A test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography and its use method

Abstract

The present application relates to the field of clenbuterol hydrochloride detection in minced meat products, and specifically discloses a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography, comprising a PVC base plate, a test sample pad, and a nitrocellulose membrane, wherein the test sample pad and the nitrocellulose membrane are sequentially connected to the PVC base plate, the test sample pad is used to drip a mixture of a fluorescently labeled clenbuterol hydrochloride antibody and a test sample, and the nitrocellulose membrane is used to coat a clenbuterol hydrochloride monoclonal antibody and goat anti-mouse IgG; wherein the fluorescently labeled clenbuterol hydrochloride monoclonal antibody comprises water-soluble quantum dots, trichloride, and clenbuterol hydrochloride monoclonal antibody. The test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography of the present application has the effects of low detection limit and high sensitivity.

Description

Technical Field

[0001] The present application relates to the field of clenbuterol hydrochloride detection in minced meat products, and more specifically, to a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography and a method for using the test strip. Background Art

[0002] Clenbuterol hydrochloride is the main component of clenbuterol. After entering the body of livestock, this type of clenbuterol can change the metabolic pathway of nutrients, promote animal muscle growth, especially promote the synthesis of skeletal muscle protein, accelerate the conversion and decomposition of fat, and increase the lean meat rate of livestock. In order to ensure the quality and safety of livestock products, the method of detecting the content of clenbuterol hydrochloride in clenbuterol is extremely important.

[0003] In the related art, a Chinese application document with application number CN201510258151.1 discloses a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography and a detection method thereof. The present invention discloses a colloidal gold immunochromatographic test strip for detecting clenbuterol hydrochloride, comprising a detection sample pad, a conjugation pad, a nitrocellulose coating, a water-absorbing pad and a PVC bottom plate, wherein the detection sample pad, the conjugation pad, the nitrocellulose coating and the water-absorbing pad are sequentially overlapped on the PVC bottom plate, wherein the conjugation pad is coated with a clenbuterol monoclonal antibody-colloidal gold marker; a detection line and a quality control line are provided on the nitrocellulose coating, wherein the detection line is coated with a clenbuterol hydrochloride antigen, and the quality control line is coated with a sheep anti-mouse IgG; when in use, the test strip is assembled and detected; the sensitivity of the test strip prepared in this experiment for detecting clenbuterol hydrochloride is 8 ng / mL.

[0004] With respect to the above-mentioned related technologies, the inventors believe that the sensitivity of the above-mentioned colloidal gold immunochromatographic test strips in detecting clenbuterol hydrochloride is low, and it is impossible to detect trace amounts of clenbuterol hydrochloride added to minced meat products. Summary of the invention

[0005] In order to reduce the detection limit of clenbuterol hydrochloride and improve the detection sensitivity, the present application provides a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography and a method for using the same.

[0006] In the first aspect, the present application provides a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography, using the following technical solution:

[0007] A test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography, comprising a PVC base plate, a test sample pad, and a nitrocellulose membrane, wherein the test sample pad and the nitrocellulose membrane are sequentially connected to the PVC base plate, the test sample pad is used for dripping a mixture of a fluorescently labeled clenbuterol hydrochloride antibody and a test sample, and the nitrocellulose membrane is used for coating a clenbuterol hydrochloride monoclonal antibody and goat anti-mouse IgG;

[0008] Among them, the fluorescently labeled clenbuterol hydrochloride monoclonal antibody includes water-soluble quantum dots, trichloride and clenbuterol hydrochloride monoclonal antibody.

[0009] By adopting the above technical solution, water-soluble quantum dots can be used as a new type of fluorescent marker in fluorescent immunochromatography. Since the surface of water-soluble quantum dots carries carboxyl, amino or hydroxyl groups that are soluble in water, they can be complexed with trivalent cations in trichloride to form stable water-soluble quantum dots-M 3+ When the water-soluble quantum dots are used as fluorescent markers for clenbuterol hydrochloride monoclonal antibodies, the fluorescence intensity of the fluorescently labeled clenbuterol hydrochloride monoclonal antibodies increases, thereby reducing the detection limit of the final detection of clenbuterol hydrochloride antigen and increasing the sensitivity.

[0010] Optionally, the trichloride is one of aluminum trichloride and ferric chloride.

