A monoclonal antibody against chloramphenicol and a magnetic-bead-based automatic chemiluminescent enzyme immunoassay method and application thereof

Abstract

The application discloses an anti-chloramphenicol monoclonal antibody and a magnetic-bead-based automatic chemiluminescence enzyme immunoassay method and application thereof. The application first provides an anti-chloramphenicol monoclonal antibody, which is prepared by immunizing mice with an artificial antigen obtained by coupling of a chloramphenicol hapten N2 and a carrier protein as an immunogen. 50 The IC50 of the monoclonal antibody to chloramphenicol is 0.23 ng / mL, the linear range is 0.05-1.04 ng / mL, and the detection limit is 0.02 ng / mL. On the basis of the anti-chloramphenicol monoclonal antibody, the application further provides a magnetic-bead-based automatic chemiluminescence enzyme immunoassay method for detecting chloramphenicol, wherein the detection limit of the method to chloramphenicol is 0.41 ng / mL, the linear range is 1.02-24.15 ng / mL, the method is simple and rapid in operation, the result is accurate and reliable, and the method can realize extensive detection of chloramphenicol.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology and immunoassay technology, and more specifically, relates to an anti-chloramphenicol monoclonal antibody and its automated chemiluminescent enzyme immunoassay method and application based on magnetic beads. Background Technology

[0002] Chloramphenicol (CAP), also known as chloramine-benzyl alcohol, is an amide class of antibiotics and a highly effective broad-spectrum antibiotic. Originally extracted from the culture medium of *Streptococcus vena cava*, it can now be synthesized chemically. Chloramphenicol is effective in inhibiting the growth of various aerobic microorganisms, including Gram-positive bacteria, anaerobic Bacteroides, rickettsiae, chlamydia, and mycoplasma. Due to its good antibacterial effect, chloramphenicol has been widely used in livestock and poultry farming, aquaculture, and clinical medicine. However, chloramphenicol can accumulate in the food chain and cause gastrointestinal diseases, oral diseases, neuritis, visual impairment, and irreversible loss of white blood cells and platelets, potentially leading to anemia and cardiovascular failure. According to my country's "Maximum Residue Limits for Veterinary Drugs in Animal-Derived Foods," chloramphenicol is a prohibited drug, and its presence in all food animals and their edible tissues must not be detected. Because chloramphenicol is inexpensive and readily available, its illegal use in production persists. Therefore, given the current status of chloramphenicol residues, there is a significant demand for chloramphenicol residue detection. Developing methods for detecting chloramphenicol residues in animal-derived foods is of great importance in order to meet this need.

[0003] Currently, commonly used methods for detecting chloramphenicol (CAP) can be broadly categorized into instrumental detection methods and immunoassay methods. Instrumental detection methods include high-performance liquid chromatography (HPLC), gas chromatography (GC), HPLC-MS / MS, and GC-MS / MS. Immunoassay is currently the most convenient and rapid detection technique, offering advantages such as high sensitivity, simple operation, and low cost. Enzyme-linked immunosorbent assay (ELISA), colloidal gold immunoassay (GICA), and chemiluminescent immunoassay (CLIA) are widely used for rapid CAP detection. However, the reported immunoassay methods for detecting chloramphenicol exhibit high sensitivity but a narrow detection range. While they can sensitively detect chloramphenicol within a certain low concentration range, this inevitably increases the difficulty of sample pretreatment and reduces the accuracy of the method. For example, if the CAP concentration in the test solution exceeds the detection range, it will affect the accuracy of the detection. To reduce the concentration to the detection range, repeated sample dilution is required, increasing the difficulty of sample pretreatment. Therefore, exploring a simple, rapid immunoassay method with a wide linear range for chloramphenicol detection is beneficial to meeting the demand for chloramphenicol residue detection.

[0004] Automated chemiluminescent enzyme immunoassay (CLIA) based on magnetic beads is a technique that combines magnetic separation technology and chemiluminescent immunoassay (CLIA). This technology fully utilizes the speed and ease of automation of magnetic separation, the sensitivity of chemiluminescence, and the specificity of immunoassay, demonstrating its advantages in detecting hazardous substances in food. Applying automated chemiluminescent enzyme immunoassay based on magnetic beads to detect hazardous substances in food can improve immunoassay performance to a certain extent, shorten detection time, achieve high-throughput detection, improve detection efficiency, simplify sample pretreatment, and facilitate automated detection operation. Because it incorporates magnetic separation technology, automated chemiluminescent immunoassay detection can be achieved. Therefore, providing a suitable monoclonal antibody for developing an automated chemiluminescent enzyme immunoassay method based on magnetic beads is of great significance for meeting the detection requirements of chloramphenicol with a wide linear range. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an anti-chloramphenicol monoclonal antibody.

[0006] A second object of the present invention is to provide the application of the aforementioned antichloramphenicol monoclonal antibody.

[0007] The third objective of this invention is to provide an automated chemiluminescent enzyme immunoassay method based on magnetic beads.

[0008] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0009] The present invention first provides an anti-chloramphenicol monoclonal antibody, wherein the light chain variable region of the monoclonal antibody has the amino acid sequence shown in SEQ ID No. 1, and the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 2.

[0010] Specifically, the monoclonal antibody 4H5 light chain variable region and heavy chain variable region each include 4 framework regions (FR1, FR2, FR3, FR4) and 3 complementarity-determining regions (CDR1, CDR2, CDR3), and the order of the 4 framework regions and 3 complementarity-determining regions is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0011] The present invention also provides a gene encoding the anti-chloramphenicol monoclonal antibody, wherein the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 4.

[0012] The amino acid sequence of the anti-chloramphenicol monoclonal antibody 4H5 and the nucleotide sequence encoding the monoclonal antibody given above should also be within the scope of protection of this invention.

