Double-quantum-dot microsphere fluorescence immunochromatography kit for detecting clenbuterol and ractopamine as well as preparation method and detection method of double-quantum-dot microsphere fluorescence immunochromatography kit

Abstract

The invention discloses a double-quantum-dot microsphere fluorescence immunochromatography kit for detecting clenbuterol and ractopamine as well as a preparation method and a detection method of the double-quantum-dot microsphere fluorescence immunochromatography kit, and relates to the technical field of immunochromatography. The fluorescent test strip based on the double quantum dot microsphere-monoclonal antibody conjugate is prepared, clenbuterol and ractopamine can be simultaneously detected on one test strip, and the detection flux of a single test strip is improved; two antibodies are respectively marked by the bicolor quantum dot microspheres, so that fluorescence interference between two T lines during reading can be effectively avoided, and meanwhile, the problem that a C line of a multi-component immunochromatography test strip is difficult to judge whether a single antibody is effective or not is solved. The detection method provided by the invention has relatively good specificity, accuracy and repeatability. Therefore, the method has an excellent application prospect in the field of high-sensitivity multi-component immunoassay.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatography, and in particular to a double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, and a preparation method and a detection method thereof. Background Art

[0002] Clenbuterol and ractopamine are β-adrenergic receptor agonists, two common types of clenbuterol, which can increase the lean meat rate of animals by regulating animal metabolism. Eating meat containing clenbuterol can cause poisoning in humans. my country prohibits the use of clenbuterol in animal breeding. High-precision detection of clenbuterol is particularly important for combating the illegal use of clenbuterol and ensuring food safety.

[0003] At present, the detection methods of clenbuterol and ractopamine mainly include instrument confirmation method and rapid detection method. The instrument confirmation method is based on liquid chromatography tandem mass spectrometry, which has high accuracy and sensitivity, but the detection cost is high, time-consuming, and has high requirements for laboratories and technicians. It is not suitable for on-site rapid detection of a large number of samples; the rapid detection method based on colloidal gold immunochromatographic test strips has the advantages of simple operation, short detection time, and visualization, and is widely used in on-site rapid screening of clenbuterol in animal urine samples. The colloidal gold test strips mainly determine the results by interpreting the color of the strips with the naked eye, and can only perform semi-quantitative detection with low sensitivity. In addition, the colloidal gold nanoparticles are coupled with the detection antibodies by physical adsorption, and have poor stability. The drug residue in animal urine is low and the matrix interference is large. The existing colloidal gold test strips are difficult to achieve high-sensitivity and accurate detection of clenbuterol and ractopamine.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine and a preparation method and a detection method thereof to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which comprises a test strip, an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate; the test strip is provided with a sample pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; and the detection line T1 line is coated with clenbuterol (CLE)-carrier protein, and the detection line T2 line is coated with ractopamine (RAC)-carrier protein; and the quality control line C line is coated with a secondary antibody, which is a secondary antibody that recognizes the species origin of the anti-clenbuterol antibody and the anti-ractopamine antibody;

[0008] The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under the excitation of a light source and an anti-clenbuterol antibody; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under the excitation of a light source and an anti-ractopamine antibody; the first wavelength and the second wavelength are different.

[0009] In a second aspect, the present invention provides a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which comprises a test strip; the test strip is provided with a sample pad, a conjugation pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction, an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate are coated on the conjugation pad, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; and the detection line T1 line is coated with clenbuterol (CLE)-carrier protein, and the detection line T2 line is coated with ractopamine (RAC)-carrier protein; and the quality control line C line is coated with a secondary antibody, which is a secondary antibody that recognizes the species origin of the anti-clenbuterol antibody and the anti-ractopamine antibody;

[0010] The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under the excitation of a light source and an anti-clenbuterol antibody; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under the excitation of a light source and an anti-ractopamine antibody; the first wavelength and the second wavelength are different.

[0011] In a third aspect, the present invention also provides a method for preparing a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which includes the preparation of an antibody-quantum dot microsphere conjugate. The preparation method of the antibody-quantum dot microsphere conjugate includes: mixing the quantum dot microspheres with a microsphere surfactant to activate the quantum dot microspheres, and then incubating the activated quantum dot microspheres with anti-clenbuterol antibodies and anti-ractopamine antibodies, respectively, and after blocking, obtaining anti-clenbuterol antibody-quantum dot microsphere conjugates and anti-ractopamine antibody-quantum dot microsphere conjugates, respectively.

