Thiamphenicol hapten, artificial antigen, antibody, and preparation and application thereof

Abstract

The present invention discloses a thiamphenicol hapten, an artificial antigen, an antibody, and a preparation method and application thereof. The thiamphenicol hapten structural formula is shown in formula (I). An artificial antigen is prepared based on the hapten to immunize an animal to obtain a monoclonal antibody; a thiamphenicol colloidal gold immunochromatographic test strip is prepared based on the antibody to detect the IC value of thiamphenicol. 50 The detection limit of the present invention is 0.70 ng / mL, the linear range is 0.16-3.17 ng / mL, and the minimum detection limit is 0.06 ng / mL, which meets the limit of thiamphenicol in GB 31650-2019. It shows that the artificial antigen prepared by the present invention has good immunogenicity, can be used to prepare thiamphenicol monoclonal antibodies and establish corresponding immunoassay methods, and is suitable for on-site high-throughput rapid detection of thiamphenicol in food. The antigen and antibody preparation process of the present invention are simple, low in cost, highly sensitive, and have a low detection limit, and have good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and more specifically to a thiamphenicol hapten, an artificial antigen, an antibody, and a preparation method and application thereof. Background Art

[0002] Thiamphenicol (TAP) is a second-generation chloramphenicol-type broad-spectrum antibacterial drug and an amide antibiotic. As an alternative to chloramphenicol, thiamphenicol overcomes the potentially fatal side effects of chloramphenicol, such as bone marrow suppression, aplastic anemia, and gray baby syndrome. It is commonly used in animal husbandry to treat and prevent bacterial infections and promote animal growth. However, thiamphenicol still has strong hemotoxicity, embryotoxicity, and immunotoxicity, inhibiting immunoglobulin synthesis and antibody production in the body, thereby impairing human immune function. According to my country's GB 31650-2019 "National Standard for Maximum Residue Limits of Veterinary Drugs in Food," thiamphenicol is a restricted drug and is prohibited from use during the egg-laying period.

[0003] In order to protect environmental safety and human life and health, it is urgent to establish a rapid and efficient method for detecting thiamphenicol.

[0004] At present, the detection methods of thiamphenicol are mainly instrumental analysis methods, including: gas chromatography, liquid chromatography tandem mass spectrometry, chromatography-mass spectrometry and surface enhanced Raman spectroscopy. Such methods are expensive and cumbersome to operate, which limits their use in high-throughput screening on site. Immunoassay methods have the advantages of low cost and high throughput, and complement the advantages of large-scale instrument confirmation methods, and have gradually become the mainstream technology for detecting veterinary drug residues. For immunoassays, antibodies are the core raw materials, and the effect of antibodies depends to a large extent on the antigen structure that causes the corresponding animals to have an immune response. Only by synthesizing and preparing suitable thiamphenicol haptens can antibodies with strong specificity and high sensitivity be obtained. Although Chinese patent CN 111592476 A has disclosed a thiamphenicol hapten and a thiamphenicol artificial antigen, and obtained monoclonal antibodies through animal immunization, and used chemiluminescence enzyme-linked immunosorbent assay to determine the IC of the antibody. 50 The value is 0.26 ng / mL, and the minimum detection limit is 0.1 ng / mL. However, this hapten is prepared in three steps, and the entire detection process takes approximately 50 minutes, making it unsuitable for high-throughput rapid testing on-site. Furthermore, the number of artificial antigens and antibodies currently available for thiamphenicol detection is relatively small. Therefore, there is a need for more rapid detection methods that can provide haptens, antigens, and antibodies for thiamphenicol immunoassays. This is of great significance for the rapid, sensitive, and high-throughput detection of thiamphenicol. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a thiamphenicol hapten.

[0006] The second object of the present invention is to provide a method for preparing the thiamphenicol hapten.

[0007] The third object of the present invention is to provide the application of the thiamphenicol hapten.

[0008] The fourth object of the present invention is to provide an artificial thiamphenicol antigen.

