Preparation method and application of antibody for detecting ovalbumin and vancomycin in biological product

Abstract

The invention discloses a preparation method of an antibody for detecting ovalbumin and vancomycin in a biological product, the ovalbumin antibody is obtained by directly immunizing an animal, the vancomycin antibody is prepared by carrying out nucleophilic substitution reaction on vancomycin and N-(P-maleimidophenyl) isocyanate to obtain a vancomycin hapten, and the vancomycin hapten is used for detecting the ovalbumin and vancomycin in the biological product. The vancomycin immunogen is obtained by coupling the vancomycin with carrier protein, and then animals are immunized to obtain the vancomycin immunogen. The antibody is prepared into an enzyme linked immunosorbent assay kit, and when the enzyme linked immunosorbent assay kit is used for detecting ovalbumin contained in a biological product, the detection limit is 200 g / L; when the kit is used for detecting vancomycin contained in a biological product, the detection limit is 0.5 g / L. The detection result is high in accuracy, especially in a vancomycin antibody specificity experiment, the detection result shows that the detection result has remarkable specificity on vancomycin, and the detection result is not interfered by drugs such as polymyxin E, kanamycin, streptomycin, gentamicin, penicillin, tetracycline, chloramphenicol, neomycin, aureomycin, oxytetracycline and doxycycline.

Description

Technical Field

[0001] This invention relates to the field of antibody preparation technology, specifically to a method for preparing and applying antibodies for detecting ovalbumin and vancomycin in biological products. Background Technology

[0002] Ovalbumin (OVA) is an allergen that poses a potential health hazard. When the immune system is exposed to an allergen, it produces an antibody called immunoglobulin E (IgE). IgE antibodies trigger immune system cells to release substances (such as histamine, prostaglandins, and leukotrienes), causing swelling or inflammation in surrounding tissues. These substances initiate a reaction that continuously stimulates and damages tissues. As a foreign substance, ovalbumin is prone to causing allergic reactions when inoculated into the human body.

[0003] In the production of biological products, especially influenza vaccines using chicken embryos as a culture medium, the detection of residual ovalbumin (OVA) content is one of the essential monitoring indicators. The detection of OVA content is related to the product's qualification, which in turn affects human health and even directly relates to life safety. Furthermore, the 2020 edition of the Chinese Pharmacopoeia requires that the residual ovalbumin level be less than or equal to 200 ng / mL. Chinese patent document CN101893636A discloses an enzyme-linked immunosorbent assay (ELISA) method for detecting ovalbumin, an egg allergen, in food. This method uses polyclonal antibodies obtained by immunizing healthy New Zealand white rabbits with high-purity ovalbumin. Using these antibodies as the detection reagent, and high-purity ovalbumin as the standard and coating antigen, an indirect ELISA method for ovalbumin in food is established.

[0004] In addition, vancomycin is a common biopharmaceutical process residue and can also be found in biological products, thus it is also an impurity that needs to be detected. Furthermore, the 2020 edition of the Chinese Pharmacopoeia requires antibiotic concentrations to be less than or equal to 100 mg / L. Currently, vancomycin detection mainly includes high-performance liquid chromatography (HPLC), immunoassay, and liquid chromatography-mass spectrometry (LC-MS). The fundamental factor affecting the quality of immunoassay detection is the specificity and affinity of the antibody, which in turn depends on the molecular structure of the immune hapten. Therefore, the design and selection of immune hapten molecules are the most critical steps in generating specific antibodies and establishing rapid detection techniques for small molecule drugs. Chinese patent document CN112500496A discloses a complete vancomycin antigen and its application in the preparation of vancomycin-specific monoclonal antibodies and immunoassay detection. This complete vancomycin antigen uses glutaraldehyde to link the protein to vancomycin.

[0005] Therefore, there is an urgent need to find a method for preparing antibodies to detect ovalbumin and vancomycin in biological products, so that they can be applied to rapid immunoassay methods. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing antibodies to detect ovalbumin and vancomycin in biological products. The prepared antibodies are used to create an enzyme-linked immunosorbent assay (ELISA) kit, which, when used to detect ovalbumin in biological products, has a detection limit of 200 μL. g / L; when used for detecting vancomycin in biological products, the detection limit is 0.5 g / L. g / L.

[0007] A method for preparing antibodies to detect ovalbumin and vancomycin in biological products, wherein the ovalbumin antibody is obtained by direct immunization of animals, and the vancomycin antibody is obtained by preparing a vancomycin immunogen and then immunizing animals, wherein the vancomycin immunogen is obtained by coupling a vancomycin hapten with a carrier protein, and the structure of the vancomycin hapten is shown in formula (I): Equation (Ⅰ).