[0012] In a fourth aspect, the present invention further provides a method for detecting clenbuterol and ractopamine, which comprises the following detection method (1) or (2):

[0013] (1) Incubate the sample to be tested with the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate in the above-mentioned dual quantum dot microsphere fluorescent immunochromatography kit to obtain an incubation mixture, then insert the test strip into the incubation mixture, read the fluorescence intensity of the test line T1, the test line T2 and the quality control line C or perform the test under ultraviolet light;

[0014] (2) Load the sample to be tested onto the test strip in the above-mentioned dual quantum dot microsphere fluorescent immunochromatography kit, read the fluorescence intensity of the test line T1, the test line T2 and the quality control line C or perform the test under ultraviolet light.

[0015] The present invention has the following beneficial effects:

[0016] The purpose of the present invention is to solve the problems of low sensitivity, poor stability and difficulty in accurate quantification of traditional clenbuterol test strips, and to prepare a dual quantum dot microsphere fluorescent immunochromatographic test strip for clenbuterol (CLE) and ractopamine (RAC). The present invention prepares a fluorescent test strip based on a dual "quantum dot microsphere-monoclonal antibody conjugate", which can detect clenbuterol and ractopamine on a test strip at the same time, thereby improving the detection flux of a single test strip; by marking two antibodies respectively with dual-color (emitting two wavelengths) quantum dot microspheres, fluorescence interference when reading between two T lines (detection line T1 and detection line T2) can be effectively avoided, and the problem that the C line of a multi-component immunochromatographic test strip is difficult to determine whether a single antibody is effective is also solved. If two antibodies are marked with monochrome quantum dot microspheres, although quantification can be performed, when a single antibody fails, the quality control line C line can also combine with another antibody, thereby causing the C line to develop color normally, and the quality control line C line cannot be used to determine whether a single antibody is effective. The present invention uses dual-color quantum dot microspheres to respectively label two antibodies, and the quality control line C line combines the two antibody conjugates, so the quality control line C line can display two fluorescences, thereby accurately determining whether a single antibody is effective.

[0017] The kit provided by the present invention can be semi-quantitatively identified by naked eye observation under ultraviolet light, and can also be quantitatively detected by using a fluorescent test strip detector. Through methodological evaluation, the detection method provided by the present invention has good specificity, accuracy and repeatability. Therefore, the present invention has excellent application prospects in the field of high-sensitivity multi-component immunoassay. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 This is the principle diagram of fluorescent immunochromatographic test strips for detecting CLE and RAC;

[0020] Figure 2 is the standard curve of CLE and RAC (n=3);

[0021] Figure 3 The experimental results of simultaneous detection of CLE and RAC at some concentrations by fluorescent test strips are shown in Figure 2.

[0022] Figure 4 This is a statistical graph of the relative fluorescence intensity of the T line when the fluorescent test strip detects different β-agonists. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0024] Quantum dots are a type of crystalline nanoparticles that exhibit size-dependent properties due to the quantum confinement effect of the electronic state. They have the excellent properties of large Stokes shift, narrow emission spectrum, and resistance to photobleaching. They can achieve single excitation and multiple emission, and there is no mutual interference between fluorescence. They are an ideal fluorescent coding material and can also achieve high-throughput detection.

[0025] Fluorescent quantum dot microspheres are a new type of nanomaterial that embeds fluorescent quantum in polymer microspheres. They have excellent fluorescent properties, adjustable wavelength, and can be stably chemically coupled with detection antibodies. Compared with traditional fluorescent quantum dots, quantum dot microspheres use polymer microspheres to embed a large number of quantum dots, which can greatly increase the fluorescence signal intensity of nanoparticles; using microspheres to protect quantum dots can reduce matrix interference and improve fluorescence stability; quantum dot microspheres have a larger particle size than quantum dots, which is very easy to chemically couple and separate and purify, and can also reduce the flow rate during chromatography and improve the adequacy of antigen-antibody reactions. Using quantum dot microspheres as markers for immunochromatographic test strips can greatly improve the output signal intensity and stability, and enhance the sensitivity and accuracy of the test strips.