[0009] The fifth object of the present invention is to provide the application of the thiamphenicol artificial antigen.

[0010] The sixth object of the present invention is to provide a thiamphenicol antibody.

[0011] The seventh object of the present invention is to provide the application of the thiamphenicol antibody.

[0012] The eighth object of the present invention is to provide a rapid immunoassay method and colloidal gold immunochromatographic test strip for detecting thiamphenicol.

[0013] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0014] A thiamphenicol hapten, whose structural formula is shown in formula (I):

[0015]

[0016] The thiamphenicol hapten of the present invention introduces a polar group on the basis of the thiamphenicol molecule and extends the spacer arm, hydrolyzes the amide bond of the thiamphenicol by potassium hydroxide to generate an amino group, and then undergoes an amide condensation reaction with succinic anhydride to introduce a four-carbon spacer arm and a carboxyl group, thereby obtaining the designed hapten.

[0017] The method for preparing the thiamphenicol hapten of the present invention comprises the following steps:

[0018] S1. Thiamphenicol and potassium hydroxide are added to an organic solution, heated to reflux, and the organic solvent is removed to obtain product I;

[0019] S2. Product I is dissolved in N,N-dimethylformamide (DMF), and succinic anhydride is added to react with stirring. After the reaction is completed, the mixture is separated and the organic solvent is removed to obtain a white powdery product II, i.e., thiamphenicol hapten.

[0020]

[0021] Preferably, in step S1, the organic solvent is an aqueous solution containing 50% methanol.

[0022] Preferably, in step S1, the molar ratio of thiamphenicol to potassium hydroxide is 1:1 to 1.2.

[0023] Preferably, in step S1, the reaction conditions are 70-90° C. for 1-2 h.

[0024] Preferably, in step S2, the molar ratio of thiamphenicol to succinic anhydride is 1:1.1-1.5.

[0025] Preferably, in step S2, the reaction conditions are 20-25° C. and the reaction time is 12-24 hours.

[0026] Preferably, in step S2, the separation is performed by 200-300 mesh silica gel column chromatography (ethyl acetate: petroleum ether = 1:1)

[0027] Preferably, in steps S1 and S2, the organic solvent is removed by rotary evaporation.

[0028] As a preferred embodiment, the method for preparing the thiamphenicol hapten comprises the following steps: thiamphenicol (710 mg, 2 mmol) and potassium hydroxide (112 mg, 2 mmol) are weighed and dissolved in 6 mL of a 1:1 methanol / water mixture, and the mixture is reacted at 80°C for 2 hours. After the reaction, the mixture is extracted with ethyl acetate (50 mL x 3), the organic phases are combined, and the solvent is evaporated to dryness to obtain a pale yellow powder, namely, Product I. Product I (245 mg, 1 mmol) and succinic anhydride (120 mg, 1.2 mmol) are dissolved in 2 mL of N,N-dimethylformamide (DMF), and the mixture is stirred and reacted at room temperature for 24 hours. After the reaction, the mixture is extracted with ethyl acetate (50 mL x 3), the organic phases are combined, the solvent is evaporated to dryness, and the mixture is separated by chromatography on a 200-300 mesh silica gel column (ethyl acetate:petroleum ether = 1:1) to obtain a white powder, namely, the thiamphenicol hapten.

[0029] The present invention also provides the use of the thiamphenicol hapten of formula (I) in the preparation of thiamphenicol artificial antigen.

[0030] A thiamphenicol artificial antigen is obtained by coupling the thiamphenicol hapten of formula (I) with a carrier protein. The structural formula of the thiamphenicol artificial antigen is shown in formula (II):

[0031]

[0032] Preferably, the carrier protein is lactoferrin (LF) or hen ovalbumin (OVA), LF is used to prepare the immunogen, and OVA is used to prepare the coating.

[0033] Preferably, the coupling method is the active ester method.