[0008] In this invention, ovalbumin antibody is obtained by direct immunization of animals, and vancomycin antibody is prepared by nucleophilic substitution reaction of vancomycin with N-(P-maleimide phenyl) isocyanate to obtain vancomycin hapten. The maleimide group in the hapten can be further coupled with a carrier protein containing a thiol group to obtain vancomycin immunogen, which is then used to immunize animals.

[0009] Preferably, the method for preparing the vancomycin hapten includes: preparing it by nucleophilic substitution reaction of vancomycin with N-(P-maleimide phenyl) isocyanate.

[0010] In this invention, the molecular formula of N-(P-maleimide phenyl)isocyanate is: C 11 H6N2O3, structural formula as follows: Molecular formula: C 11 H6N2O3, CAS number 123457-83-0, has the following structural formula: , The specific reaction route is as follows: .

[0011] By utilizing the highly reactive isocyanate group in the N-(P-maleimide-phenyl)isocyanate molecule, a nucleophilic addition reaction is carried out with the amino group on the vancomycin molecule to form a stable urea bond, thereby generating a vancomycin derivative that still retains the maleimide group at the end. This allows it to be further coupled to a carrier protein containing a thiol group through the efficient reaction between the maleimide group and the thiol group.

[0012] Preferably, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin, or keyhole hemocyanin.

[0013] More preferably, the carrier protein is keyhole hemocyanin.

[0014] The present invention also provides an enzyme-linked immunosorbent assay (ELISA) kit, comprising: an ELISA plate coated with a coating agent, a standard solution, an antibody, an enzyme conjugate concentrate, an enzyme conjugate diluent, a substrate chromogenic solution, a stop solution, and a washing solution, wherein the coating agents are vancomycin coating agent and ovalbumin, the antibodies are vancomycin antibody and ovalbumin antibody, and the enzyme conjugates are enzyme-labeled vancomycin antibody and ovalbumin antibody.

[0015] Preferably, the labeled enzyme of the enzyme conjugate is horseradish peroxidase or bacterial-extracted alkaline phosphatase.

[0016] More preferably, the labeled enzyme of the enzyme conjugate is horseradish peroxidase; the enzyme conjugate is obtained by conjugating the labeled enzyme with the above-mentioned vancomycin antibody and ovalbumin antibody, respectively.

[0017] More preferably, when the labeling enzyme is horseradish peroxidase, the substrate colorimetric solution consists of substrate solution A and substrate solution B, where substrate solution A is urea peroxide and substrate solution B is tetramethylbenzidine; the stop solution is a 1-2 mol / L sulfuric acid solution.

[0018] Preferably, the standard solutions are vancomycin standard solution and ovalbumin standard solution, wherein the concentrations of the vancomycin standard solution are 0, 0.075, 0.225, 0.675, 2.025, and 6.075 μg / L, and the concentrations of the ovalbumin standard solution are 0, 0.05, 0.15, 0.45, 1.35, and 4.05 μg / L.

[0019] Preferably, the washing solution has a pH of 7.4 and contains 0.5%~1.0% Tween 20, 0.01‰~0.03‰ sodium azide preservative, and 0.1~0.3 mol / L phosphate buffer, wherein the percentages are weight-volume percentages.

[0020] This invention also provides a method for using the above-mentioned enzyme-linked immunosorbent assay (ELISA) kit, specifically as follows: Number the microwells corresponding to the test biological product and the standard in sequence. Run two parallel wells for each biological product and the standard, and record the positions of the standard wells and the wells containing the test biological product. Add 20–80 μL of the test biological product and the standard to each well. Then add 20–80 μL of enzyme conjugate working solution (the above enzyme conjugate concentrate is diluted with enzyme conjugate diluent at a volume ratio of 1:10 to obtain the enzyme conjugate working solution). Gently shake to mix, cover with a cover plate, and incubate at room temperature in the dark. Shake off the liquid in the wells, add washing buffer, and wash thoroughly 4–5 times. Add substrate chromogenic solution, gently shake to mix, cover with a cover plate, and incubate at room temperature in the dark. Add stop solution, gently shake to mix, and measure the absorbance of each well using a microplate reader. Calculate the concentrations of vancomycin and ovalbumin in the test biological product using the absorbance values.

[0021] Preferably, the enzyme-linked immunosorbent assay kit has a detection limit of 0.5 μg / L for vancomycin and a detection limit of 200 μg / L for ovalbumin.