[0026] In a first aspect, the present invention provides a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which comprises a test strip, an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate; the test strip is provided with a sample pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; and the detection line T1 line is coated with clenbuterol (CLE)-carrier protein, and the detection line T2 line is coated with ractopamine (RAC)-carrier protein; and the quality control line C line is coated with a secondary antibody, which is a secondary antibody that recognizes the species origin of the anti-clenbuterol antibody and the anti-ractopamine antibody;

[0027] The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under the excitation of a light source and an anti-clenbuterol antibody; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under the excitation of a light source and an anti-ractopamine antibody; the first wavelength and the second wavelength are different.

[0028] In order to solve the problems of low sensitivity, poor stability and difficulty in accurate quantification of traditional clenbuterol test strips, a dual quantum dot microsphere fluorescent immunochromatographic test strip is prepared for clenbuterol (CLE) and ractopamine (RAC). The present invention prepares a fluorescent test strip based on a dual "quantum dot microsphere-monoclonal antibody conjugate", which can detect clenbuterol and ractopamine on a test strip at the same time, thereby improving the detection flux of a single test strip; by marking two antibodies respectively with dual-color (emitting two wavelengths) quantum dot microspheres, fluorescence interference when reading between two T lines (detection line T1 and detection line T2) can be effectively avoided, and the problem that the C line of a multi-component immunochromatographic test strip is difficult to determine whether a single antibody is effective is also solved. The inventor found that if two antibodies are marked with monochrome quantum dot microspheres, although quantification can be performed, when a single antibody fails, the quality control line C line can bind to the antibody on another T line, thereby causing the C line to still show color normally, and the quality control line C line cannot be used to determine whether a single antibody is effective. The present invention respectively labels two antibodies by dual-color quantum dot microspheres, and the quality control line C line is combined with the two antibody conjugates, so the quality control line C line can display two fluorescences. By detecting whether the quality control line C line has two fluorescences, it is possible to accurately determine whether a single antibody is effective, which completely solves the problem that it is difficult for the C line of a multi-component immunochromatography test strip to determine whether a single antibody is effective.

[0029] The kit provided by the present invention can be semi-quantitatively identified by naked eye observation under ultraviolet light, and can also be quantitatively detected using a fluorescent test strip detector. Through methodological evaluation, the detection method provided by the present invention has good specificity, accuracy and repeatability (between batches and within batches).

[0030] In a preferred embodiment of the present invention, the quantum dot microspheres emitting fluorescence of the first wavelength refer to: quantum dots capable of emitting fluorescence of the first wavelength are embedded in polymethyl methacrylate (PMMA), polyglycidyl methacrylate microspheres or polystyrene microspheres.

[0031] The quantum dot microspheres emitting fluorescence of the second wavelength refer to: quantum dots capable of emitting fluorescence of the second wavelength are embedded in polymethyl methacrylate (PMMA), polyglycidyl methacrylate microspheres or polystyrene microspheres.

[0032] In a preferred embodiment of the present invention, the quantum dots are selected from at least one of substance I having a chemical formula shown in formula I, substance II having a chemical formula shown in formula II, and substance III having a chemical formula shown in formula III;

[0033] AM type Ⅰ;

[0034] EFQ2 type II;

[0035] GHX3 type III;

[0036] Wherein, A is selected from at least one of Cd, Zn, Pb and In; M and Q are independently selected from at least one of S, Se, Te and P; E is selected from at least one of Cu, Ag and Zn; F is selected from at least one of Al, Ga and In; G is selected from at least one of CH3NH3, CH(NH)NH3 and Cs; H is selected from at least one of Ag, Sb, Bi, Pb, In and Al; and X is selected from at least one of halogens.

[0037] In a preferred embodiment of the present invention, the quantum dots are selected from substances having a CdSe / ZnS core-shell structure;

[0038] In a preferred embodiment of the present invention, the quantum dot microspheres are green light quantum dot microspheres or red light quantum dot microspheres.

[0039] In a preferred embodiment of the present invention, the particle size of the quantum dot microspheres is 100 nm to 200 nm.

[0040] The particle size of the quantum dot microspheres is selected to be 100nm-200nm. If the particle size is too large, the quantum dot microspheres will be chromatographed too slowly, and will be stuck under the NC membrane and unable to run up or have non-specific signals; if the particle size of the quantum dot microspheres is too small, the signal will become weak and the sensitivity will decrease. Preferably, the average particle size of the quantum dot microspheres is 150-200nm, preferably 200nm.

