Time-resolved fluorescence immunochromatography method and kit for detecting thiamphenicol

Abstract

The invention provides a time-resolved fluorescence immunochromatography method and kit for detecting thiamphenicol, and belongs to the field of analytical chemistry. The kit mainly comprises a time-resolved fluorescent microsphere labeled antibody probe and a test strip, the time-resolved fluorescent microspheres are prepared by embedding europium complexes into polystyrene microspheres through a swelling method. Performing chemical coupling on the time-resolved fluorescent microspheres and a thiamphenicol monoclonal antibody to prepare a time-resolved fluorescent microsphere labeled antibody probe; the test strip comprises a nitrocellulose membrane, a sample pad and an absorption pad which are arranged on a bottom plate, and the nitrocellulose membrane is coated with a detection line T of thiamphenicol antigen and a quality control line C of goat anti-mouse IgG antibody. The invention provides a time-resolved fluorescence immunochromatography method for detecting thiamphenicol and a kit, which can be applied to detection of thiamphenicol in food samples and have the characteristics of simplicity, convenience, high sensitivity, rapidness in quantification and the like.

Description

Technical Field

[0001] The present invention relates to the technical fields of analytical chemistry and food safety detection, and particularly relates to a time-resolved fluorescence immunochromatography method and kit for detecting thiamphenicol. Background Art

[0002] Thiamphenicol (TAP) is a broad-spectrum bacteriostatic antibiotic with the chemical formula C 12 H 15 C 12 NO5S, and is widely used in the prevention and treatment of intestinal infections and other diseases during the breeding processes of livestock, poultry, and aquatic products. However, thiamphenicol has toxicities such as blood toxicity, neurotoxicity, and embryotoxicity, and the residue of thiamphenicol in animal-derived foods may pose a potential threat to human health. Therefore, it is of great significance to establish a rapid detection method for thiamphenicol in animal-derived foods.

[0003] Currently, the detection methods for thiamphenicol mainly include high performance liquid chromatography, gas chromatography-mass spectrometry, thin layer chromatography, enzyme-linked immunosorbent assay, colloidal gold strip method, etc. Instrument methods have advantages in terms of sensitivity and accuracy, but have problems such as long detection time, expensive instruments, and high requirements for operators. The colloidal gold strip method has the characteristics of being simple, rapid, highly specific, high throughput, and suitable for on-site detection, but the disadvantage is low sensitivity.

[0004] Lanthanide elements - europium (Eu), samarium (Sm), etc. have characteristics such as high fluorescence intensity, large Stokes shift, long fluorescence lifetime, narrow emission spectrum, and strong anti-background interference, and are excellent fluorescent materials. Packaging lanthanide elements in polystyrene microspheres to prepare time-resolved fluorescence microspheres can further improve the fluorescence intensity and stability of the materials. Using time-resolved fluorescence microspheres to replace colloidal gold to establish a fluorescence immunochromatography method for thiamphenicol can effectively improve the detection sensitivity of the traditional immunochromatography method. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a time-resolved fluorescence immunochromatography method and kit for detecting thiamphenicol. The present invention synthesizes Eu-time-resolved fluorescence microspheres (Eu-TRFM), couples the time-resolved fluorescence microspheres with anti-thiamphenicol monoclonal antibody to prepare a fluorescent probe (TRFM-mAb), and provides a time-resolved fluorescence immunochromatography method and kit for detecting thiamphenicol, which have the advantages of being simple, rapid, highly sensitive, and accurate, and can be applied to the on-site detection of thiamphenicol in animal-derived foods.

[0006] To solve the technical problems, the present invention provides the following technical solutions:

[0007] The time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol provided by the present invention includes a test strip and a probe. The test strip includes a base plate, a sample pad, a nitrocellulose membrane, and a water absorption pad. The water absorption pad, the nitrocellulose membrane, and the sample pad are sequentially pasted on the base plate from top to bottom along the length direction of the test strip, and the sample pad and the water absorption pad each press a certain length of the nitrocellulose membrane.

[0008] On the detection area of the nitrocellulose membrane, a T line and a C line are provided. The T line is coated with thiamphenicol antigen, and the C line is coated with goat anti-mouse IgG antibody. The probe is a time-resolved fluorescence microsphere-labeled antibody probe. The preparation method of the probe includes: embedding europium complex in polystyrene microspheres to prepare time-resolved fluorescence microspheres; chemically conjugating the time-resolved fluorescence microspheres with thiamphenicol monoclonal antibody to obtain the probe.