[0013] This invention provides a method for preparing the anti-chloramphenicol monoclonal antibody, which uses an artificial antigen obtained by conjugating chloramphenicol hapten N2 with a carrier protein as an immunogen and an artificial antigen obtained by conjugating chloramphenicol hapten NI-6 with a carrier protein as a coating antigen, and then immunizes animals to obtain the antibody; the structural formulas of chloramphenicol hapten N2 and NI-6 are shown in formula (I) and formula (III), respectively:

[0014] Furthermore, the carrier protein is keyhole hemocyanin (KLH) or ovalbumin (OVA).

[0015] Furthermore, the method for preparing the anti-chloramphenicol monoclonal antibody is as follows: the artificial antigen obtained by conjugating the above-mentioned chloramphenicol hapten N2 with the carrier protein is used as an immunogen to immunize mice, mouse spleen cells are fused with SP2 / 0 myeloma cells to obtain hybridoma cells, the hybridoma cells are inoculated into female Balb / c mice for culture to obtain ascites containing a high concentration of monoclonal antibody, and the ascites is purified to prepare a highly specific anti-chloramphenicol monoclonal antibody.

[0016] Furthermore, the chloramphenicol hapten N2 is obtained by using the amino group on the purified levorotatory amino group of the chloramphenicol intermediate as an arm extension site, and introducing an arm containing a carboxylic acid through a substitution reaction between the amino group and succinic anhydride. The high degree of overlap between the chloramphenicol hapten N2 and the analyte chloramphenicol backbone structure, along with the exposure of chloramphenicol's characteristic structure, lays the foundation for generating highly specific antibodies. Furthermore, an artificial antigen obtained by conjugating the carboxyl terminus of the chloramphenicol hapten N2 to a carrier protein is used as an immunogen for animal immunization experiments. Monoclonal cell lines are prepared through cell fusion to obtain highly specific, highly sensitive anti-chloramphenicol monoclonal antibodies with a lower detection limit.

[0017] Preferably, the method for preparing the chloramphenicol hapten N2 includes the following steps:

[0018] S1. Dissolve purified L-amino compound (Ni) and anhydrous potassium carbonate (K2CO3) in an organic solvent, add succinic anhydride (C4H4O3), and heat to 80℃ for reflux reaction while stirring;

[0019] S2. After the reaction is complete, the organic solvent is evaporated, water is added, the pH is adjusted to 3-4, extraction is performed, the organic phase is mixed and then subjected to vacuum distillation to obtain a pale yellow oily target substance, which is the N2 hapten.

[0020] Preferably, the organic solvent is anhydrous pyridine (Pyr).

[0021] Preferably, the succinic anhydride is added after being dissolved in anhydrous pyridine.

[0022] Preferably, the reflux reaction is carried out for 30 minutes.

[0023] Preferably, the extraction is performed with ethyl acetate and followed by washing with saturated saline solution.

[0024] Furthermore, the chloramphenicol hapten NI-6 is obtained by using the amino group on the purified levorotatory amino group of the chloramphenicol intermediate as an arm extension site, and introducing a carboxylic acid arm through a substitution reaction between the amino group and ethyl 6-bromohexanoate. The high degree of overlap between the chloramphenicol hapten NI-6 and the analyte chloramphenicol backbone structure, along with the exposure of chloramphenicol's characteristic structure, lays the foundation for identifying chloramphenicol-specific antibodies. Furthermore, the artificial antigen obtained by conjugating the carboxyl terminus of the chloramphenicol hapten NI-6 with a carrier protein can be used as a coating agent for antibody performance detection.

[0025] Preferably, the method for preparing the chloramphenicol hapten NI-6 includes the following steps:

[0026] S1. Take purified L-amino compound (Ni) and triethylamine (C6H) 15 N) is dissolved in an organic solvent, and ethyl 6-bromohexanoate (C8H) is added. 15 BrO2), and during stirring, the mixture is heated to 80°C and refluxed.

[0027] S2. After the reaction is complete, extract the organic phase, mix the organic phases, and then distill under reduced pressure to obtain a yellow oily substance, which is the intermediate product.

[0028] S3. The intermediate product was dissolved in an organic solvent, and then water and lithium hydroxide (LiOH) were added. The mixture was stirred at room temperature for 12 hours.

[0029] S4. After the reaction is complete, extract the aqueous layer, adjust the pH of the aqueous layer to 3-4, and then extract the organic phase.

[0030] S5. After mixing the organic phases, perform vacuum distillation to obtain a deep yellow oily compound, which is the NI-6 hapten.

[0031] Preferably, the organic solvent in step S1 is N,N-dimethylformamide (DMF).

[0032] Preferably, the reflux reaction in step S1 is carried out for 24 hours.

[0033] Preferably, the extraction in steps S2 and S4 is performed using saturated brine and ethyl acetate.

[0034] Preferably, the organic solvent in step S3 is methanol.

[0035] Preferably, step S3 involves dissolving the intermediate product in methanol, then adding tertiary water and 5 equivalents of lithium hydroxide (LiOH), and stirring at room temperature for 12 hours.

[0036] Furthermore, the structural formula of the artificial antigen obtained by coupling the chloramphenicol hapten N2 with the carrier protein is shown in formula (II): The carrier protein is keyhole hemocyanin.

[0037] Furthermore, the structural formula of the artificial antigen obtained by conjugating the chloramphenicol hapten NI-6 with a carrier protein is shown in formula (Ⅳ): The carrier protein is ovalbumin.

[0038] Furthermore, the carrier protein in the immunogen is keyhole hemocyanin, and the carrier protein in the coating antigen is ovalbumin.

[0039] Preferably, the artificial antigen is prepared by conjugating the hapten N2 and NI-6 with a carrier protein using an active ester method. Specifically, the preparation method is as follows: first, chloramphenicol hapten N2 and NI-6 are dissolved in DMF, and EDC and NHS are added and stirred at 4°C for activation, denoted as solution A. Then, carrier proteins KLH and OVA are dissolved in phosphate buffer, denoted as solution B. Solutions A and B are mixed and stirred at 4°C. After the reaction, the mixture is collected and dialyzed at 4°C for 3 days to obtain artificial antigens N2-KLH and NI-6-OVA.