[0011] By adopting the above technical solution, since aluminum chloride and ferric chloride are both soluble in water, and chloride ions in water will not affect the effect of the fluorescently labeled clenbuterol hydrochloride monoclonal antibody, Al 3+ with Fe 3+ In water, Al can exist in the form of ions. 3+ with Fe 3+ They can all be connected to water-soluble quantum dots with negative charges, increasing the number of water-soluble quantum dots connected to a single clenbuterol hydrochloride monoclonal antibody and enhancing the detection fluorescence intensity, thereby reducing the detection limit and improving sensitivity.

[0012] Optionally, the water-soluble quantum dots are one of ZnSe / ZnS quantum dots, InP / ZnS quantum dots, CdS / ZnS quantum dots, and water-soluble quantum dots made from CdSe / ZnS quantum dots.

[0013] By adopting the above technical scheme, ZnSe / ZnS quantum dots, InP / ZnS quantum dots, CdS / ZnS quantum dots, and CdSe / ZnSde quantum dots have higher luminescence intensity among luminescent quantum dots and are moderately priced; water-soluble quantum dots prepared from ZnSe / ZnS quantum dots, InP / ZnS quantum dots, CdS / ZnS quantum dots, and CdSe / ZnSde quantum dots have high stability, and when used as fluorescent markers for clenbuterol hydrochloride monoclonal antibodies, the final detection results have high stability.

[0014] Optionally, the surface of the water-soluble quantum dots carries carboxyl groups.

[0015] By adopting the above technical scheme, water-soluble quantum dots with carboxyl groups on the surface are more stable in aqueous solution than water-soluble quantum dots with amino groups on the surface, and the hydrogen ions of carboxyl groups are more easily dissociated in aqueous solution than the hydrogen ions of hydroxyl groups; therefore, the complex formed by the water-soluble quantum dots with carboxyl groups on the surface and the cations in the trichloride is more stable, so that the fluorescence stability of the fluorescently labeled clenbuterol hydrochloride monoclonal antibody is higher, and the stability of the final detection result is high.

[0016] Optionally, the concentration of the water-soluble quantum dots is 0.5-2 nmol / mL; the concentration of the clenbuterol hydrochloride monoclonal antibody is 3.5-6 mg / mL.

[0017] By adopting the above technical solution, water-soluble quantum dots of appropriate concentration can be better connected with clenbuterol hydrochloride monoclonal antibodies, and it is difficult to waste water-soluble quantum dots and clenbuterol hydrochloride monoclonal antibodies.

[0018] Optionally, the concentration of the trichloride is 10-30nmol / mL.

[0019] By adopting the above technical solution, the appropriate trichloride concentration makes it difficult to produce precipitation when the trichloride is added to the fluorescently labeled clenbuterol hydrochloride monoclonal antibody while connecting the water-soluble quantum dots, thereby ensuring the smooth flow of the fluorescently labeled clenbuterol hydrochloride monoclonal antibody on the nitrocellulose membrane, thereby ensuring a shorter detection time.

[0020] In a second aspect, the present application provides a method for using a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography, using the following technical solution:

[0021] A method for using a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography comprises the following steps:

[0022] Step S1: Preparation of a mixture of fluorescently labeled clenbuterol hydrochloride antibody and test sample: adding water-soluble quantum dots, coupling agent, and clenbuterol hydrochloride monoclonal antibody to serum, adjusting the pH to 7-8, vortexing and letting stand; then adding the test sample, vortexing; then adding BSA solution and aluminum chloride aqueous solution, vortexing and sealing at 0-4° C., letting stand, to obtain a mixture of fluorescently labeled clenbuterol hydrochloride antibody and test sample;

[0023] Step S2: Assembling the test strip: stick the nitrocellulose coating membrane and the absorbent pad on the PVC bottom plate respectively; coat the clenbuterol hydrochloride monoclonal antibody and goat anti-mouse IgG on the nitrocellulose coating membrane in sequence as the detection line and control line respectively.

[0024] By adopting the above technical scheme, a mixture of fluorescently labeled clenbuterol hydrochloride antibodies and test samples is prepared, and test strips are assembled, and the competitive detection in the prior art is changed to double antibody sandwich detection, so that the mixture of fluorescently labeled clenbuterol hydrochloride antibodies and test samples only needs to be combined with the clenbuterol hydrochloride monoclonal antibodies, and the reaction can be completed in one step.