[0034] As a preferred embodiment, the method for preparing the artificial thiamphenicol antigen comprises the following steps: dissolving 5 mg of thiamphenicol hapten, 10 mg of EDC and 5 mg of NHS in 200 μL of DMF, stirring overnight at 4°C, and taking the supernatant after centrifugation as liquid A. Weigh 10 mg of carrier protein and dissolve it in 2 mL of coating buffer solution to obtain liquid B. Under magnetic stirring, liquid A is slowly added dropwise to liquid B, and the reaction is stirred at 4°C for 12 hours. After centrifugation, the supernatant is taken and placed in a dialysis bag. It is dialyzed with PBS (0.01 M, pH 7.4) at 4°C for 3 days, and the dialysate is changed 3 times a day to obtain the artificial antigen, which is stored at -20°C for use.

[0035] The present invention also provides a thiamphenicol antibody, which is prepared using the thiamphenicol artificial antigen described in formula (II) as an immunogen. The antibody includes but is not limited to monoclonal antibodies, polyclonal antibodies, nanobodies, etc.

[0036] Preferably, the antibody is a thiamphenicol monoclonal antibody, using a thiamphenicol artificial antigen with lactoferrin (LF) as a carrier protein as the immunogen; female Balb / C mice of appropriate age are selected for immunization, and the anti-serum titer and inhibition rate are measured after the fourth and fifth immunizations, and a shock immunization is performed 3 days before cell fusion; spleen cells of the mice after the shock immunization are taken and fused with myeloma cells, hybridoma cells that can secrete specific antibodies are screened, and the expanded culture is carried out; the expanded cultured hybridoma cells are injected into mice that have been injected with paraffin in advance, and the ascites is collected and purified to obtain monoclonal antibodies.

[0037] The present invention also provides application of the thiamphenicol artificial antigen and thiamphenicol antibody in immunoassay detection of thiamphenicol.

[0038] The present invention also provides a rapid immunoassay method for detecting thiamphenicol, which uses the thiamphenicol artificial antigen whose carrier protein is chicken ovalbumin as the coating source, and the antibody prepared by immunizing an animal with the thiamphenicol artificial antigen whose carrier protein is lactoferrin as the immunogen as the detection antibody for detection.

[0039] The present invention also provides a colloidal gold immunochromatographic test strip for detecting thiamphenicol, which uses the thiamphenicol artificial antigen whose carrier protein is chicken ovalbumin as a coating source, and the antibody prepared by immunizing an animal with the thiamphenicol artificial antigen whose carrier protein is lactoferrin as an immunogen as a detection antibody.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention first provides a thiamphenicol hapten, whose structural formula is shown in formula (I). An artificial antigen is prepared based on the hapten, and then an animal is immunized with the artificial antigen and a monoclonal antibody against thiamphenicol is prepared by combining hybridoma technology. A colloidal gold immunochromatographic method for detecting thiamphenicol is established based on the antibody, and the IC value of thiamphenicol is 0.04. 50 The concentration of the artificial antigen prepared by the present invention is 0.70 ng / mL, with a linear range of 0.16 to 3.17 ng / mL and a minimum detection limit of 0.06 ng / mL. The entire detection process takes only 10 minutes, indicating that the artificial antigen prepared by the present invention has good immunogenicity and can be used to prepare monoclonal antibodies to thiamphenicol and establish corresponding immunoassay methods, suitable for on-site high-throughput rapid determination of thiamphenicol in food. The antigen and antibody preparation process of the present invention is simple, low-cost, and has a lower detection limit, showing good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a mass spectrometry identification diagram of the hapten of thiamphenicol formula (I) of the present invention;

[0043] Figure 2 This is an ultraviolet scan of the hapten, carrier protein and artificial antigen of the present invention;

[0044] Figure 3 The figure is a standard curve of the indirect competition ELISA of the thiamphenicol monoclonal antibody of the present invention.