[0040] Preferably, the molar ratio of the hapten to the carrier protein is 60:1.

[0041] Preferably, the molar ratio of the hapten, NHS and EDC is 1:1.5:1.5.

[0042] This invention provides the application of any of the above-described anti-chloramphenicol monoclonal antibodies in the detection of chloramphenicol or in the preparation of chloramphenicol detection kits.

[0043] This invention provides an automated chemiluminescent enzyme immunoassay kit for detecting chloramphenicol based on magnetic beads, containing any of the anti-chloramphenicol monoclonal antibodies described above.

[0044] Furthermore, the kit contains magnetic bead antibody, enzyme-labeled hapten, substrate solution, and washing solution; the magnetic bead antibody is a conjugate of the aforementioned anti-chloramphenicol monoclonal antibody and NHS magnetic beads (NHS-IMBs-CAP-mAb); the enzyme-labeled hapten is a conjugate of alkaline phosphatase and the chloramphenicol hapten NI-6 (NI-6-ALP); the substrate solution is APS-5 solution; and the washing solution is PBST.

[0045] More preferably, the kit contains magnetic bead reagent bottles, reagent bottle 1, reagent bottle 2, reagent bottle 3, several 2mL centrifuge tubes, and reaction cups; the magnetic bead reagent bottles contain magnetic bead antibody diluent; reagent bottle 1 contains enzyme-labeled hapten diluent; reagent bottle 2 contains substrate solution; reagent bottle 3 contains washing solution; the 2mL centrifuge tubes contain sample diluent; the reaction cups serve as containers for the detection reaction; the magnetic bead antibody is the anti-chloramphenicol monoclonal antibody conjugate with NHS magnetic beads described above, and the enzyme-labeled hapten is a conjugate with the chloramphenicol hapten NI-6; the substrate solution is APS-5 solution; the washing solution is PBST; and the diluent for each substance is PB solution.

[0046] A method for detecting chloramphenicol based on any of the above-described kits includes the following steps:

[0047] S1. Mix the magnetic bead antibody, enzyme-labeled hapten, and the sample solution to be tested, and perform a direct competitive reaction;

[0048] S2. After the reaction, the magnetic beads are separated from the solution, the magnetic beads are washed with washing solution, and substrate solution is added. The magnetic bead antibody, which is bound to the enzyme-labeled hapten, catalyzes the reaction of the substrate solution and outputs a luminescent signal. The luminescent signal is read for quantitative detection.

[0049] As a preferred embodiment, the above-mentioned chloramphenicol detection method is as follows: A fully automated chemiluminescence immunoassay analyzer automatically aspirates magnetic bead antibodies, enzyme-labeled hapten, and free test solution into a reaction vessel for a direct competitive reaction. After the reaction, the magnetic beads are separated from the solution by the attraction of a magnet. The magnetic beads are then washed with washing solution. APS-5 substrate solution is then added to the reaction vessel. The magnetic bead antibodies bound to the enzyme-labeled hapten catalyze the decomposition reaction of the APS-5 substrate solution, releasing photons and generating a signal output. The fully automated chemiluminescence immunoassay analyzer reads the luminescence signal, and the data is processed by the data processing software for quantitative detection.

[0050] This invention combines the aforementioned anti-chloramphenicol monoclonal antibody with an automated chemiluminescent enzyme immunoassay method based on magnetic beads. The fully automated chemiluminescence analyzer eliminates the need for manual sample loading and washing, allowing for the simultaneous detection of up to 40 samples. This high degree of automation shortens detection time, effectively improving detection efficiency and avoiding cumbersome procedures. Furthermore, the provided detection method has a detection limit of 0.41 ng / mL for chloramphenicol and a linear range of 1.02–24.15 ng / mL, fulfilling the requirements for simple, rapid, and wide-linear-range detection.

[0051] This invention provides the application of the detection method in the field of molecular detection, which includes food quality and safety testing and environmental pollution monitoring.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] This invention provides an anti-chloramphenicol monoclonal antibody, wherein the light chain variable region has the amino acid sequence shown in SEQ ID No. 1, and the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 2. The monoclonal antibody is prepared by immunizing mice with an artificial antigen obtained by conjugating chloramphenicol hapten N2 with KLH, obtaining hybridoma cells through cell fusion, preparing ascites fluid by in vivo inoculation of hybridoma cells, and purifying the ascites fluid to obtain the anti-chloramphenicol monoclonal antibody 4H5. The IC50 of the monoclonal antibody 4H5 against chloramphenicol is detected by indirect competitive ELISA. 50 The detection limit (LOD) is 0.23 ng / mL, the linear range is 0.05–1.04 ng / mL, and the LOD is 0.02 ng / mL. Based on the chloramphenicol monoclonal antibody 4H5, this invention also provides an automated chemiluminescent enzyme immunoassay method for detecting chloramphenicol based on magnetic beads. The method has a detection limit of 0.41 ng / mL and a linear range of 1.02–24.15 ng / mL. This method is simple, rapid, accurate, and reliable, enabling the detection of a wide range of chloramphenicol. Attached Figure Description

[0054] Figure 1 This is the mass spectrum of the hapten N2.

[0055] Figure 2 This is the mass spectrum of the hapten NI-6.

[0056] Figure 3 Ultraviolet scan images of haptens (N2, NI-6), carrier proteins (KLH, OVA), and artificial antigens (N2-KLH, NI-6-OVA).

[0057] Figure 4 This is a schematic diagram of the amino acid sequence and domain division of the variable region of the 4H5 light chain of the anti-chloramphenicol monoclonal antibody.

[0058] Figure 5 This is a schematic diagram of the amino acid sequence and domain division of the variable region of the 4H5 heavy chain of the anti-chloramphenicol monoclonal antibody.