[0025] Optionally, in step S1, BSA solution is added, and the step of reacting at 35-37° C. for 10-30 minutes is also included.

[0026] By adopting the above technical solution and reacting at a suitable temperature and time, BSA can play a better blocking role, preventing the clenbuterol hydrochloride monoclonal antibody from binding to non-specific proteins, thereby forming a one-to-one connection between the clenbuterol hydrochloride monoclonal antibody and the clenbuterol hydrochloride antigen, and the final test result is accurate.

[0027] Optionally, the coupling agent is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0028] By adopting the above technical solution, the addition of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride can stably connect the clenbuterol hydrochloride monoclonal antibody with the water-soluble quantum dots to form a highly stable fluorescent-labeled clenbuterol hydrochloride monoclonal antibody, and the final detection result is accurate.

[0029] In summary, this application has the following beneficial effects:

[0030] 1. In the present application, since the surface of the water-soluble quantum dots has carboxyl groups, hydroxyl groups or amino groups that are soluble in water, they can be complexed with trivalent cations in trichloride to form stable water-soluble quantum dots-M 3+When the water-soluble quantum dots are used as fluorescent markers for clenbuterol hydrochloride monoclonal antibodies, the fluorescence intensity of the fluorescently labeled clenbuterol hydrochloride monoclonal antibodies increases, so that the detection limit of the final detection of clenbuterol hydrochloride antigen is reduced and the sensitivity is increased;

[0031] 2. In the present application, in the water-soluble quantum dots, the hydrogen ions on -COOH are more easily dissociated than the hydrogen ions on -OH, and -COOH is more stable in water than -NH4. Therefore, the water-soluble quantum dots carrying carboxyl groups on the surface are more likely to form stable complexes with cations in trichloride, so that the fluorescence stability of the fluorescently labeled clenbuterol hydrochloride monoclonal antibody is higher, and the stability of the final detection result is high;

[0032] 3. The appropriate trichloride concentration makes it difficult to produce precipitation when the trichloride is added to the fluorescently labeled clenbuterol hydrochloride monoclonal antibody while connecting the water-soluble quantum dots, thereby ensuring the smooth flow of the fluorescently labeled clenbuterol hydrochloride monoclonal antibody on the nitrocellulose membrane, thereby ensuring a shorter detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a linear relationship diagram in Example 3. DETAILED DESCRIPTION

[0034] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0035] The sources of raw materials for the following embodiments and comparative examples are provided: the raw materials for the embodiments and comparative examples can be purchased commercially, L-cysteine-CdS / ZnS quantum dots, L-cysteine-ZnSe / ZnS quantum dots and mercaptoethylamine-CdS / ZnS were purchased from Taizhou Juna New Energy Co., Ltd.; clenbuterol hydrochloride monoclonal antibody, clenbuterol hydrochloride antigen and sheep anti-mouse IgG were purchased from Sanofi, and clenbuterol hydrochloride antigen was the test sample; calf serum was purchased from Kejing Bio.

[0036] Embodiment of a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography Example

[0037] A method for using a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography comprises the following steps:

[0038] Step S1: Preparation of a mixture of fluorescently labeled clenbuterol hydrochloride antibody and test sample: L-cysteine-CdS / ZnS quantum dots, coupling agent N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and clenbuterol hydrochloride monoclonal antibody were added to calf serum diluted with 10-fold volume of 0.1M boric acid solution, and the pH was adjusted to 7.5 with phosphate buffer solution. After vortexing at 1000 rpm for 30 seconds, the mixture was allowed to stand for 10 minutes; then the test sample clenbuterol hydrochloride antigen was added, and vortexed at 1000 rpm for 30 seconds; then B was added. SA and aluminum chloride were vortexed and sealed at 4°C, and allowed to stand for 30 minutes to obtain a mixture of fluorescently labeled clenbuterol hydrochloride antibody and test sample; wherein the concentration of L-cysteine-CdS / ZnS quantum dots was 1 nmol / mL, the concentrations of coupling agent N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were both 5 μL / mL, the concentration of clenbuterol hydrochloride monoclonal antibody was 4.5 mg / mL, and the concentration of clenbuterol hydrochloride monoclonal antigen was 6 mg / mL; the concentration of aluminum chloride was 20 nmol / mL; and the concentration of BSA was 10wt%;

[0039] Step S2: Assembly of the test strip: stick the nitrocellulose coating membrane and the absorbent pad on the PVC bottom plate respectively; coat the clenbuterol hydrochloride monoclonal antibody and goat anti-mouse IgG on the nitrocellulose coating membrane in sequence as the test line and control line respectively; unit spray volume: the test line is 1 μL / cm, the quality control line is 1.0 μL / cm, the coating concentration of the clenbuterol hydrochloride monoclonal antibody is 0.4 mg / mL; the coating concentration of the goat anti-mouse IgG is 1.0 mg / mL.