[0009] According to the preferred embodiment of the present invention, the embedding of europium complex in polystyrene microspheres to prepare time-resolved fluorescence microspheres includes the following steps:

[0010] 1) Dissolve europium chloride hexahydrate, 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione, and o-phenanthroline in absolute ethanol, and heat and stir to synthesize europium-containing fluorescent complex Eu(NTA)3Phen;

[0011] 2) Under nitrogen protection, dissolve styrene, ammonium persulfate, and α-methylacrylic acid in an ethanol aqueous solution, and heat and stir to synthesize polystyrene microspheres;

[0012] 3) Embed the europium-containing fluorescent complex synthesized in step 1) into the polystyrene microspheres synthesized in step 2) to prepare time-resolved fluorescence microspheres, wash with absolute ethanol and deionized water, and resuspend with ultrapure water for standby.

[0013] According to the preferred embodiment of the present invention, the molar ratio of europium chloride hexahydrate, 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione, and o-phenanthroline is 1:3:1.

[0014] According to the preferred embodiment of the present invention, step 3) is specifically: weigh the europium-containing fluorescent complex and dissolve it in dichloromethane, and dropwise add it to the aqueous solution of the polystyrene microspheres in step 2), and stir and react; then distill under reduced pressure to remove dichloromethane; wash the product with absolute ethanol several times until there is no fluorescence in the supernatant, and then wash with deionized water; finally, disperse the product in ultrapure water to obtain time-resolved fluorescence microspheres, and store them at 4°C for standby.

[0015] According to the preferred embodiment of the present invention, the chemical conjugation of the time-resolved fluorescence microspheres with thiamphenicol monoclonal antibody to obtain the probe includes the following steps:

[0016] After washing the time-resolved fluorescence microspheres with boric acid buffer, EDC and NHS were added for activation respectively. After activation, chloramphenicol monoclone antibody was added for conjugation, and then bovine serum albumin was added for blocking. After centrifugation, the precipitate was resuspended with boric acid buffer containing 0.5 wt% sucrose, 0.05 wt% bovine serum albumin, 0.09 wt% NaCl and 0.05 wt% Tween-20 to obtain the time-resolved fluorescence microsphere conjugated antibody probe; the mass ratio of the time-resolved fluorescence microspheres to the chloramphenicol monoclone antibody was 10:1.

[0017] According to the preferred embodiment of the present invention, the pH of the boric acid buffer for washing the time-resolved fluorescence microspheres is 7.0 and the concentration is 50 mM; the concentrations of both EDC and NHS are 50 mg / mL, and the addition amounts are both 4 μL; the addition amount of the chloramphenicol monoclone antibody is 100 μg.

[0018] According to the preferred embodiment of the present invention, the test strip is prepared by the following method: The chloramphenicol antigen TAP-BSA and the goat anti-mouse IgG antibody were successively sprayed onto the positions of the test line T and the quality control line C on the nitrocellulose membrane with a membrane scribing instrument, and the scribed nitrocellulose membrane was dried and then adhered to the sample pad and the absorbent pad on the bottom plate in sequence.

[0019] According to the preferred embodiment of the present invention, the concentration of the chloramphenicol antigen TAP-BSA is 0.8 mg / mL; the sample pad and the absorbent pad each press 1 mm of the nitrocellulose membrane at both ends; and the test strip is cut into a width of 4 mm.

[0020] The present invention also provides a time-resolved fluorescence immunochromatography method for detecting chloramphenicol based on the kit, which comprises the following steps:

[0021] a) Mix different concentrations of chloramphenicol standard solutions with the time-resolved fluorescence microsphere labeled antibody probe respectively and add them to the sample pad of the test strip. Then measure the signal value of the T line. Taking the concentration of the chloramphenicol standard as the abscissa and the ratio of the signal value of the T line of different standard concentrations to the blank control as the ordinate, a standard curve is established.