[0059] Figure 6 Ultraviolet scans of the hapten (NI-6), alkaline phosphatase (ALP), and NI-6-ALP.

[0060] Figure 7 A standard curve was established for an automated chemiluminescent enzyme immunoassay method based on magnetic beads to detect chloramphenicol. Detailed Implementation

[0061] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0062] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0063] Example 1: Preparation of Hapten

[0064] 1. Preparation of hapten N2

[0065] The structure of the hapten N2 is shown in formula (Ⅰ): The preparation method involves using the amino group on a purified levorotatory amino compound as the extension site for the carboxylic acid arm, and introducing the carboxylic acid arm through a substitution reaction between the amino group and succinic anhydride. The specific steps include the following:

[0066] S1. Take 1 mmol (212.2 mg) of purified L-amino compound (Ni) and 5 mmol (691.05 mg) of anhydrous potassium carbonate (K2CO3) and dissolve them in 5 mL of anhydrous pyridine (Pyr). Stir for 10 min. Then slowly add 0.9 mmol (90.063 mg) of succinic anhydride (C4H4O3) dissolved in 2 mL of anhydrous Pyr. During stirring, heat to 80 °C and reflux for 30 min.

[0067] S2. After the reaction is complete, Pyr is evaporated, tertiary water is added, the pH is adjusted to 3-4 with 1M HCl, extraction is performed with ethyl acetate, and the mixture is washed with saturated saline solution.

[0068] S3. After mixing the three organic phases, perform vacuum distillation to obtain a pale yellow oily target substance, which is the N2 hapten.

[0069] The mass spectrum of chloramphenicol hapten N2 is as follows: Figure 1 As shown in the mass spectrum, 311.1 is the negative ion molecular peak of hapten N2, and its relative molecular mass is calculated to be 312, which is consistent with the actual relative molecular mass, indicating that chloramphenicol hapten N2 was successfully prepared.

[0070] 2. Preparation of hapten NI-6

[0071] The structure of the hapten NI-6 is shown in formula (Ⅲ): The carboxylic acid arm is introduced by using the amino group on the purified levorotatory amino compound as the arm extension site, and by reacting the amino group with ethyl 6-bromohexanoate. The specific preparation method includes the following steps:

[0072] S1. Take 1 mmol (212.2 mg) of purified L-amino compound (Ni) and 2 mmol (140 μL) of triethylamine (C6H2O). 15 N) was dissolved in 5 mL of DMF and stirred for 10 min. Then, 2 mmol (356 μL) of ethyl 6-bromohexanoate (C8H) dissolved in 2 mL of DMF was added. 15 BrO2) is added slowly, and the mixture is heated to 80°C and refluxed for 24 hours while stirring.

[0073] S2. After the reaction is complete, extract with saturated brine and ethyl acetate;

[0074] S3. After mixing the three organic phases, vacuum distillation was performed to obtain a yellow oily substance, which was the intermediate product.

[0075] S4. Dissolve the intermediate product in a small amount of methanol, then add tertiary water and 5 equivalents of lithium hydroxide (LiOH), and stir at room temperature for 12 hours.

[0076] S5. After the reaction is complete, extract with saturated brine and ethyl acetate, retain the aqueous layer, adjust the pH of the aqueous layer to 3-4 with 6M HCl, and then extract with ethyl acetate, retaining the organic phase;

[0077] S6. After mixing the three organic phases, vacuum distillation was performed to obtain a deep yellow oily compound, which is the NI-6 hapten.

[0078] The mass spectrum of chloramphenicol hapten NI-6 is shown below. Figure 2 As shown in the mass spectrum, 325.3 is the negative ion molecular peak of the hapten NI-6. Its calculated relative molecular mass is 326, which is consistent with the actual relative molecular mass, indicating that the chloramphenicol hapten NI-6 was successfully prepared.

[0079] Example 2: Preparation of Chloramphenicol Artificial Antigen

[0080] The chloramphenicol hapten N2 and NI-6 were prepared by coupling carrier proteins with keyhole hemocyanin (KLH) and ovalbumin (OVA) respectively using the active ester method. The preparation method is as follows:

[0081] Chloramphenicol haptens N2 and NI-6 were dissolved in DMF, and EDC and NHS (molar ratio N2 / NI-6:NHS:EDC = 1:1.5:1.5) were added. The mixture was stirred at 4°C for 8 hours to activate the reaction, and this was recorded as solution A. The carrier protein was then dissolved in phosphate buffer, and this was recorded as solution B. Solutions A and B were mixed and stirred at 4°C for 8 hours (molar ratio of hapten to carrier protein = 60:1). After the reaction, the mixture was collected and dialyzed at 4°C for 3 days. After dialysis, artificial antigens N2-KLH and NI-6-OVA were obtained, and their structural formulas are shown in formulas (II) and (IV).

[0082]

[0083] Among them, the carrier protein of hapten N2 is keyhole hemocyanin (KLH), and the carrier protein of hapten NI-6 is ovalbumin (OVA).

[0084] To verify the success of the artificial antigen synthesis, the absorption peaks of the N2 and NI-6 haptens before and after conjugation, the carrier protein used for conjugation, and the conjugated artificial antigen were measured under ultraviolet light. The results are as follows: Figure 3 As shown, in the ultraviolet wavelength range of 200–400 nm, the characteristic absorption peaks of the successfully synthesized conjugated artificial antigens under ultraviolet light differ from those of the hapten and carrier proteins. Compared to the characteristic absorption peaks of the hapten and carrier proteins, the conjugated artificial antigens also exhibit an overall blue shift in their absorption peaks. Therefore, it can be confirmed that the two artificial antigens, N2-KLH and NI-6-OVA, have been successfully synthesized.