[0045] Figure 4 The figure is a standard curve diagram of the thiamphenicol colloidal gold immunochromatographic test strip of the present invention. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0048] Example 1 Preparation of Thiamphenicol Hapten

[0049] (1) Thiamphenicol (710 mg, 2 mmol) and potassium hydroxide (112 mg, 2 mmol) were dissolved in 6 mL of a 1:1 methanol / water mixture and reacted at 80°C for 2 h. After the reaction, the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and the solvent was evaporated to dryness to obtain a light yellow powder, i.e., Product I.

[0050] (2) Product I (245 mg, 1 mmol) and succinic anhydride (120 mg, 1.2 mmol) were weighed and dissolved in 2 mL of N,N-dimethylformamide (DMF). The mixture was stirred at room temperature for 24 h. After the reaction, the solvent was evaporated and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, the solvent was evaporated, and the mixture was separated by 200-300 mesh silica gel column chromatography (ethyl acetate: petroleum ether = 2:1) to obtain a white solid, i.e., product II.

[0051] The chemical reaction equation above is as follows:

[0052]

[0053] The ESI-MS spectrum of product 2 is as follows Figure 1 As shown, ESI-MS analysis (negative) m / z 344.4; 1 HNMR (400MHz, d6-DMSO)δ7.97(d,J=0.6Hz,1H),7.75(m,2H),7.37(m,2H),5.81(d,J=5.0Hz,1H),5.14(dt,J=4.9,1.0,1.0Hz,1H),4.83(t,J=5.5,5.5Hz,1H),4.60(d,J=0.6Hz,1H),3.81(dd,J=12.4,5.5Hz,1H),3.31(dd,J=12.5,5.6Hz,1H),3.28(s,3H),2.49(m,4H), indicating that the thiamphenicol hapten represented by formula (I) was successfully prepared.

[0054] Example 2 Preparation and Identification of Thiamphenicol Artificial Antigen

[0055] (1) Immunogen preparation

[0056] Dissolve 5 mg of Formula I hapten, 10 mg of EDC, and 5 mg of NHS in 200 μL of DMF, stir overnight at 4°C, and centrifuge. The supernatant is referred to as Solution A. Weigh 10 mg of lactoferrin (LF) and dissolve it in 2 mL of coating buffer to form Solution B. Under magnetic stirring, slowly add Solution A dropwise to Solution B. Stir and react at 4°C for 12 hours. After centrifugation, remove the supernatant, transfer it to a dialysis bag, and dialyze against PBS (0.01 M, pH 7.4) at 4°C for 3 days, changing the dialysate three times daily to obtain the immunogen. Adjust the concentration to 1 mg / mL with PBS, aliquot 500 μL into 1.5 mL centrifuge tubes, and store at -20°C until needed.

[0057] (2) Preparation of coating material

[0058] Dissolve 5 mg of Formula I hapten, 10 mg of EDC, and 5 mg of NHS in 200 μL of DMF, stir overnight at 4°C, and centrifuge to obtain the supernatant (Solution A). Weigh 10 mg of hen ovalbumin (OVA) and dissolve each in 2 mL of coating buffer (Solution B). Under magnetic stirring, slowly add Solution A dropwise to Solution B. Stir and react at 4°C for 12 hours. After centrifugation, remove the supernatant, transfer it to a dialysis bag, and dialyze against PBS (0.01 M, pH 7.4) at 4°C for 3 days, changing the dialysate three times daily to obtain the coating source. Adjust the concentration to 1 mg / mL with PBS, aliquot 500 μL into 1.5 mL centrifuge tubes, and store at -20°C until needed.

[0059] The hapten prepared in Example 1 and the artificial antigen prepared in Example 2 were respectively subjected to ultraviolet wavelength scanning (150-400 nm) for identification. Figure 2 As shown, the maximum absorption peak of the conjugate thiamphenicol hapten-carrier protein has changed compared with the position and characteristic peak of the maximum absorption peak of thiamphenicol hapten and carrier protein, indicating that the synthesis of thiamphenicol hapten-carrier protein is successful.