[0041] In a preferred embodiment of the present invention, the volume mass ratio of quantum dot microspheres to the anti-clenbuterol antibody mass is 1:0.5-1.2; the unit of quantum dot microspheres is μL, and the unit of antibodies is μg. If the volume mass ratio of quantum dot microspheres to the anti-clenbuterol antibody mass is too large (more than 1.66), that is, too little antibody, the T line signal will be weakened. If the ratio is too small, that is, the antibody is excessive, the quantum dot microsphere-antibody will bind to the target and the coated antigen at the same time, which will lead to reduced sensitivity. The volume mass ratio of quantum dot microspheres to the anti-clenbuterol antibody mass includes but is not limited to 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0 or 1:1.2.

[0042] In a preferred embodiment of the present invention, the volume mass ratio of quantum dot microspheres to the anti-ractopamine antibody mass is 1:0.5 to 1.2. If the volume mass ratio of quantum dot microspheres to the anti-ractopamine antibody mass is too large (more than 1.66), that is, too little antibody, the T line signal will become weaker. If the ratio is too small, that is, the antibody is excessive, the quantum dot microsphere-antibody will bind to the target and the coated antigen at the same time, which will lead to reduced sensitivity. The volume mass ratio of quantum dot microspheres to the anti-ractopamine antibody mass includes but is not limited to 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0 or 1:1.2.

[0043] In a preferred embodiment of the present invention, the coating concentrations of the detection line T1 and the detection line T2 are 0.1-0.3 mg / mL and 0.1-0.3 mg / mL, respectively. For example, the coating concentrations of the detection line T1 and the detection line T2 are 0.1, 0.2 or 0.3 mg / mL and 0.1, 0.2 or 0.3 mg / mL, respectively. The above coating concentrations have a good detection effect.

[0044] In a preferred embodiment of the present invention, the coating concentration of the quality control line C is 0.08 mg / mL.

[0045] In a preferred embodiment of the present invention, the carrier protein is diphtheria toxin cross-reactive material 197, diphtheria toxoid, tetanus toxoid or bovine serum albumin.

[0046] In a second aspect, the present invention provides a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which comprises a test strip; the test strip is provided with a sample pad, a conjugation pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction, an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate are coated on the conjugation pad, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; and the detection line T1 line is coated with clenbuterol (CLE)-carrier protein, and the detection line T2 line is coated with ractopamine (RAC)-carrier protein; and the quality control line C line is coated with a secondary antibody, which is a secondary antibody that recognizes the species origin of the anti-clenbuterol antibody and the anti-ractopamine antibody;

[0047] The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under the excitation of a light source and an anti-clenbuterol antibody; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under the excitation of a light source and an anti-ractopamine antibody; the first wavelength and the second wavelength are different.

[0048] In the kit of the above embodiment, the antibody-quantum dot microsphere conjugate is coated on the conjugate pad, which is easy to carry, store and transport.

[0049] In a preferred embodiment of the present invention, the coating volume ratio of the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate on the conjugate pad is 1:0.5-1, such as 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 or 1:1.

[0050] In a third aspect, the present invention also provides a method for preparing a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which includes the preparation of an antibody-quantum dot microsphere conjugate. The preparation method of the antibody-quantum dot microsphere conjugate includes: mixing the quantum dot microspheres with a microsphere surfactant to activate the quantum dot microspheres, and then incubating the activated quantum dot microspheres with anti-clenbuterol antibodies and anti-ractopamine antibodies, respectively, and after blocking, obtaining anti-clenbuterol antibody-quantum dot microsphere conjugates and anti-ractopamine antibody-quantum dot microsphere conjugates, respectively.

[0051] The microsphere surfactant is, for example, selected from EDC. The quantum dot microspheres are modified by the microsphere surfactant so that the surface is modified with carboxyl groups, which can react with the amino groups on the antibody under certain conditions to undergo an amidation reaction, thereby coupling the antibody to the microspheres. The activation conditions are, for example, a metal bath, shaking and incubating for 30 minutes (1,000 rpm, 37°C), centrifuging and discarding the supernatant, re-dissolving the precipitate in 150 μL of labeling buffer, and ultrasonically dispersing it evenly. Then, the two antibodies with a mass ratio of 1:1 are mixed with the activated quantum dot microspheres, shaken and incubated in a metal bath for 30 minutes (1,000 rpm, 37°C), centrifuged (8,000 rpm, 6 minutes) and the supernatant is discarded. The unreacted carboxyl groups are blocked by a blocking reaction.