[0085] Example 3: Preparation of anti-chloramphenicol monoclonal antibody

[0086] 1. Experimental Methods

[0087] (1) Animal immunization: Healthy 6-week-old Balb / c female mice were used as experimental animals. The complete antigen N2-KLH was used as the immunogen and an equal amount of immune adjuvant (Frederick complete adjuvant was used for the first immunization, and Frederick incomplete adjuvant was used for subsequent booster immunizations). The mice were immunized by various injection methods, including subcutaneous injection on the back, subcutaneous injection at various sites, intraperitoneal injection, and injection into the feet. The mice were immunized once every 2 weeks. After the fourth immunization, a small amount of blood was taken from the tail vein for antibody quality identification. After the antibody stabilized, the mice with the best performance were selected for cell fusion. Three days before cell fusion, 0.5 mg of immunogen was directly injected into the intraperitoneal cavity of the mice for a booster immunization.

[0088] (2) Antibody quality identification: Using chloramphenicol complete antigen NI-6-OVA as the coating antigen, mouse venous blood collected above was used as the detection antibody. The antiserum titer and inhibition rate of mouse serum were determined by indirect competitive ELISA. The titer and inhibition rate of each antiserum were comprehensively considered for evaluation. The specific operation steps are as follows:

[0089] NI-6-OVA and NI-6-BSA were used as heterocoating agents to coat ELISA plates, and the titer and inhibition rate of the antiserum were evaluated using icELISA. The specific evaluation steps were as follows:

[0090] ① Coating: Dilute the chloramphenicol complete antigen NI-6-OVA to 1 μg / mL with 0.05M carbonate buffer (pH 9.6), add 100 μL to each well of the ELISA plate, and incubate in a water bath at 37℃ for 12 h. Then, add 300 μL of PBST to each well for washing, repeat twice, and pat dry. Then, add 120 μL of blocking buffer (5% skim milk) to each well and block at 37℃ for 3 h. After blocking, spin dry the ELISA plate and dry it in an oven at 37℃ for 1 h.

[0091] ② Serum titer and inhibition assay: For the enzyme-labeled plate prepared in step ①, add 50 μL of PBS and 50 μL of serum diluted sequentially (1K, 3K, 9K, 27K, 81K, 243K, 729K) to each well in the titer column; add 50 μL of diluted 10 ng / mL chloramphenicol and 50 μL of serum diluted sequentially (1K, 3K, 9K, 27K, 81K, 243K, 729K) to each well in the inhibition column; add only 100 μL of PBS to each well in the blank column. Perform 3 replicates for each group. After reacting in a 37°C water bath for 40 min, the plate was washed 5 times with PBST and patted dry. Then, 100 μL of horseradish peroxidase-labeled goat anti-mouse solution diluted 5000 times was added, and the plate was incubated at 37°C for 30 min. The plate was washed 5 times with PBST, and 100 μL of TMB substrate chromogenic solution was added. The plate was incubated at 37°C in the dark for 10 min, and the reaction was terminated by adding 50 μL of stop solution (10% H2SO4).

[0092] ⑦ Reading measurement: Use an ELISA reader to read the absorbance of each well at a wavelength of A450nm.

[0093] The inhibition results of the antiserum obtained from immunizing Balb / c female mice are shown in Table 1. It can be seen that all immunized mice produced mouse polyclonal antibodies, and the resulting antiserum had an inhibitory effect on the target analyte, chloramphenicol, with the most significant inhibitory effect observed in mouse number 3. This indicates that the two haptens prepared in this invention can be used for the subsequent preparation of chloramphenicol monoclonal antibodies and the establishment of immunoassay methods.

[0094] Table 1. Determination of antiserum efficacy

[0095]

[0096] (3) Cell fusion and antibody preparation: Mice with the best performance were selected for cell fusion. Spleen cells were mixed with mouse SP2 / 0 myeloma cells in the logarithmic growth phase using electrofusion. The cells were suspended evenly in HAT medium, and an appropriate amount of feeder cells were added. The mixture was cultured in 96-well plates at 37°C in a 5% CO2 incubator. After 7 days of fusion, the medium was partially replaced with HT medium, and completely replaced after 9 days. After subcloning and four limiting dilutions, a portion of the positive hybridoma was cryopreserved, and the other portion was injected into the peritoneal cavity of mice to generate ascites. The ascites was collected after 7 days and purified using a Protein G immunoaffinity column, yielding the monoclonal antibody 4H5.

[0097] (4) Establishment of the standard curve: Carbonate buffer (CBS, pH = 9.6) was used as the dilution buffer for the coating agent NI-6-OVA, and phosphate buffer (PBS, pH = 7.4) was used as the dilution buffer for antibody 4H5, standards, and horseradish peroxidase-labeled goat anti-mouse solution. The coating agent was diluted to 1 μg / mL and added to each well of a 96-well microplate at 100 μL. The plate was incubated overnight at 4°C. After washing the plate twice with PBST, 120 μL of blocking buffer (5% skim milk powder) was added to each well. The plate was blocked at 37°C for 3 h, and then dried at 37°C for 1 h after spin drying. Add 50 μL of anti-chloramphenicol monoclonal antibody 4H5 and a series of 50 μL of chloramphenicol standards at different concentrations to each well. Incubate at 37°C for 40 min, wash 5 times with PBST, add 100 μL of horseradish peroxidase-labeled goat anti-mouse solution diluted 5000 times, incubate at 37°C for 30 min, wash 5 times with PBST, add 100 μL of TMB substrate chromogenic solution, and develop at 37°C in the dark for 10 min. Stop the reaction by adding 50 μL of stop solution (10% H2SO4), and read the absorbance at 450 nm on a microplate reader. Construct an indirect competition standard curve with chloramphenicol standard concentration on the x-axis and B / B0 (absorbance of wells with chloramphenicol added / absorbance of wells without chloramphenicol added) on the y-axis.