[0022] b) Mix the sample to be detected with the time-resolved fluorescence microsphere labeled antibody probe and add it to the sample pad of the test strip. Then, qualitative judgment or quantitative detection is carried out according to the following method:

[0023] Qualitative determination method: Observe under an ultraviolet lamp. If both the test line T and the quality control line C have colors, it is a negative sample without chloramphenicol; if the T line has no color and the C line has color, it is a positive sample containing chloramphenicol; Quantitative determination method: Take a photo under an ultraviolet lamp to measure the signal value of the test line T, and perform quantitative detection according to the standard curve established in step a).

[0024] The present invention further provides an application of the above-mentioned time-resolved fluorescence immunochromatographic kit in detecting thiamphenicol in food samples.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The time-resolved fluorescence immunochromatographic test strip provided by the present invention has higher detection sensitivity than the traditional colloidal gold test strip. Based on the characteristics of Eu-TRFM, the T line of the test strip shows red fluorescence, thus amplifying the detection signal intensity. The lowest detection limit of the test strip reaches 0.02 ng / mL, and the linear detection range is 0.05 - 33.09 ng / mL.

[0027] The time-resolved fluorescence immunochromatographic method and kit for detecting thiamphenicol provided by the present invention can be applied to the detection of thiamphenicol in food samples, and have the advantages of simplicity, convenience, high sensitivity, and rapid quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the detection principle of the time-resolved fluorescence immunochromatographic method of the present invention;

[0029] Figure 2 It is the characterization result of time-resolved fluorescence microspheres; (A) TEM image of TRFM, (B) fluorescence spectrum of TRFM, (C) Zeta potential of TRFM and TRFM-mAb, (D) DLS of TRFM and TRFM-mAb;

[0030] Figure 3 It is the optimization result of the coupling conditions of time-resolved fluorescence microspheres and thiamphenicol monoclonal antibody; (A) Optimization of the dosage of EDC / NHS, (B) Optimization of the antibody addition amount, (C) Optimization of the coupling buffer, (D) Optimization of the pH value;

[0031] Figure 4 It is the optimization result of the conditions of the time-resolved immunochromatographic test strip; (A) Optimization of the chromatography time, (B) Optimization of the Tween-20 concentration, (C) Optimization of the antigen coating concentration, (D) Optimization of the probe addition amount;

[0032] Figure 5 It is the sensitivity analysis result of the time-resolved immunochromatographic test strip; (A) Test strip detection result, (B) Standard curve of the test strip;

[0033] Figure 6 It is the specificity analysis result of the time-resolved immunochromatographic test strip. DETAILED DESCRIPTION OF THE INVENTION

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1. Synthesis of time-resolved fluorescence microspheres and preparation of fluorescence probes

[0036] 1. Experimental materials

[0037] Thiamphenicol (TAP), florfenicol (FF), florfenicol amine (FFA), chloramphenicol (CAP), chlortetracycline (AM), and gentamicin (GM), 2-(N-morpholino)ethanesulfonic acid (MES), 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), absolute ethanol, methanol, acetonitrile, styrene, α-methylacrylic acid, europium chloride hexahydrate (EuCl3·6H2O), 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione (NTA), 1,10-phenanthroline (Phen), n-hexane, ethyl acetate, ammonium hydroxide, and anhydrous sodium sulfate were purchased from Aladdin. Bovine serum albumin (BSA) and goat anti-mouse immunoglobulin (IgG) antibody were purchased from Sigma. Anti-TAP monoclonal antibody (mAb) and antigen TAP-BSA were purchased from Kejie Biotechnology Co., Ltd. (Shenzhen, China). NC membrane (CN140), PVC rubber plate (SM31-25), sample pad (glass fiber membrane, SB08), and absorbent pad (CH27) were purchased from Shanghai Goldbio (Shanghai, China).

[0038] 2. Synthesis of time-resolved fluorescence microspheres

[0039] Weigh 692 mg of EuCl3·6H2O and dissolve it in 20 mL of absolute ethanol, and stir well to dissolve. Weigh 798 mg of NTA and 180 mg of Phen and dissolve them in 10 mL of absolute ethanol, adjust the pH value to 7.0, and gradually add the ethanol solution containing EuCl3·6H2O dropwise. Stir and react at 60 °C and 400 rpm for 6 h. Let the reaction product stand for 24 h, then carry out vacuum filtration, wash it several times with deionized water, and put it in an oven at 50 °C to dry to obtain the europium-containing fluorescent complex Eu(NTA)3Phen.

