol-resistant monoclonal antibody is a sequence 2;

the chloramphenicol detection line T is formed by a chloramphenicol antigen or a solution thereof;

or the coating concentration of the chloramphenicol antigen solution is specifically 0.3 mg/ml;

the quality control line C is formed by IgG antibody or IgG antibody solution;

or the concentration of the IgG antibody solution is specifically 0.875 mg/mL;

the nitrocellulose membrane containing the chloramphenicol detection line T and the quality control line C is prepared by coating a chloramphenicol antigen solution on a nitrocellulose membrane to form a detection line T, and coating an IgG antibody solution on a nitrocellulose membrane to form a quality control line C.

The application of the reagent card in the first object in the preparation of a product for detecting or assisting in detecting chloramphenicol is also the protection scope of the invention;

or the application of the reagent card in the first purpose in the preparation of a product for detecting or assisting in detecting whether a sample to be detected contains chloramphenicol is also the protection scope of the invention;

or the application of the reagent card in the first purpose in preparing a product for detecting or assisting in detecting the content of chloramphenicol in a sample to be detected is also the protection scope of the invention;

or the use of said reagent card in the detection or the auxiliary detection of chloramphenicol in the above first object is also within the scope of protection of the present invention;

or the application of the reagent card in the first purpose in detecting or assisting in detecting whether the sample to be detected contains chloramphenicol is also the protection scope of the invention;

or the application of the reagent card in the first purpose in detecting or assisting in detecting the content of chloramphenicol in a sample to be detected is also the protection scope of the invention;

or the application of the europium microsphere-labeled anti-chloramphenicol antibody in the reagent card in the first object in detecting or assisting in detecting whether the sample to be detected contains chloramphenicol or the content of chloramphenicol in the sample to be detected is also within the protection scope of the present invention.

It is a further object of the present invention to provide a method of:

the invention provides a method for detecting or assisting in detecting whether a sample to be detected contains chloramphenicol, which comprises the following steps: extracting a sample to be detected by using an organic solvent, and collecting an extracting solution; adding the extracting solution to a sample pad of the reagent card for reaction, and detecting a reaction product by using a fluorescence immunoassay quantitative analyzer;

the organic solvent comprises ethyl acetate and n-hexane;

if the fluorescence signal value of the detection line T of the reagent card is less than or equal to the fluorescence signal value of the quality control line C, the sample to be detected contains or is candidate to contain chloramphenicol;

and if the fluorescence signal value of the detection line T of the reagent card is greater than the fluorescence signal value of the quality control line C, the sample to be detected does not contain or candidate does not contain chloramphenicol.

Or, the invention provides a method for detecting or detecting the content of chloramphenicol in a sample to be detected in an auxiliary way, which comprises the following steps:

1) preparing a standard curve;

taking a sample which has the same source as the sample to be detected and has the chloramphenicol content of less than 0.1 mug/kg, extracting the sample by using an organic solvent, and collecting an extracting solution; the organic solvent comprises ethyl acetate and n-hexane;

adding chloramphenicol with different concentrations into the extracting solution to obtain standard solutions with different concentrations;

adding the standard substance solutions with different concentrations onto the sample pad of the reagent card for reaction, and detecting the reaction product by using a fluorescence immunoassay quantitative analyzer to obtain the fluorescence signal values of the detection line T and the quality control line C of the standard substance solutions with different concentrations;

taking different concentrations of the added chloramphenicol as an X axis, and taking the ratio of the fluorescence signal value of the detection line T corresponding to the different concentrations to the fluorescence signal value of the quality control line C as a Y axis to make a standard curve;

2) extracting a sample to be detected by using an organic solvent, and collecting a sample extracting solution to be detected; the organic solvent comprises ethyl acetate and n-hexane;

adding the sample extracting solution to be detected on a sample pad of the reagent card for reaction, and detecting a reaction product by using a fluorescence immunoassay quantitative analyzer to obtain a detection line T fluorescence signal value and a quality control line C fluorescence signal value of the sample to be detected;

and substituting the fluorescence signal value of the detection line T and the fluorescence signal value of the quality control line C of the sample to be detected into the standard curve obtained in the step 1) to obtain the content of chloramphenicol in the sample to be detected.

In the above method, the organic solvent extraction sample comprises the following steps: homogenizing edible muscle tissue of the sample, extracting with ethyl acetate, collecting ethyl acetate layer, drying, adding n-hexane, removing upper n-hexane and middle layer impurities, and collecting lower layer solution as sample extractive solution.

In the above method, the reaction is carried out at 40 ℃ for 5 min.