[0052] In a fourth aspect, the present invention further provides a method for detecting clenbuterol and ractopamine, which comprises the following detection method (1) or (2):

[0053] (1) Incubate the sample to be tested with the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate in the above-mentioned dual quantum dot microsphere fluorescent immunochromatography kit to obtain an incubation mixture, then insert the test strip into the incubation mixture, read the fluorescence intensity of the test line T1, the test line T2 and the quality control line C or perform the test under ultraviolet light;

[0054] (2) Load the sample to be tested onto the test strip in the above-mentioned dual quantum dot microsphere fluorescent immunochromatography kit, read the fluorescence intensity of the test line T1, the test line T2 and the quality control line C or perform the test under ultraviolet light.

[0055] The fluorescence intensity of the test line T1, the test line T2 and the quality control line C can be read by a fluorescence analyzer. In quantitative analysis, the mixed standard solutions of the CLE and RAC series gradients are incubated with the antibody-quantum dot microsphere conjugates to establish the standard curves of CLE and RAC concentration and fluorescence intensity, and the concentration of the sample to be tested is obtained according to the fluorescence intensity of the sample to be tested after loading.

[0056] In an alternative embodiment, a standard curve of CLE and RAC concentrations and fluorescence intensity ratios (ratio of the fluorescence intensity of the test line (F) to the fluorescence intensity of the test line after adding a negative control (F0)) is established.

[0057] In a preferred embodiment of the present invention, in the detection method (1), the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate are first mixed at a volume ratio of 1:0.5-1 to obtain a mixed conjugate; then the sample to be tested is mixed with the mixed conjugate at a volume ratio of 1:1.

[0058] In a preferred embodiment of the present invention, in the detection method (1), the sample to be tested and the mixed conjugate are incubated at 37°C ± 1°C for 1-1.5 min.

[0059] In a preferred embodiment of the present invention, in the detection method (1), the incubation time of the test strip inserted into the incubation mixture is 8-10 minutes.

[0060] In a preferred embodiment of the present invention, in the detection method (2), the incubation time of loading the sample on the test strip is 10-12 minutes.

[0061] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0062] Example 1

[0063] This embodiment provides a dual quantum dot microsphere fluorescent immunochromatography kit, which includes a test strip and a quantum dot microsphere-monoclonal antibody conjugate (QBs-mAbs). The detection principle is as follows Figure 1 shown.

[0064] (1) Preparation of quantum dot microsphere-monoclonal antibody conjugates (QBs-mAbs)

[0065] The quantum dot microspheres are prepared by respectively embedding CdSe / ZnS red light quantum dots and green light quantum dots in polystyrene microspheres. The surface of the microspheres is modified with carboxyl groups, which can undergo amidation reaction with the amino groups on the antibody under certain conditions, thereby coupling them together.

[0066] The red and green quantum dots of CdSe / ZnS are QBs630 and QBs530 respectively. QBs630 is a quantum dot submicrobead (QBS) that emits red light under 630nm excitation light. QBs530 is a quantum dot submicrobead (QBS) that emits green light under 530nm excitation light.

[0067] The coupling steps of green light quantum dot microsphere-CLE monoclonal antibody conjugate (QBs630-CLE mAbs) and red light quantum dot microsphere-RAC antibody conjugate (QBs530-RAC mAbs) are as follows: 50 μL QBs (QBs630 and QBs530, 10 mg / mL) and 3 μL EDC (20 mg / mL) were added to 100 μL labeling buffer, incubated in a metal bath for 30 min (1,000 rpm, 37°C), centrifuged (15,000 rpm, 15 min), the supernatant was discarded, the precipitate was redissolved in 150 μL labeling buffer, and ultrasonically dispersed evenly. Then, 50 μg of RAC monoclonal antibody (Shenzhen Antibiotech Co., Ltd.) and 50 μg of CLE monoclonal antibody (Shenzhen Antibiotech Co., Ltd.) were added to the activated QBs630 and QBs530 solutions, respectively, and incubated in a metal bath for 30 min (1,000 rpm, 37°C), centrifuged (8,000 rpm, 6 min), the supernatant was discarded, and the precipitate was redissolved in 100 μL of BSA (20 mg / mL) solution, and incubated in a metal bath for 120 min (1,000 rpm, 37°C) to block the unreacted carboxyl groups. After the blocking was completed, 100 μL of preservation solution (PBS buffer with a pH of 7.4 containing 1% BSA, 10% sucrose, 0.1% pc300, and 2% Tween 20) was added to the above solution and stored at 4°C for use.