[0040] Add 50 mL of 30% ethanol solution to a three-necked flask. Using the soap-free polymerization method, under nitrogen purging, add 3 mL of styrene. After heating the reaction system to 85 °C, gradually add 0.5 mL of an aqueous sodium persulfate solution with a concentration of 80 mg / mL, and stir the reaction at 400 rpm for 3 h; add 0.5 mL of α-methylacrylic acid, and continue the reaction for 8 h under nitrogen purging; after centrifuging and filtering the reaction product, wash it twice with anhydrous ethanol, then wash it twice with deionized water, and place it in a vacuum drying oven to dry for 8 h to obtain carboxylated polystyrene microspheres.

[0041] Weigh 30 mg of europium-containing fluorescent complex and dissolve it in 1 mL of dichloromethane. Gradually add it to 10 mL of an aqueous solution of polystyrene microspheres with a concentration of 10 mg / mL, and stir the reaction at 50 °C and 400 rpm for 3 h; then perform vacuum distillation for 15 min at a water bath temperature of 45 °C to remove dichloromethane; wash the product several times with anhydrous ethanol until there is no fluorescence in the supernatant, and then wash it once with deionized water; finally, disperse the product in ultrapure water to obtain time-resolved fluorescent microspheres, and store them at 4 °C for later use.

[0042] 3. Preparation of time-resolved fluorescent microsphere-coupled antibody probe

[0043] (1) Wash the microspheres: Disperse 100 μL of time-resolved fluorescent microspheres with a concentration of 10 mg / mL in 2 mL of boric acid buffer (50 mM, pH 7.0), and centrifuge at 14000 rpm for 15 min.

[0044] (2) Activation: Sequentially add 4 μL of an EDC solution with a concentration of 50 mg / mL and 4 μL of an NHS solution with a concentration of 50 mg / mL to the microsphere solution, and shake the reaction at room temperature for 15 min.

[0045] (3) Wash the microspheres again: Disperse the time-resolved fluorescent microspheres in 2 mL of boric acid buffer (50 mM, pH 7.0), and centrifuge at 14000 rpm for 15 min, and repeat the washing twice.

[0046] (4) Coupling: Add 100 μg of chloramphenicol succinate monoclonal antibody, and shake the reaction for 2.5 h.

[0047] (5) Blocking: Add 200 μL of a 10% BSA (w / v) solution, and shake the reaction for 1 h. Centrifuge the mixture at 14000 rpm for 10 min at 4 °C.

[0048] (6) Re-dissolution: Resuspend the precipitate with 1 mL of boric acid buffer (50 mM, pH 7.0) containing 0.5 wt% sucrose, 0.05 wt% BSA, 0.09 wt% NaCl, and 0.05 wt% Tween-20. Store the resuspended solution in the dark at 4 °C for later use.

[0049] 4. Characterization of Time-Resolved Fluorescent Microspheres and Fluorescent Probes

[0050] The prepared time-resolved fluorescent microspheres and fluorescent probes were characterized. The TEM electron microscopy results are shown in A of Figure 2 . The synthesized time-resolved fluorescent microspheres were evenly distributed and had relatively uniform morphological particle sizes; the fluorescence spectrum scanning results are shown in B of Figure 2 . The maximum emission wavelength of the time-resolved fluorescent microspheres was 614 nm; the Zeta potential results are shown in C of Figure 2 . The Zeta potential of the time-resolved fluorescent microspheres was -33.0 mV, and the Zeta potential of the time-resolved fluorescent microsphere probe after coupling was -29.5 mV; the DLS results are shown in D of Figure 2 . The average particle size of the time-resolved fluorescent microsphere-coupled antibody probe was 188.4 nm, slightly larger than the particle size of the time-resolved fluorescent microspheres before coupling, which was 179.4 nm, indicating that thiamphenicol monoclonal antibody was coupled to the surface of the time-resolved fluorescent microspheres. Based on the experimental results of the comprehensive characterization, time-resolved fluorescent microspheres with good uniformity and dispersibility were successfully synthesized, and time-resolved fluorescent microsphere-labeled antibody probes were successfully coupled.