[0040] Example 2

[0041] The difference from Example 1 is that in step S1, the concentrations of L-cysteine-CdS / ZnS quantum dots, clenbuterol hydrochloride monoclonal antibody, clenbuterol hydrochloride monoclonal antigen, and aluminum chloride are different;

[0042] Among them, the concentration of L-cysteine-CdS / ZnS quantum dots is 0.5nmol / mL, the concentration of clenbuterol hydrochloride monoclonal antibody is 6mg / mL, the concentration of clenbuterol hydrochloride monoclonal antigen is 8mg / mL; and the concentration of aluminum chloride is 30nmol / mL.

[0043] Example 3

[0044] The difference from Example 1 is that in step S1, the concentrations of L-cysteine-CdS / ZnS quantum dots, clenbuterol hydrochloride monoclonal antibody, clenbuterol hydrochloride monoclonal antigen, and aluminum chloride are different;

[0045] Among them, the concentration of L-cysteine-CdS / ZnS quantum dots is 2nmol / mL, the concentration of clenbuterol hydrochloride monoclonal antibody is 3.5mg / mL, the concentration of clenbuterol hydrochloride monoclonal antigen is 5mg / mL; and the concentration of aluminum chloride is 10nmol / mL.

[0046] Example 4

[0047] The difference from Example 3 is that in step S1, L-cysteine-CdS / ZnS quantum dots are replaced with L-cysteine-ZnSe / ZnS quantum dots of equal concentration.

[0048] Example 5

[0049] The difference from Example 3 is that in step S1, L-cysteine-CdS / ZnS quantum dots are replaced with mercaptoethylamine-CdS / ZnS quantum dots of equal concentration.

[0050] Example 6

[0051] The difference from Example 3 is that in step S1, aluminum chloride is replaced by ferric chloride of equal concentration.

[0052] Example 7

[0053] The difference from Example 3 is that in step S1, aluminum trichloride is replaced by boron trichloride of equal concentration.

[0054] Comparative Example 1

[0055] The difference from Example 3 is that in step S1, aluminum chloride is not added.

[0056] Performance testing

[0057] A. The test strips prepared in Examples 1 to 7 and Comparative Example 1 were used to detect clenbuterol hydrochloride in minced meat products by the photoimmunochromatography method to perform performance tests, and the fluorescence intensity of the detection line of each test strip was detected. The test results are shown in Table 1.

[0058] Table 1

[0059] project Fluorescence intensity (AU) project Fluorescence intensity (AU) Example 1 53231 Example 5 48138 Example 2 53246 Example 6 52843 Example 3 54325 Example 7 50996 Example 4 52469 Comparative Example 1 24245

[0060] Combining Examples 1, 2 and 3, it can be seen that the difference between Examples 1, 2 and 3 is that the raw material amounts of L-cysteine-CdS / ZnS quantum dots, clenbuterol hydrochloride monoclonal antibodies and aluminum trichloride are different, but the fluorescence detection results are all good and the fluorescence intensity is high, proving that the raw material amounts of L-cysteine-CdS / ZnS quantum dots, clenbuterol hydrochloride monoclonal antibodies and aluminum trichloride within the protection scope of the claims of the present application are appropriate.

[0061] Combining Examples 3, 4 and 5, it can be seen that L-cysteine-CdS / ZnS quantum dots, L-cysteine-ZnSe / ZnS quantum dots and mercaptoethylamine-CdS / ZnS quantum dots are used in Examples 3, 4 and 5 respectively. The fluorescence intensity finally detected in Examples 3 and 4 is higher, and the fluorescence intensity detected in Example 5 is slightly lower; therefore, the best choice of quantum dots in the present application is quantum dots carrying carboxyl groups on the surface.