[0068] (2) Preparation of immunochromatographic test strips

[0069] First, the sample pad, NC membrane and absorbent pad were respectively pasted to the PVC base plate. RAC-BSA, CLE-BSA and goat anti-mouse secondary antibody were diluted to 0.3, 0.3 and 0.08 mg / mL with diluent, and then the goat anti-mouse secondary antibody (C line), RAC-BSA (T1 line) and CLE-BSA (T2 line) solutions were applied to the NC membrane in the order from top to bottom at a spray rate of 1 μL / cm as two detection lines and quality control lines, with a spacing of 7.0 mm between each two lines. Finally, the assembled immunochromatography plate was dried in a 45°C oven for 2 hours, cut into 4.0 mm wide test strips, kept dry and placed at room temperature for use.

[0070] (3) Quantitative detection of clenbuterol and ractopamine

[0071] Before use, the QBs-mAbs conjugates of clenbuterol and ractopamine were diluted 150 times with 0.01M phosphate buffer and mixed at a volume ratio of 1:0.6. Then, 50 μL of the mixed conjugate was added to a 96-well plate and mixed with 50 μL of the test substance (standard solution or sample solution). The mixture was incubated at 37°C for 1 min, and the test strips were inserted into the corresponding wells. After incubation at 37°C for 10 min, the test strips were immediately taken out and placed in a fluorescence analyzer to read the fluorescence intensity data of the two test lines and the quality control line. In the quantitative analysis, the CLE and RAC standard solutions were diluted with phosphate buffer (0.01M, pH 7.0) containing 1% Tween to prepare a series of mixed standard solutions and mixed with QBs-mAbs solution. The concentrations of CLE were 0.02, 0.05, 0.2, 0.4, 0.8, 1.0, and 1.5 ng / mL, respectively; the concentrations of RAC were 0.01, 0.02, 0.05, 0.2, 0.4, 0.8, and 1.0 ng / mL, respectively. The standard curve was drawn with the logarithm of the concentration of clenbuterol as the abscissa and the ratio of the fluorescence intensity of the test line after adding the positive sample (F) to the fluorescence intensity of the test line after adding the negative control (F0) as the ordinate.

[0072] The detection limit is defined as the sample concentration when F / F0 = 0.9. The fluorescence test strip and standard curve are as follows Figure 2 , Figure 3 Among them, CLE showed a good linear relationship in the range of 0.02-1.5 ng / mL, and the calculated detection limit was 0.031 ng / mL; RAC showed a good linear relationship in the range of 0.01-1.0 ng / mL, and the calculated detection limit was 0.015 ng / mL.

[0073] The detection principle is as follows: First, CLE-BSA and RAC-BSA are sprayed on the NC membrane of the test strip as capture antigens (detection line, T line), and goat anti-mouse secondary antibody is used as the quality control line (C line). Then, the target and the prepared quantum dot microsphere-monoclonal antibody conjugate (QBs-mAbs) are mixed and incubated on a 96-well plate for 30 seconds, and then one end of the prepared test strip sample pad is inserted into the corresponding well and incubated for 10 minutes. At this time, the capture antigen and target will competitively bind to the QBs-mAbs. For negative samples, QBs-mAbs will be captured by the antigen on the T line, so that the T line emits strong red / green fluorescence under 305nm wavelength excitation. For positive samples, a part of QBs-mAbs will first bind to the target. When the target content gradually increases, the QBs-mAbs that can be captured by the coated antigen will gradually decrease, causing the fluorescence intensity of the T line to gradually weaken. Regardless of whether QBs-mAbs binds to the target or not, they can bind to the secondary antibody to keep the C line always bright. As a quality control of the test strip, if the C line is not bright, it means that the QBs-mAbs of the test strip is invalid. Compared with ordinary multi-component test strips, the present invention can also judge whether both antibodies are effective by the fluorescence color of the C line. Quantitative detection of targets can be achieved by the corresponding relationship between the fluorescence intensity of the T line and the target concentration in the sample.

[0074] Example 2

[0075] Methodological validation.