[0051] Example 2. Establishment of a Time-Resolved Fluorescent Immunoassay for Thiamphenicol

[0052] 1. Assembly of the Time-Resolved Fluorescent Immunoassay Strip

[0053] Thiamphenicol antigen TAP-BSA (0.4 mg / mL) and goat anti-mouse IgG (0.05 mg / mL) were sequentially sprayed onto the detection line T and the quality control line C positions on the NC membrane with a spraying volume of 0.5 μL / cm using a membrane scribing instrument. The scribed NC membrane was placed in an oven at 37 °C and dried overnight. The sample pad was fully soaked in a 2% BSA solution and then dried for later use. The dried NC membrane, sample pad, and absorbent pad were sequentially adhered to the PVC bottom plate, and the sample pad and absorbent pad each pressed the NC membrane by 1 mm at both ends. The assembled bottom plate was cut into test strips with a width of 4 mm using a strip cutting machine and placed in a dry and sealed aluminum foil bag for later use.

[0054] The time-resolved fluorescent immunoassay strip of the present invention can be used for qualitative determination or quantitative detection of thiamphenicol in the sample to be detected, and can be carried out according to the following procedure:

[0055] The sample to be detected is mixed with the time-resolved fluorescent microsphere-labeled antibody probe and then added to the sample pad of the test strip. Subsequently, qualitative determination or quantitative detection is carried out according to the following method:

[0056] Qualitative determination method: Observe under an ultraviolet lamp. If both the test line T and the control line C show color, it is a negative sample without thiamphenicol; if the T line has no color and the C line has color, it is a positive sample containing thiamphenicol.

[0057] Quantitative determination method: Take a photo under ultraviolet light to measure the signal value of the test line T, and perform quantitative detection according to the standard curve.

[0058] 2. Optimization of time-resolved fluorescence immunochromatography method

[0059] 2.1. Optimization of the dosage of EDC / NHS

[0060] When coupling the probe, add 0.25 μL, 0.5 μL, 1 μL, 2 μL, 4 μL, 8 μL, and 16 μL of EDC and NHS solutions (50 mg / mL) respectively. Other reaction conditions are as described above. After coupling, perform immunochromatographic analysis. The results are as Figure 3 shown in A of [reference]. As the dosage of EDC / NHS increases, the signal value of the T line gradually increases. However, when the added volume exceeds 4 μL, microsphere aggregation begins to occur. Therefore, the final dosage of EDC / NHS selected is 4 μL each.

[0061] 2.2. Optimization of the antibody addition amount

[0062] Under the same other conditions, the antibody addition amount during coupling was optimized. The antibody addition amounts were set to 12 μg, 25 μg, 50 μg, 100 μg, 200 μg, and 400 μg respectively. The results are as Figure 3 shown in B of [reference]. As the antibody addition amount increases, the signal value of the T line gradually increases. When the antibody addition amount increases to 100 μg, the signal value of the T line reaches the peak. Therefore, 100 μg is selected as the optimal antibody addition amount.

[0063] 2.3. Optimization of the coupling buffer

[0064] The coupling buffer was optimized, and five commonly used coupling buffers (BB, HEPES, MES, H2O, Tris-HCl) were selected for coupling. The results are as Figure 3 shown in C of [reference]. When using BB buffer as the coupling buffer, the signal value of the T line is the highest. Therefore, boric acid is finally selected as the coupling buffer.

[0065] 2.4. Optimization of the pH value

[0066] Coupling was performed using boric acid buffers with pH values of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 respectively, with the same other conditions. The results are as Figure 3 shown in D of [reference]. When the pH is 7.0, the signal value of the test line T is the strongest. Therefore, the optimal pH of the boric acid buffer finally selected is 7.0.

[0067] 2.5. Optimization of chromatography time

[0068] After sample loading, starting from the 1st minute, take pictures every 2 minutes to measure the signal values of the test line T and the control line C of the test strip. As shown in A of Figure 4 As time increases, the signal value of the T line gradually increases. When the time exceeds 10 minutes, the signal value tends to balance. Therefore, the final chromatography time of the test strip is selected as 10 minutes.