[0062] Combining Examples 3, 6 and 7, it can be seen that aluminum chloride, ferric chloride and boron chloride are used in Examples 3, 6 and 7, respectively, and the fluorescence intensity finally detected in Examples 3 and 6 is higher than the fluorescence intensity detected in Example 7; therefore, the best trichlorides selected in the present application are ferric chloride and aluminum chloride.

[0063] Combining Example 3 with Comparative Example 1, it can be seen that aluminum chloride is not added in Comparative Example 1. Under the same experimental conditions, the fluorescence intensity detected in Example 3 is 2-3 times that of Comparative Example 1, which proves that the addition of aluminum chloride can enhance the fluorescence intensity of clenbuterol hydrochloride monoclonal antigen detection at the same concentration.

[0064] B. Fluorescence detection was performed on Example 3 and Comparative Example 1 at antigen concentrations of 0 mg / mL, 1 mg / mL, 3 mg / mL, 6 mg / mL, 10 mg / mL, 30 mg / mL, 60 mg / mL, and 100 mg / mL, and a linear relationship was drawn to calculate the detection limit; the test results are shown in Table 2 and the attached specification Figure 1 As shown;

[0065] Table 2 Six parallel experiments of fluorescence intensity detection in Example 3

[0066] 1 2 3 4 5 6 RSD(%) 53105 55261 54052 50987 52655 54011 2.74

[0067] The linear relationship diagram of Example 3 is shown in the attached specification. Figure 1 As shown, the linear relationship of Example 3 is Y=3347.8X+13550, R 2 =0.9971; calculated by 3RSD / k (k is the slope), the detection limit of clenbuterol hydrochloride in minced meat products by the fluorescent immunochromatography test strip of Example 3 is 2.45ng / mL; using the same method as above, it is calculated that the detection limit of clenbuterol hydrochloride in minced meat products by the fluorescent immunochromatography test strip of Comparative Example 1 is 6.5ng / mL; it is proved that the fluorescent immunochromatography test strip for detecting clenbuterol hydrochloride in minced meat products using semiconductor quantum dots as fluorescent markers and adding aluminum chloride can reduce the detection limit of clenbuterol hydrochloride and improve the sensitivity.

[0068] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for using a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography, characterized in that: The test strip comprises a PVC bottom plate, a detection sample pad, and a nitrocellulose membrane. The detection sample pad and the nitrocellulose membrane are sequentially connected to the PVC bottom plate. The detection sample pad is used to drip a mixture of a fluorescently labeled clenbuterol hydrochloride antibody and a detection sample. The nitrocellulose membrane is used to coat the clenbuterol hydrochloride monoclonal antibody and goat anti-mouse IgG. The fluorescently labeled clenbuterol hydrochloride antibody comprises water-soluble quantum dots, aluminum chloride and clenbuterol hydrochloride monoclonal antibody, wherein the water-soluble quantum dots are one of L-cysteine-CdS / ZnS quantum dots or L-cysteine-ZnSe / ZnS quantum dots, and the concentration of the aluminum chloride is 10-30 nmol / mL; The method of use comprises the following steps: Step S1: Preparation of a mixture of fluorescently labeled clenbuterol hydrochloride antibody and test sample: adding water-soluble quantum dots, coupling agent, and clenbuterol hydrochloride monoclonal antibody to serum, adjusting the pH to 7-8, vortexing and letting stand; then adding the test sample, vortexing; then adding BSA solution and aluminum chloride aqueous solution, vortexing and sealing at 0-4° C., letting stand, to obtain a mixture of fluorescently labeled clenbuterol hydrochloride antibody and test sample; Step S2: Assembling the test strip: stick the nitrocellulose coating membrane and the absorbent pad on the PVC bottom plate respectively; coat the clenbuterol hydrochloride monoclonal antibody and goat anti-mouse IgG on the nitrocellulose coating membrane in sequence as the detection line and control line respectively.

2. The method for using a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography according to claim 1, characterized in that: The surface of the water-soluble quantum dots carries carboxyl groups.

3. The method for using the test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography according to claim 1, characterized in that: The concentration of the water-soluble quantum dots is 0.5-2 nmol / mL; the concentration of the clenbuterol hydrochloride monoclonal antibody is 3.5-6 mg / mL.

4. The method for using a test strip for detecting clenbuterol hydrochloride in minced meat products by fluorescent immunochromatography according to claim 1, characterized in that: The coupling agent is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

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