[0060] Example 3 Preparation of monoclonal antibodies to thiamphenicol artificial antigen

[0061] Animal immunization: The prepared immunogen was fully emulsified with an equal amount of Freund's complete adjuvant and injected subcutaneously into the abdomen of 5-week-old BALB / c mice, 0.20 mL per mouse. Starting from the first immunization, booster immunizations were performed at weeks 2, 4, and 6 using Freund's incomplete adjuvant instead of Freund's complete adjuvant. The method and dosage were the same as those for the first immunization.

[0062] Cell fusion: Splenocytes from immunized mice were mixed with SP2 / 0 myeloma cells. 1 mL of warmed PEG2000 was added dropwise over 1 minute to initiate fusion. HAT culture medium was added and shaken evenly. The cells were then added to eight pre-prepared 96-well culture plates containing feeder cells and cultured in a 37°C, 5% CO2 incubator. On the 5th day, the medium was half-changed with HT culture medium, and on the 8th day, the medium was fully changed with complete culture medium. On the 10th day, 120 μL of cell supernatant was collected from each well and analyzed using an indirect competitive enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0063] (1) Coating: Dilute the coating agent to 1000 ng / mL with coating buffer and add 100 μL per well to the microwells of a 96-well ELISA plate. Incubate in a 37°C water bath overnight. Pour the liquid from the wells and add 300 μL of PBST to each well. Wash twice and spin dry. Add 120 μL of blocking solution to each well and block in a 37°C water bath for 3 h. Spin dry the liquid in the wells and dry in a 37°C oven. After 1 h, remove from the wells and store at 4°C for later use.

[0064] (2) Cell supernatant titer and inhibition assay: For the ELISA plate prepared in step (1), add 50 μL of PBS and 50 μL of cell supernatant to each well of the titer column; for the inhibition column, add 50 μL of diluted 1000 ng / mL drug (thiamphenicol) and 50 μL of cell supernatant to each well. Incubate at 37°C for 40 min, wash five times with PBST, pat dry the wells, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP), incubate at 37°C for 30 min, wash five times with PBST, pat dry the wells, add 100 μL of TMB substrate solution, and develop at 37°C in the dark for 10 min; add 50 μL of stop solution (10% H2SO4) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader.

[0065] Coating buffer: pH 9.6, 0.1 M sodium carbonate-sodium bicarbonate buffer (the solvent is distilled water, and the solutes and their concentrations are as follows: Na2CO3 1.65 g / L and NaHCO3 2.65 g / L).

[0066] PBST: 0.01M PBS and 0.06% Tween-20 (v / v) (solvent is distilled water)

[0067] Blocking solution: phosphate buffer containing 1% (volume percentage) skimmed milk powder, pH 7.4.

[0068] (3) Screening of positive hybridomas: Select positive hybridoma cells with high titer and strong drug inhibition for the next step of limiting dilution. Under sterile conditions, use a microscope to pick out the cells growing in clusters in the positive wells and transfer them to a 96-well culture plate pre-plated with feeder cells. Each original well is cloned into 8 wells. After the cells adhere to the wall and cover 1 / 2 to 1 / 3 of the bottom of the well, take the supernatant and perform icELISA detection. Also using titer and inhibition rate as measurement indicators, take the strong positive ones and use the limiting dilution method for subcloning. Repeat this 3-4 times until each well on each plate is positive and the titer / inhibition rate is similar after detection. At this point, the hybridoma cell line is successfully established, and a hybridoma cell line that can stably secrete uniform antibodies is obtained. Pick single cell clones, and those that test positive are transferred to 24-well cell culture plates or cell culture dishes for expansion and freezing in time.