[0069] 2.6. Optimization of Tween-20 concentration

[0070] The concentration of Tween-20 in the loading buffer was optimized. 0%, 1.5wt%, 3wt%, 4.5wt%, 6wt%, 7.5wt% and 9wt% of Tween-20 were added to the loading buffer respectively. After reacting for 10 minutes, take pictures and measure the signal values of the test line T and the control line C of each test strip respectively. The results are shown in B of Figure 4 When the loading solution contains 3wt% of Tween-20, the signal value of the test line T is relatively high, and the ratio of the signal values of the test line T and the control line C is closest to 1. Therefore, 3wt% is selected as the optimal concentration of Tween-20.

[0071] 2.7. Optimization of antigen coating concentration

[0072] Spray different concentrations of TAP-BSA antigen at the test line T of the test strip: 0.1mg / mL, 0.2mg / mL, 0.4mg / mL, 0.8mg / mL and 1.6mg / mL respectively. The results are shown in C of Figure 4 When the antigen concentration is 0.8mg / mL, the ratio of the signal values of the test line T and the control line C is closest to 1. Therefore, the optimal concentration of the coated antigen is selected as 0.8mg / mL.

[0073] 2.8. Optimization of probe addition amount

[0074] Add different amounts of time-resolved fluorescence microsphere probes (concentration 1mg / mL) respectively. The results are shown in D of Figure 4 As the probe addition amount increases, the signal value of the T line gradually increases. When the addition amount reaches 5μL, the signal value of the T line tends to balance, and at this time, the ratio of the signal value of the test line T and the control line C is closest to 1. Therefore, 5μL is selected as the optimal addition amount of the probe.

[0075] 3. Method sensitivity

[0076] The thiamphenicol standard was diluted to 0, 0.01 ng / mL, 0.05 ng / mL, 0.20 ng / mL, 0.78 ng / mL, 3.13 ng / mL, 12.5 ng / mL, 50 ng / mL, and 200 ng / mL respectively. After mixing with the time-resolved fluorescence microsphere-labeled antibody probe, it was added to the sample pad of the test strip. Subsequently, the signal value of the T line was measured, and each gradient was repeated 3 times. With the concentration of thiamphenicol as the abscissa and B / B0 as the ordinate, a standard curve was fitted. The specific steps were as follows:

[0077] (1) Pipette 5 μL of the time-resolved fluorescence microsphere probe (concentration 1 mg / mL) into 100 μL of thiamphenicol standard solutions with different concentrations, pipette and mix repeatedly, and incubate at 37 °C for 5 min;

[0078] (2) Pipette the mixed solution of the fluorescence microsphere probe and the thiamphenicol standard and add it to the sample pad of the test strip, and react for 10 min;

[0079] (3) Place the test strip in a dark box, take a photo with a smartphone under an ultraviolet lamp, convert the fluorescence intensity of the T line into an R value through RGB color analysis. With the ratio of the T line signal value (B / B0) as the ordinate and the concentration of the thiamphenicol standard as the abscissa, a standard curve was established.

[0080] The results were as Figure 5 shown. The linear equation of the standard curve established in the present invention was Y = -21.2185lg(X) + 52.2462, and the IC 50 was 1.28 ng / mL, the linear range was 0.05 - 33.09 ng / mL, and the detection limit was 0.02 ng / mL.

[0081] 4. Method specificity

[0082] To verify the specificity of the established method, several other common antibiotics (florfenicol, florfenicol amine, chloramphenicol, chlortetracycline, and gentamicin) were detected, and the concentration of each antibiotic was 200 ng / mL. The results were as Figure 6 shown. The time-resolved fluorescence immunoassay method for thiamphenicol established in the present invention had a cross-reaction with florfenicol, an analogue of thiamphenicol, and no cross-reaction with the other several antibiotics.

[0083] 5. Sample detection

[0084] 5.1 Sample pretreatment

[0085] Weigh 6 g of the samples (pork, chicken, fish) into centrifuge tubes respectively, add 18 mL of ethyl acetate, mix well by shaking, centrifuge at 4000 rpm for 5 min, take 12 mL of the supernatant, rotary evaporate until nearly dry, dissolve with 1 mL of n - hexane, then add 3 mL of PBS solution (10 mM) for extraction, centrifuge at 4000 rpm for 5 min, take the PBS solution layer, filter through a 0.45 - μm filter membrane and store at 4°C for later use.