[0098] 2. Experimental Results

[0099] A standard curve was established based on the anti-chloramphenicol monoclonal antibody 4H5 prepared using chloramphenicol complete antigen NI-6-OVA as the coating antigen. The results showed that the IC50 of monoclonal antibody 4H5 against chloramphenicol was [missing value]. 50 The effective concentration was 0.23 ng / mL, the linear range was 0.05–1.04 ng / mL, and the limit of detection (LOD) was 0.02 ng / mL.

[0100] Example 4: Sequencing and determination of the amino acid sequence of the gene encoding the anti-chloramphenicol monoclonal antibody 4H5.

[0101] 1. Total RNA extraction

[0102] Total RNA was extracted using the Trizol reagent method from Guangzhou Jiebes Biotechnology Co., Ltd.

[0103] The specific steps are as follows:

[0104] Culture chloramphenicol 4H5 monoclonal cells. Transfer the cells from the culture flask to a sterile 50mL centrifuge tube. Adjust the volume to 30mL. Take 10μL for cell counting. Estimate the total cell count and then take approximately 1×10⁻⁶ cells. 6 Transfer the cells to another sterile 50mL centrifuge tube, centrifuge at 1000r / min for 7min to completely remove the supernatant, and gently tap the bottom of the centrifuge tube to loosen the cell pellet; add 2mL of lysis buffer (TRNsol) to the cell pellet, disperse it, and collect it in a 2mL centrifuge tube; add 0.2mL of chloroform to every 1mL of the above lysis buffer. Cover the centrifuge tube, gently shake up and down for 15 seconds, incubate on ice for 5 minutes, centrifuge at 12000 rpm for 10 minutes at room temperature; transfer the upper aqueous phase to a new 2 mL centrifuge tube, slowly add 0.7 times the volume of anhydrous ethanol, and mix well; transfer the resulting solution and precipitate together into a GBC adsorption column, centrifuge at 12000 rpm for 30 seconds, and remove the waste liquid; add 500 μL of Wash Buffer I to the GBC adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; add 600 μL of Wash Buffer II to the GBC adsorption column, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and finally centrifuge at 12000 rpm for 1 minute to thoroughly remove the waste liquid; open the centrifuge tube cover under a clean bench and let the residual washing solution in the adsorption column air dry. Transfer the GBC adsorption column into a new centrifuge tube, add 30–100 μL of RNase-free ddH2O, incubate at room temperature for 2 min, and centrifuge at 12000 r / min for 1 min to obtain the RNA solution.

[0105] 2. cDNA Synthesis

[0106] Using RNA as a template, cDNA first-strand synthesis was performed according to the instructions of the Takara First-Strand Reverse Transcription Kit. Specifically, 3 μg of RNA was used as a template, and 1 μL of Oligo(dT) 18 primer was added to a 1.5 mL nuclease-free centrifuge tube. RNase-free ddH2O was added to bring the total volume to 12 μL. The mixture was incubated at 65°C for 5 min, followed by cooling on ice for 2 min. After the reaction, 4 μL of 5×Reaction Buffer, 1 μL of RNase inhibitor, 2 μL of 10 mM dNTP Mix, and 1 μL of M-MuLV reverse transcriptase (200 U / μL) were added to the final volume, bringing the total volume to 20 μL. The mixture was incubated at 42°C for 60 min, followed by incubation at 70°C for 5 min to inactivate the reverse transcriptase. After the reaction, the mixture was stored at -80°C for later use.

[0107] 3. Amplification of antibody variable region genes

[0108] (1) PCR cloning of V H V L Gene

[0109] Using cDNA synthesized by reverse transcription as a template, genes in the variable regions of the antibody heavy and light chains were cloned using universal primers. The universal primers are shown in Table 2.

[0110] Table 2. Universal primers for amplifying the variable regions of the heavy and light chains of single-chain antibodies.

[0111]

[0112] ① Cloning of light chain variable region genes

[0113] Using the first strand of cDNA as a template, the variable region of the light chain was amplified using the LB4 / LF1 primer set. The reaction system and specific parameters are shown in Table 3. After PCR, the bands were identified by gel electrophoresis at 120V for 25min. The bands in the range of 300bp to 500bp were recovered by gel excision using a kit.

[0114] Table 3. PCR amplification system and reaction conditions for the light chain variable region

[0115]

[0116] ② Cloning of heavy chain variable region genes

[0117] Using the first strand of cDNA as a template, the variable region of the heavy chain was amplified using the HB2 / HF1 primer set. The reaction system and specific parameters are shown in Table 4. After PCR, the bands were identified by gel electrophoresis at 120V for 25min. The bands in the range of 300bp to 500bp were recovered by gel excision using a kit.

[0118] Table 4. PCR amplification system and reaction conditions for the heavy chain variable region

[0119]

[0120] (2) DNA gel recovery kit was used to recover the amplified V H V L Gene

[0121] The OMEGA gel extraction kit was used to recover PCR gel products. The specific steps are as follows: Cut off the target band with a clean blade and weigh it. Add an equal volume of Binding Buffer (XP2) and incubate at 55-60 °C for 10 min until the gel is completely melted, shaking every 2-3 minutes. Transfer the melted gel to a HiBind DNA column and centrifuge at 10000 r / min for 1 min. Discard the liquid in the collection tube, add 300 μL of Binding Buffer (XP2), and centrifuge at 10000 r / min for 1 min. Discard the liquid in the collection tube, add 700 μL of SPW Wash Buffer (ethanol should be added to SPW Wash Buffer for first use), and centrifuge at 10000 r / min for 1 min. Repeat the washing with SPW Wash Buffer. Discard the liquid in the collection tube and centrifuge at 10000 r / min for 2 min. Transfer the adsorbed membrane to a 1.5 mL centrifuge tube, add 15 μL of sterile water, and centrifuge at 10000 r / min. Centrifuge at r / min for 2 min, collect the liquid in the tube, measure the DNA concentration using a nanodrop micro-UV spectrophotometer, and store at -20℃. The recovered V... H V L Genes were ligated into an 18-T Vector Cloning vector (TakaRa).