[0076] 1. Accuracy and repeatability verification

[0077] Three concentrations of clenbuterol and ractopamine standards were added to blank bovine urine, and the accuracy of the method was evaluated by detecting the recovery rate, which was calculated as follows: recovery rate (%) = (detected value / added value) × 100%. The precision of the method was evaluated by the relative standard deviation (RSD) of the test results of 4 parallel samples, which was calculated as follows: relative standard deviation (%) = standard deviation (SD) / arithmetic mean of the test results × 100%. The repeatability of the method was evaluated by the precision of the test results between batches and within batches. The recovery rate results are shown in Table 1. The recovery rate is between 81.2% and 108.4%, and the RSD is less than 13.5%, indicating that this method has good accuracy and repeatability.

[0078] Table 1 Statistical table of the recovery rates of CLE and RAC in urine (n=4)

[0079]

[0080] 2. Specificity test

[0081] The kit provided in Example 1 was used to detect CLE and RAC mixed standards (1 ng / mL), as well as the other 7 β-agonists (10 ng / mL), and the T-line fluorescence intensity (F) of the test strips with added β-agonists and the T-line fluorescence intensity (F0) of the test strips with added blank samples were measured using a card reader to calculate F / F0 (refer to Example 1) to evaluate the specificity of the method.

[0082] The results are as follows Figure 4 As shown. Figure 4 It can be seen that the two T lines of the test strip showed an obvious decrease in fluorescence when detecting CLE and RAC mixed standards, and the two T lines did not show a decrease in fluorescence after adding the other 7 β-agonists, indicating that the kit and detection method provided by the present invention have good specificity.

[0083] Example 3

[0084] This embodiment provides a dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, which includes a test strip; compared with Example 1, the only difference is that the antibody-quantum dot microsphere conjugate is coated on the binding pad, and the other steps and the composition of the test strip are the same as Example 1.

[0085] The test strip is provided with a sample pad, a conjugate pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction; the conjugate pad is coated with an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; the detection line T1 line is coated with clenbuterol (CLE)-BSA, the detection line T2 line is coated with ractopamine (RAC)-BSA; and the quality control line C line is coated with sheep anti-mouse antibody.

[0086] The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under the excitation of a light source and an anti-clenbuterol antibody, and in this embodiment it is QBs630-CLE mAbs; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under the excitation of a light source and an anti-ractopamine antibody, and in this embodiment it is QBs530-RAC mAbs.

[0087] In the kit of this embodiment, the antibody-quantum dot microsphere conjugate is coated on the conjugate pad, which is easy to carry, store and transport.

[0088] The coating volume ratio of the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate on the conjugate pad is 1:0.5.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, characterized in that: It comprises a test strip, an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate; the test strip is provided with a sample pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; the detection line T1 line is coated with a clenbuterol (CLE)-carrier protein, and the detection line T2 line is coated with a ractopamine (RAC)-carrier protein; the quality control line C line is coated with a secondary antibody, and the secondary antibody is a secondary antibody corresponding to the species of the anti-clenbuterol antibody and the anti-ractopamine antibody; The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under excitation of a light source and an anti-clenbuterol antibody; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under excitation of a light source and an anti-ractopamine antibody; the first wavelength and the second wavelength are different.

2. The double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to claim 1, characterized in that: The quantum dot microspheres emitting fluorescence of the first wavelength refer to: quantum dots capable of emitting fluorescence of the first wavelength are embedded in polymethyl methacrylate (PMMA), polyglycidyl methacrylate microspheres or polystyrene microspheres; The quantum dot microspheres emitting fluorescence of the second wavelength refer to: quantum dots capable of emitting fluorescence of the second wavelength are embedded in polymethyl methacrylate (PMMA), polyglycidyl methacrylate microspheres or polystyrene microspheres; Preferably, the quantum dots are selected from at least one of substance I having a chemical formula shown in formula I, substance II having a chemical formula shown in formula II, and substance III having a chemical formula shown in formula III; AM type Ⅰ; EFQ2 type II; GHX3 type III; Wherein, A is selected from at least one of Cd, Zn, Pb, and In; M and Q are independently selected from at least one of S, Se, Te, and P; E is selected from at least one of Cu, Ag, and Zn; F is selected from at least one of Al, Ga, and In; G is selected from at least one of CH3NH3, CH(NH)NH3, and Cs; H is selected from at least one of Ag, Sb, Bi, Pb, In, and Al; X is selected from at least one of halogens; Preferably, the quantum dots are selected from substances having a CdSe / ZnS core-shell structure; Preferably, the quantum dot microspheres are green light quantum dot microspheres or red light quantum dot microspheres.