[0069] (4) Mass preparation of monoclonal antibodies: Use indirect competitive enzyme-linked immunosorbent assay to measure the cell supernatant and screen the positive wells. Use limiting dilution method to subclone the positive wells and screen out single cells that can stably secrete uniform antibodies. Expand the culture. The specific steps are as follows:

[0070] Inject 500 μL of liquid paraffin into the peritoneal cavity of Balb / c mice aged 8 weeks or more. After 7-10 days, transfer the expanded hybridoma cells into 2 mL of culture medium and dilute the cells to 2 × 10 6Cells were expressed in 96 cells / mL. 0.5 mL of the cell suspension was injected into the peritoneal cavity of mice until the abdominal cavity was distended, the fur was frizzy and dull, and the mice were in a state of low spirits. The mice were then killed by cervical dislocation and the ascites collected. The ascites were centrifuged at 12,000 rpm for 15 minutes at 4°C to remove the upper fat layer and impurities below. The clear, pale yellow fluid in the middle was collected and the potency and inhibitory rate were determined using the icELISA assay. After purification, the fluid was stored at -20°C for future use.

[0071] Example 4 Sensitivity determination of thiamphenicol monoclonal antibody

[0072] (1) Experimental methods

[0073] To the ELISA plate prepared in step (1) of Example 3, add a series of 50 μL of thiamphenicol standard and 50 μL of thiamphenicol monoclonal antibody (5.86 ng / mL) of different concentrations to each well, incubate at 37°C for 40 min, wash five times with PBST, pat dry the liquid in the wells, add 1:5000 diluted enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP), incubate at 37°C for 40 min, wash five times with PBST, pat dry the liquid in the wells, add 100 μL of TMB substrate solution, develop at 37°C in the dark for 10 min; add 50 μL of stop solution (2M H2SO4) to terminate the reaction; read the absorbance at 450 nm using an ELISA reader. The concentration of thiamphenicol standard is plotted against the horizontal axis, B / B0 (OD value of the wells with thiamphenicol added) 450 / OD of the well without thiamphenicol 450 ) as the vertical axis, and establish an indirect competition standard curve.

[0074] (2) Experimental results

[0075] The standard curve of indirect competitive ELISA established based on monoclonal antibodies is shown in the figure below. Figure 3 As shown in the figure, it can be seen that the standard curve is S-shaped and has a good linear correlation. 50 The detection sensitivity is high, with a minimum detection limit of 0.1 ng / mL and a linear range of 0.20-2.42 ng / mL.

[0076] Example 5 Preparation of colloidal gold immunochromatographic test strips for thiamphenicol monoclonal antibodies

[0077] (1) Preparation of colloidal gold solution

[0078] Take 500 mL of double-distilled deionized water and place it in a round-bottom flask. Heat it to boiling using a constant temperature electromagnetic stirrer. Quickly add 5 mL of 1% chloroauric acid solution. After boiling again, add 20 mL of 1% trisodium citrate solution. Keep boiling for 15 minutes. Stop heating when the color changes from purple to clear and transparent wine red and no longer changes. Cool to room temperature to obtain a colloidal gold solution, which is stored at 4°C.

[0079] (2) Preparation of thiamphenicol monoclonal antibody-colloidal gold probe

[0080] Under magnetic stirring, 1 mL of colloidal gold solution was added with an appropriate amount of 0.2 mol / L K2CO3 solution, the pH was adjusted to 7.4, 3-5 μg of the thiamphenicol monoclonal antibody prepared in step (4) of Example 3 was added, and the mixture was stirred for 5 minutes. 10 μL of 10% bovine serum albumin (BSA) was then added, and the mixture was stirred for 5 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 10 minutes, the supernatant was discarded, and the solution was resuspended in 200 μL of reconstitution buffer to prepare a thiamphenicol monoclonal antibody-colloidal gold probe, which was then stored at 4°C for later use.

[0081] Reconstitution buffer: pH 7.4, 0.01 M PB (solvent is distilled water, solutes and their concentrations are as follows: BSA 5 g / L, sucrose 25 g / L, Tween-20 1 g / L)

[0082] (3) Preparation of sample pad

[0083] The sample pad was immersed in a 0.01 M PB solution containing 0.5% Tween-20 at a pH of 7.4 for 30 minutes, and then dried in a 37° C. oven for 6 hours before use.