[0122] (3) Transformation

[0123] Competent DH5α cells were placed on ice and allowed to thaw for 5 minutes. 5 μL of the target vector was added, gently mixed, and incubated on ice for 25 minutes. The cells were then heat-shocked in a 42 °C water bath for 45 seconds, immediately returned to ice, and incubated for 2 minutes. 400 μL of preheated LB broth (37 °C) was added, and the cells were incubated at 37 °C and 250 rpm for 1 hour. 100 μL of the culture was then evenly spread onto LB-A plates and incubated upside down at 37 °C overnight. The following day, multiple single colonies were randomly selected from the plates for colony PCR and DNA sequencing identification and analysis.

[0124] 4. Antibody heavy chain and light chain gene sequence analysis

[0125] After adjusting the sequencing results using DNAman software, the complete forward sequence was obtained and entered into IMGT in FASTA format. https: / / www.imgt.org / IMGT_vquest / analysis ) Perform murine antibody variable region gene sequence analysis. Through analysis, the heavy chain variable region (V) was obtained. H -4H5) and light chain variable region (V L The amino acid sequence after translation of (-4H5).

[0126] 5. Experimental Results

[0127] The amino acid sequence of the variable region of the 4H5 light chain of the anti-chloramphenicol monoclonal antibody is shown in SEQ ID No. 1:

[0128] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIY

[0129] LVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPS

[0130] WKSN

[0131] The amino acid sequence of the variable region of the 4H5 heavy chain of the anti-chloramphenicol monoclonal antibody is shown in SEQ ID No. 2:

[0132] EVQLQQSGAELARPGASVKLSCKASGYSFTTYWMQWVKQRPGQGLEWIGAI

[0133] CPGADDTRYTQKFKGKATLTADKSSNTVYMQLTSLASEDSAVYYCARGGYG

[0134] RSYLYFDVWGAGTTVTVSS

[0135] The nucleotide sequence encoding the light chain variable region of the anti-chloramphenicol monoclonal antibody 4H5 is shown in SEQ ID No. 3:

[0136] GACATTTGTGTTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAG

[0137] AGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTA

[0138] TAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCC

[0139] TCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTG

[0140] GCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAG

[0141] GAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTC

[0142] GGAGGGGGACCAAGCTGGAAATCAAAC

[0143] The nucleotide sequence encoding the heavy chain variable region of the anti-chloramphenicol monoclonal antibody 4H5 is shown in SEQ ID No. 4:

[0144] GAGGTGCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTC

[0145] AGTGAAGTTGTCCTGCAAGGCTTCTGGCTACAGCTTCACTACCTACTGGAT

[0146] GCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCT

[0147] ATTTGTCCTGGAGCTGATGATACTAGGTACACTCAGAAGTTCAAGGGCAA

[0148] GGCCACATTGACTGCAGATAAAATCCTCCAATACAGTCTACATGCAACTCA

[0149] CCAGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGGGGC

[0150] TACGGTAGAAGTTACTTATACTTCGATGTCTGGGGCGCAGGGACCACGGT

[0151] CACCGTTTCCTCG

[0152] The amino acid sequences of the light chain variable region and heavy chain variable region of the antichloramphenicol monoclonal antibody 4H5 prepared according to Example 3 are as follows: Figure 4 and Figure 5 As shown.

[0153] Example 5: Preparation of chloramphenicol antibody-magnetic bead conjugate based on magnetic beads

[0154] Antibody-magnetic bead conjugates were prepared according to the instructions for use of Beaver Beads Mag NHS. The specific method is as follows:

[0155] 1. Preparation of protein solution: Take an appropriate amount of the CAP-mAb to be coupled and dilute it with MES (0.1M, pH 4.8) to prepare a protein solution of 3.0 mg / mL. If the antibody is already stored in another buffer, be sure to remove substances such as primary amino groups from the buffer. Store the prepared protein solution at 4℃ for later use.

[0156] 2. Cleaning of magnetic beads: Take 500 μL of NHS magnetic beads into a 1.5 mL centrifuge tube, vortex thoroughly, then place the centrifuge tube in a magnetic separator to enrich the magnetic beads and remove the supernatant; add 1 mL of 1 mM hydrochloric acid solution pre-cooled to 4 °C, vortex to mix; then place the centrifuge tube in a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0157] 3. Immobilization of biological ligands: Add 500 μL of the prepared chloramphenicol antibody solution (concentration of 3 mg / mL) to the centrifuge tube and vortex to mix thoroughly. Incubate at room temperature for 1 hour on a vertical mixer; enrich magnetic beads using a magnetic separator and preserve the flow-through solution.

[0158] 3. Magnetic bead sealing: Add 500 μL of 3M ethanolamine to the centrifuge tube, vortex to mix, enrich the magnetic beads using a magnetic separator, discard the supernatant, and repeat the washing process 4 times. Finally, add 500 μL of 3M ethanolamine to the centrifuge tube, vortex to mix, and seal the reaction in a vertical mixer at room temperature for 2 hours. After the reaction, enrich the magnetic beads using a magnetic separator and remove the supernatant. Then add 1 mL of ultrapure water to the centrifuge tube, vortex to mix, enrich the magnetic beads using a magnetic separator, and discard the supernatant.

[0159] 4. Storage: Add 1 mL of PBS buffer solution containing 0.05% sodium azide to a centrifuge tube, vortex to mix, place on a magnetic separator to enrich magnetic beads, and discard the supernatant; finally, add 500 μL of PBS buffer solution containing 0.05% sodium azide to a centrifuge tube, vortex to mix, and store at 4°C for later use.