In the above, the sample to be tested is a food animal or a tissue sample of the food animal;

or, the food animal is specifically a marine animal; the food animal tissue sample is specifically aquatic animal muscle;

or, the aquatic animal is specifically a fish, shrimp, or crab; the fish, shrimp or crab tissue sample is specifically fish muscle, shrimp muscle or crab muscle.

The invention aims to establish a simple, rapid and high-sensitivity fluorescence quantitative immunochromatography detection method for chloramphenicol residues in fish, shrimps and crabs by using europium microspheres as an antibody marker, and evaluates the indexes of sensitivity, accuracy and precision of the established method, so as to provide a rapid detection method suitable for chloramphenicol trace residues in aquatic products, and guarantee the quality safety of the aquatic products and the health of consumers.

In order to detect chloramphenicol residues in an aquatic product more simply and efficiently, the invention provides a method for quantitatively detecting chloramphenicol in an aquatic product by using a time-resolved fluorescence immune layer. The invention uses carboxyl microspheres of lanthanide europium (365/610nm, lambda ex/lambda em) as antibody markers to establish an immunochromatography method. Europium element has larger Stokes shift (>200nm), so that an excited signal does not cause interference on a fluorescence signal of emitted light in a detection process; in addition, the fluorescence lifetime of lanthanide is relatively long, which can reach 1-2ms, so that detection can be performed after other fluorescence signals disappear, so as to achieve the purpose of reducing to background signal interference. The advantages of the two aspects greatly improve the sensitivity of the time-resolved fluoroimmunoassay method.

Compared with the traditional colloidal gold immunochromatographic assay, the established time-resolved fluorescence immunochromatographic assay has higher sensitivity, can be used for quantitatively detecting chloramphenicol in aquatic products, and has the quantitative detection limit of 0.1 mu g/kg, which is obviously lower than the qualitative detection limit (0.5 mu g/kg) of colloidal gold.

The invention adopts the time-resolved fluoroimmunoassay technology to establish a rapid detection method for the residual quantity of chloramphenicol in aquatic products, the detection sensitivity (the limit of quantitation is 0.1 mug/kg) of the method is superior to the traditional colloidal gold fluorescence immunochromatography technology, and the sensitivity level of an enzyme-linked immunosorbent assay method and an instrument method is reached; the method can meet the detection requirements on the addition recovery rate (73.5-114.2%) and the variation coefficient (3.9-11.5%) of chloramphenicol in aquatic products, and has high coincidence rate with the detection method of national standard instruments. Therefore, the time-resolved fluorescence immunochromatographic assay established by the invention has higher application value and application prospect in the detection work of the chloramphenicol in the aquatic products.

Drawings

FIG. 1 is a schematic diagram of a time-resolved fluorescence immunoassay rapid detection reagent card.

FIG. 2 is a graph showing the linear correlation between TRFIA and HPLC detection results.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Some of the materials in the following examples are as follows:

micropipettor (Eppendorf, Germany), a celestial balance (Tianjin Demant sensor technology Co., Ltd.), a refrigerated centrifuge (Thermo Fisher Scientific, Germany), an ultrasonic cleaning instrument KQ-100E (ultrasonic instruments Co., Ltd., Kunshan), a liquid chromatography-tandem mass spectrometer (Thermo Fisher Scientific, USA), and a sample grinder (Jiuyang JYL-C090); the detection card incubator (WH-400) and the time-resolved fluoroimmunoassay quantitative analyzer (FQ-S2, 365/610nm) were developed by Vidervican Biotechnology Ltd.

Methanol, acetonitrile, dipotassium hydrogen phosphate, hydrochloric acid, sodium hydroxide, tween 20, ethyl acetate, n-hexane and the like are purchased from national drug group chemical reagent limited; bovine Serum Albumin (BSA) was purchased from Amresco, usa; 200nm carboxylated europium microspheres (365/610nm, lambda)_ex/λ_em) Purchased from Creative Diagnostics, USA; nitrocellulose membranes (NC membranes) were purchased from Millipore, usa; the sample pad, the absorbent pad, and the PVC base plate were all purchased from shanghai liang xin science and technology limited.

Chloramphenicol standards were purchased from Sigma, usa, and standard solutions were prepared: accurately weighing 1.0mg of chloramphenicol standard, dissolving with methanol, diluting to 10mL, preparing into 100mg/kg standard solution, and storing at-20 deg.C.

The goat anti-mouse secondary antibody is obtained by Beijing Weideweikang biotechnology limited.

Fish, shrimp and crab aquatic products are purchased from supermarkets and farmer markets in the lake areas and Changping areas of Beijing City.