[0084] (4) Preparation of reaction membrane

[0085] The coating material prepared in step (2) of Example 2 was sprayed onto the T zone detection line (T line) of the nitrocellulose membrane using a gold standard streaking instrument, and 10 mg / mL goat anti-mouse antibody was sprayed onto the C zone detection line (C line) of the nitrocellulose membrane. The sprayed reaction membrane was placed in a 45°C oven to dry for 6 h.

[0086] (5) Test strip assembly

[0087] Overlap the sample pad and absorbent filter paper on both sides of the reaction membrane by 2 mm, stick them to the PVC base plate, and cut the test paper strip to a width of 3.5 mm.

[0088] Example 6 Application of Thiamphenicol Colloidal Gold Immunochromatographic Test Strips

[0089] (1) Detection steps

[0090] 10 μL of the thiamphenicol monoclonal antibody-colloidal gold probe prepared in step (2) of Example 5 was placed in a microwell. 100 μL of 0.01 M PB or 100 μL of a series of 50 μL thiamphenicol standards of varying concentrations was then added and mixed thoroughly. After standing at room temperature for 5 minutes, the assembled colloidal gold test strip was inserted and reacted at room temperature for 5 minutes. The test strip was removed, the sample pad was removed, and the results were interpreted using an immunochromatographic reader.

[0091] (2) Analysis of test results

[0092] An immunochromatographic reader was used to read the T / C value of each test strip. Specifically, a four-parameter S function was used to fit and draw a standard curve. The logarithm of the drug concentration to be tested was used as the horizontal axis, and the standard curve was fitted with B / B0 as the vertical axis (B0 is the T / C value when no drug is added, and B is the T / C value when the drug concentration is added). The detection limit of the instrument was defined as 90% of B / B0.

[0093] (3) Standard curve

[0094] The standard curve of thiamphenicol is obtained by analyzing the test results of the standard solution, as shown in Figure 4 As shown, the colloidal gold immunochromatographic test strip of the present invention is IC 50 The detection limit is 0.70 ng / mL, the linear range is 0.16-3.17 ng / mL, and the minimum detection limit is 0.06 ng / mL. The detection limit is low and the sensitivity is high.

[0095] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A thiamphenicol hapten, characterized in that Its structural formula is shown in formula (I): 。 Formula (I) 2. The method for preparing the hapten according to claim 1, characterized in that: First, thiamphenicol is hydrolyzed under alkaline conditions to derive an amino group, which is then subjected to an amide condensation reaction with succinic anhydride to obtain a white powdery product, which is the thiamphenicol hapten shown in formula (I).

3. Use of the thiamphenicol hapten according to claim 1 in the preparation of thiamphenicol artificial antigen.

4. A thiamphenicol artificial antigen, characterized in that It is obtained by coupling the thiamphenicol hapten carrier protein according to claim 1, and its structural formula is shown in formula (II): 。 Formula (II) 5. The thiamphenicol artificial antigen according to claim 4, characterized in that The carrier protein is lactoferrin or chicken ovalbumin.

6. The thiamphenicol artificial antigen according to claim 4, characterized in that The coupling method is the active ester method.

7. Use of the artificial thiamphenicol antigen according to any one of claims 4 to 6 in the preparation of thiamphenicol antibodies.

8. A thiamphenicol antibody, characterized in that The invention is prepared by immunizing an animal with the artificial thiamphenicol antigen as claimed in any one of claims 4 to 6 as an immunogen.

9. Use of the thiamphenicol artificial antigen according to any one of claims 4 to 6 or the thiamphenicol antibody according to claim 8 in the preparation of a thiamphenicol immunoassay product.

10. A colloidal gold immunochromatographic test strip for detecting thiamphenicol, characterized in that: The antibody prepared by immunizing an animal with the artificial antigen whose carrier protein is chicken ovalbumin as the coating source and the artificial antigen whose carrier protein is bovine serum albumin as the immunogen is the detection antibody.

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