[0160] Example 6 Preparation of enzyme-labeled hapten

[0161] The specific method for preparing the enzyme-labeled hapten described in this invention is as follows:

[0162] The chloramphenicol hapten NI-6 was prepared by coupling it with alkaline phosphatase (ALP) using an active esterification method. The preparation method is as follows:

[0163] Chloramphenicol hapten NI-6 was dissolved in DMF, and EDC and NHS (molar ratio NI-6:NHS:EDC = 1:1.5:1.5) were added and stirred for 8 hours to activate the reaction. This solution is designated as solution A. Alkaline phosphatase was then dissolved in phosphate buffer, designated as solution B. Solutions A and B were mixed and stirred for 8 hours (molar ratio NI-6:ALP = 60:1). After the reaction, the mixture was collected and dialyzed at 4°C in the dark for 3 days. The enzyme-labeled hapten NI-6-ALP was obtained after dialysis, and its structural formula is shown in formula (Ⅳ).

[0164]

[0165] The coupled protein is alkaline phosphatase (ALP).

[0166] To verify the success of NI-6-ALP synthesis, the absorption peaks of the hapten (NI-6), alkaline phosphatase (ALP), and the prepared NI-6-ALP were measured under ultraviolet light before and after coupling.

[0167] The results are as follows Figure 6 As shown, within the ultraviolet wavelength range of 200–400 nm, the characteristic absorption peak of the successfully synthesized NI-6-ALP under ultraviolet light differs from that of the hapten and alkaline phosphatase. Compared to the characteristic absorption peaks of the hapten and alkaline phosphatase, the conjugated NI-6-ALP also exhibits an overall blue shift in its absorption peak. Therefore, it can be confirmed that the NI-6-ALP enzyme-labeled hapten has been successfully synthesized.

[0168] Example 7: An automated chemiluminescent enzyme immunoassay method based on magnetic beads for detecting chloramphenicol.

[0169] 1. Testing steps

[0170] First, turn on the fully automated chemiluminescence immunoassay analyzer. Install the reagent bottle containing the substrate solution into the corresponding position on the instrument. Place a sufficient amount of reaction cup in the reaction cup compartment. Pour the prepared washing solution into the washing bottle. Start the instrument self-test. After the self-test is completed, it is ready for instrument detection. Dilute the prepared magnetic bead antibody and enzyme-labeled hapten to an appropriate concentration with PB buffer and put them into the magnetic bead reagent bottle and reagent bottle No. 1, respectively, and load them into the instrument. Load the test solution into a 2mL centrifuge tube and place it in the instrument sample rack. Load it into the instrument. In the instrument program settings, set to mix 50μL of NI-6-ALP solution, 100μL of NHS-IMBs-CAP-mAb solution and 50μL of the test solution. Mix thoroughly for 6 seconds, incubate for 20 minutes, then enrich with magnetic beads. Wash 4 times with washing solution, then add 200μL of substrate solution, mix thoroughly for 6 seconds, react for 5 minutes, and then detect the luminescence intensity with the instrument.

[0171] 2. Establishment of the standard curve

[0172] First, the CAP standard solution was serially diluted. The initial concentration of the CAP standard solution was 1000 ng / mL. It was diluted 3 times and serially diluted 11 times. The concentration of each was measured according to the detection procedure. The instrument read the luminescence value (RLU) in each reaction vessel. The luminescence value of the blank reaction vessel without chloramphenicol was RLU0. The standard curve was plotted using Origin 2022 software with chloramphenicol concentration as the x-axis and RLU / RLU0 as the y-axis.

[0173] 3. Quantitative analysis

[0174] The sample with unknown chloramphenicol concentration and the validated blank sample were placed into the instrument and tested according to the detection procedure. The luminescence value detected and analyzed in the fully automated chemiluminescence immunoassay analyzer was then read. The RLU / RLU0 was substituted into the established standard curve to calculate the chloramphenicol content in the tested sample.

[0175] 4. Experimental Results

[0176] The automated chemiluminescent enzyme immunoassay method based on magnetic beads for the detection of chloramphenicol of this invention has a detection limit of 0.41 ng / mL and a linear range of 1.02–24.15 ng / mL. The standard curve is shown below. Figure 7 As shown.

[0177] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against chloramphenicol, characterized in that, The light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID No. 1, and the heavy chain variable region is the amino acid sequence shown in SEQ ID No.

2.

2. A gene encoding the anti-chloramphenicol monoclonal antibody of claim 1, characterized in that, The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 3, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

4.

3. The use of the anti-chloramphenicol monoclonal antibody according to claim 1 in detecting chloramphenicol for non-disease diagnosis and treatment purposes or in preparing chloramphenicol detection kits.

4. An automated chemiluminescent enzyme immunoassay kit for detecting chloramphenicol based on magnetic beads, characterized in that, It contains the anti-chloramphenicol monoclonal antibody as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The product contains magnetic bead antibody, enzyme-labeled hapten, substrate solution, and washing solution; the magnetic bead antibody is a conjugate of the anti-chloramphenicol monoclonal antibody according to claim 1 and NHS magnetic beads; the enzyme-labeled hapten is a conjugate of alkaline phosphatase and chloramphenicol hapten NI-6; the substrate solution is APS-5 solution; the washing solution is PBST; the structural formula of the chloramphenicol hapten NI-6 is shown in formula (III). 。 6. A method for detecting chloramphenicol for non-disease diagnosis and treatment purposes, characterized in that, Includes the following steps: S1. Mix the magnetic bead antibody, enzyme-labeled hapten, and the sample solution to be tested, and perform a direct competitive reaction; S2. After the reaction, the magnetic beads are separated from the solution, the magnetic beads are washed with washing solution, and substrate solution is added. The magnetic bead antibody, which is bound to the enzyme-labeled hapten, catalyzes the reaction of the substrate solution and outputs a luminescent signal. The luminescent signal is read for quantitative detection. The magnetic bead antibody is a conjugate of the anti-chloramphenicol monoclonal antibody according to claim 1 and NHS magnetic beads; the enzyme-labeled hapten is a conjugate of alkaline phosphatase and chloramphenicol hapten NI-6; the washing solution is PBST; the substrate solution is APS-5 solution; the structural formula of the chloramphenicol hapten NI-6 is shown in formula (III): 。

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