3. The double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to claim 2, characterized in that: The particle size of the quantum dot microspheres is 100nm to 200nm.

4. The double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to claim 3, characterized in that: The volume-to-mass ratio of the quantum dot microspheres to the anti-clenbuterol antibody is 1:0.5-1.2; the unit of the quantum dot microspheres is μL, and the unit of the antibody is μg; Preferably, the volume-to-mass ratio of the quantum dot microspheres to the anti-ractopamine antibody is 1:0.5-1.

2.

5. The double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to claim 1, characterized in that: The coating concentrations of the detection line T1 line and the detection line T2 line are 0.1-0.3 mg / mL and 0.1-0.3 mg / mL respectively; Preferably, the coating concentration of the quality control line C is 0.08 mg / mL.

6. The double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to claim 1, characterized in that: The carrier protein is diphtheria toxin cross-reactive material 197, diphtheria toxoid, tetanus toxoid or bovine serum albumin.

7. A dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine, characterized in that: It comprises a test strip; the test strip is provided with a sample pad, a conjugation pad, an NC membrane and a water absorbent pad in sequence along the fluid chromatography direction; the conjugation pad is coated with an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate, and a detection line T1 line, a detection line T2 line and a quality control line C line are provided on the NC membrane; the detection line T1 line is coated with clenbuterol (CLE)-carrier protein, and the detection line T2 line is coated with ractopamine (RAC)-carrier protein; the quality control line C line is coated with a secondary antibody, and the secondary antibody is a secondary antibody corresponding to the species of the anti-clenbuterol antibody and the anti-ractopamine antibody; The anti-clenbuterol antibody-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a first wavelength under excitation of a light source and an anti-clenbuterol antibody; the anti-ractopamine-quantum dot microsphere conjugate refers to: a conjugate of quantum dot microspheres that emit fluorescence of a second wavelength under excitation of a light source and an anti-ractopamine antibody; the first wavelength and the second wavelength are different.

8. The double quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to claim 7, characterized in that: The coating volume ratio of the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate on the conjugate pad is 1:0.5-1.

9. The method for preparing the dual quantum dot microsphere fluorescent immunochromatography kit for detecting clenbuterol and ractopamine according to any one of claims 1 to 6, characterized in that: The invention comprises the preparation of an antibody-quantum dot microsphere conjugate, wherein the preparation method of the antibody-quantum dot microsphere conjugate comprises: mixing the quantum dot microspheres with a microsphere surfactant to activate the quantum dot microspheres, and then incubating the activated quantum dot microspheres with an anti-clenbuterol antibody and an anti-ractopamine antibody respectively, and after blocking, obtaining an anti-clenbuterol antibody-quantum dot microsphere conjugate and an anti-ractopamine antibody-quantum dot microsphere conjugate respectively.

10. A method for detecting clenbuterol and ractopamine, characterized in that: It includes the following detection method (1) or (2): (1) Incubate the sample to be tested with the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate in the dual quantum dot microsphere fluorescent immunochromatography kit according to any one of claims 1 to 6 to obtain an incubation mixture, then insert the test strip into the incubation mixture, read the fluorescence intensity of the test line T1, the test line T2 and the quality control line C or perform the test under ultraviolet light; (2) Loading the sample to be tested onto the test strip in the dual quantum dot microsphere fluorescent immunochromatography kit according to any one of claims 7 to 8, reading the fluorescence intensity of the test line T1, the test line T2 and the quality control line C or performing the test under ultraviolet light; Preferably, in the detection method (1), the anti-clenbuterol antibody-quantum dot microsphere conjugate and the anti-ractopamine antibody-quantum dot microsphere conjugate are first mixed at a volume ratio of 1:0.5-1 to obtain a mixed conjugate; and then the sample to be tested is mixed with the mixed conjugate at a volume ratio of 1:1; Preferably, in the detection method (1), the sample to be tested and the mixed conjugate are incubated at 37°C ± 1°C for 1-1.5 min; Preferably, in the detection method (1), the incubation time of the test strip inserted into the incubation mixture is 8-10 min; Preferably, in the detection method (2), the incubation time of loading the sample to be tested on the test strip is 10-12 minutes.

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