[0086] Weigh 5 g of milk and dissolve it in 20 mL of ethyl acetate, mix well by shaking, centrifuge at 4000 rpm for 5 min, take the supernatant and place it in a pear - shaped flask. Add 20 mL of ethyl acetate to the residue and repeat the extraction once. Combine the two extraction solutions and rotary evaporate them in a water bath at 45°C until nearly dry. Add 5 mL of water to the pear - shaped flask, sonicate for 5 min to dissolve it completely, transfer it to a centrifuge tube, and then add 5 mL of water to dissolve it again. Add 20 mL of n - hexane, shake and react for 5 min, centrifuge at 4000 rpm for 2 min, take the lower - layer solution, filter through a 0.45 - μm filter membrane and store at 4°C for later use.

[0087] Weigh 5 g of the homogenized egg sample into a centrifuge tube, add 20 mL of ethyl acetate, 0.9 mL of ammonium hydroxide and 5 g of anhydrous sodium sulfate in sequence, shake and extract for 30 s, centrifuge at 4000 rpm for 5 min, transfer the supernatant to another centrifuge tube, shake well, take the ethyl acetate extract and put it into a pear - shaped flask, place it in a water bath at 45°C and rotary evaporate until nearly dry. Add 5 mL of water to dissolve the residue in the pear - shaped flask, sonicate for 5 min, add 3 mL of n - hexane and vortex - mix for 30 s, let it stand for layer separation, discard the upper - layer n - hexane solution, add 3 mL of n - hexane again and vortex - mix for 30 s, let it stand for layer separation, transfer the aqueous phase to a centrifuge tube, centrifuge at 8000 rpm for 5 min, filter through a 0.45 - μm filter membrane and store at 4°C for later use.

[0088] 5.2 Spike recovery test

[0089] To verify the reliability of the established time - resolved fluorescence immunochromatography method in the detection of actual samples, pork, chicken, fish, milk and egg samples with different spiked amounts (10, 25 and 50 μg / kg) were detected by immunochromatography and high - performance liquid chromatography respectively. Before immunochromatographic analysis, the sample extract was moderately diluted.

[0090] The results are shown in Table 1. The spike recoveries of thiamphenicol in food samples were 92.48% - 115.10%, and the coefficient of variation (CV) was 2.79% - 12.77%. Both the recovery rate and the coefficient of variation were within a reasonable range, and the detection results were consistent with those of high - performance liquid chromatography. The results indicate that the time - resolved fluorescence immunochromatography method for thiamphenicol described in the present invention has good accuracy and can be applied to the rapid detection of thiamphenicol in actual samples.

[0091] Table 1 - Results of spike recovery experiment

[0092]

[0093] The above - described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol, comprising a test strip and a probe. The test strip includes a base plate, a sample pad, a nitrocellulose membrane, and a water-absorbing pad. The water-absorbing pad, the nitrocellulose membrane, and the sample pad are sequentially pasted on the base plate from top to bottom along the length direction of the test strip, and the sample pad and the water-absorbing pad each press a certain length of the nitrocellulose membrane. It is characterized in that On the detection area of the nitrocellulose membrane, a T line and a C line are provided. The T line is coated with thiamphenicol antigen, and the C line is coated with goat anti-mouse IgG antibody. The probe is a time-resolved fluorescence microsphere-labeled antibody probe. The preparation method of the probe includes: embedding europium complex in polystyrene microspheres to prepare time-resolved fluorescence microspheres; chemically conjugating the time-resolved fluorescence microspheres with thiamphenicol monoclonal antibody to obtain the probe.

2. The time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol according to claim 1, wherein The embedding of europium complex in polystyrene microspheres to prepare time-resolved fluorescence microspheres includes the following steps: 1) Dissolve europium chloride hexahydrate, 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione, and o-phenanthroline in absolute ethanol, and heat and stir to synthesize europium-containing fluorescent complex Eu(NTA)3Phen. 2) Under nitrogen protection, dissolve styrene, ammonium persulfate, and α-methylacrylic acid in an ethanol aqueous solution, and heat and stir to synthesize polystyrene microspheres. 3) Embed the europium-containing fluorescent complex synthesized in step 1) into the polystyrene microspheres synthesized in step 2) to prepare time-resolved fluorescence microspheres, wash with absolute ethanol and deionized water, and resuspend with ultrapure water for later use.

3. The time-resolved fluorescence immunoassay chromatographic kit for detecting thiamphenicol according to claim 2, wherein: The molar ratio of europium chloride hexahydrate, 4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione, and o-phenanthroline is 1:3:

1.

4. The time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol according to claim 1, characterized in that, The chemical conjugation of the time-resolved fluorescence microspheres with thiamphenicol monoclonal antibody to obtain the probe includes the following steps: After washing the time-resolved fluorescence microspheres with boric acid buffer solution, add EDC and NHS for activation respectively. After activation, add thiamphenicol monoclonal antibody for conjugation, then add bovine serum albumin for blocking. After centrifugation, the precipitate is resuspended with boric acid buffer solution containing 0.5 wt% sucrose, 0.05 wt% bovine serum albumin, 0.09 wt% NaCl, and 0.05 wt% Tween-20 to obtain the time-resolved fluorescence microsphere-conjugated antibody probe. The mass ratio of the time-resolved fluorescence microspheres to the thiamphenicol monoclonal antibody is 10:

1.

5. The time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol according to claim 4, characterized in that, The pH of the boric acid buffer solution for washing the time-resolved fluorescence microspheres is 7.0, and the concentration is 50 mM. The concentrations of EDC and NHS are both 50 mg / mL, and the addition amounts are both 4 μL. The addition amount of the thiamphenicol monoclonal antibody is 100 μg.

6. The time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol according to claim 1, characterized in that: The test strip is prepared by the following method: Spray the positions of the detection line T line and the quality control line C line on the nitrocellulose membrane with thiamphenicol antigen TAP-BSA and goat anti-mouse IgG antibody in sequence using a membrane scribing instrument, dry the scribed nitrocellulose membrane, and stick it to the base plate with the sample pad and the water-absorbing pad in sequence.

7. The time-resolved fluorescence immunochromatographic kit for detecting thiamphenicol according to claim 6, characterized in that: The concentration of the thiamphenicol antigen TAP-BSA is 0.8 mg / mL; the sample pad and the water-absorbing pad each press 1 mm of the nitrocellulose membrane at both ends; cut into test strips with a width of 4 mm.

8. A time-resolved fluorescence immunochromatographic method for detecting thiamphenicol based on the kit according to any one of claims 1-7, characterized in that, Including the following steps: a) Mix the thiamphenicol standard solutions with different concentrations with the time-resolved fluorescence microsphere-labeled antibody probes respectively, then add them to the sample pad of the test strip. Subsequently, measure the signal value of the T line. Use the concentration of the thiamphenicol standard as the abscissa and the ratio of the signal value of the T line at different standard concentrations to the blank control as the ordinate to establish a standard curve; b) Mix the sample to be detected with the time-resolved fluorescence microsphere-labeled antibody probes and then add them to the sample pad of the test strip. Subsequently, conduct qualitative judgment or quantitative detection according to the following methods: Qualitative determination method: Observe under an ultraviolet lamp. If both the test line T line and the control line C line have colors, it is a negative sample without thiamphenicol; if the T line has no color and the C line has color, it is a positive sample containing thiamphenicol; Quantitative determination method: Take a photo under an ultraviolet lamp to measure the signal value of the test line T line, and conduct quantitative detection based on the standard curve established in step a).

9. The time-resolved fluorescence immunochromatography method according to claim 8, characterized in that, The specific steps of step a) include: a1) Pipette 5 μL of the time-resolved fluorescence microsphere probe into each group and add them to 100 μL of thiamphenicol standard solutions with different concentrations respectively. Pipette and mix repeatedly, and incubate at 37 °C for 5 min; a2) Pipette the mixed solution of the probe and the standard and add it to the sample pad of the test strip, and react for 5 - 10 min; a3) Place the test strip under an ultraviolet lamp to take a photo, read the signal intensity value of the T line, compare it with the concentration of the thiamphenicol standard. Use the concentration of the thiamphenicol standard as the abscissa and the ratio of the signal value of the T line at different standard concentrations to the blank control as the ordinate to establish a standard curve.

10. An application for detecting thiamphenicol, characterized in that, Use the time-resolved fluorescence immunochromatography kit according to any one of claims 1 - 7 to detect thiamphenicol in food samples.

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