[0022] Preferably, the enzyme-linked immunosorbent assay (ELISA) kit has an average recovery rate of 85% to 100% for vancomycin and ovalbumin in biological products, with intra-batch and inter-batch coefficients of variation both less than 10%; the cross-reactivity rate for vancomycin is 100%, and the cross-reactivity rates for polymyxin E, kanamycin, streptomycin, gentamicin, penicillin, tetracycline, chloramphenicol, neomycin, chlortetracycline, oxytetracycline, and doxycycline are all less than or equal to 1%.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The vancomycin hapten of the present invention uses vancomycin as the starting material and undergoes a nucleophilic substitution reaction with N-(P-maleimide phenyl) isocyanate to obtain maleimide-vancomycin. Maleimide-vancomycin is used as the hapten and coupled with a carrier protein to prepare a monoclonal antibody for immunizing animals. When the obtained antibody is made into an enzyme-linked immunosorbent assay kit for the detection of vancomycin in vaccines, the detection limit is 0.5 μg / L, the detection results are accurate and precise. In addition, in the specificity experiment, it shows significant specificity for vancomycin, and the detection results are not affected by the addition of polymyxin E, kanamycin, streptomycin, gentamicin, penicillin, tetracycline, chloramphenicol, neomycin, chlortetracycline, oxytetracycline, doxycycline and other drugs in the sample. When the ovalbumin antibody of the present invention is used to make an enzyme-linked immunosorbent assay kit for detecting ovalbumin contained in vaccines, the detection limit is 200 μg / L, and the detection results have high accuracy and high precision.

[0024] (2) The vancomycin antibody in this invention can react specifically with vancomycin. When it is applied to an enzyme-linked immunosorbent assay kit, the detection time is short, the operation is simple, the cost is low, and it is suitable for testing units to detect a large number of samples at the same time. Attached Figure Description

[0025] Figure 1 The image shows the 1H NMR spectrum of the vancomycin hapten prepared in Example 1. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0027] All raw materials used in this invention are commercially available.

[0028] Example 1: Preparation of vancomycin hapten 1.4 g of vancomycin was dissolved in 80 mL of pyridine, and 0.32 g of N-(P-maleimide-phenyl)isocyanate was added. The mixture was stirred at room temperature for 3 h, and the reaction was stopped. The pyridine was removed by rotary evaporation, and the mixture was recrystallized with 10 mL of anhydrous ethanol to obtain 0.62 g of maleimide-vancomycin, i.e., vancomycin hapten, with a yield of 37.34%. Its 1H NMR spectrum is shown below. Figure 1 As shown, 1 HNMR (500 MHz, Chloroform- d ) δ 9.23 (s, 1H), 9.19 (s, 1H), 8.84 (s, 1H), 8.14– 8.08 (m, 2H), 7.82 (s, 1H), 7.77 – 7.62 (m, 6H), 7.57 (dd, J = 8.7, 5.8 Hz,2H), 7.42 – 7.36 (m, 2H), 7.32 (tdd, J = 7.8, 2.0, 1.0 Hz, 2H), 7.15 – 7.08(m, 4H), 7.08 – 7.02 (m, 2H), 6.83 – 6.77 (m, 2H), 6.73 (d, J = 8.6 Hz, 1H), 6.28 (s, 2H), 6.16 (s, 1H), 5.64 (ddd, J = 8.2, 2.4, 1.5 Hz, 2H), 5.55 (d, J = 9.3 Hz, 1H), 5.36 (dq, J= 6.0, 4.6 Hz, 1H), 5.22 – 5.15 (m, 2H), 5.10(dddt, J = 22.2, 7.1, 5.1, 1.0 Hz, 2H), 4.83 (dd, J = 8.6, 7.0 Hz, 1H), 4.76(d, J = 5.9 Hz, 1H), 4.74 – 4.69 (m, 2H), 4.72 – 4.66 (m, 1H), 4.53 (d, J =5.1 Hz, 1H), 4.30 (d, J = 6.3 Hz, 1H), 4.08 (ddd, J = 9.2, 8.0, 2.6 Hz, 1H),3.92 (dd, J = 6.6, 4.2 Hz, 1H), 3.90 – 3.83 (m, 1H), 3.83 (d, J = 1.5 Hz,1H), 3.83 – 3.80 (m, 1H), 3.78 – 3.69 (m, 2H), 3.67 – 3.59 (m, 1H), 3.62 –3.56 (m, 1H), 3.45 (t, J = 4.5 Hz, 1H), 3.16 (tdq, J = 8.8, 6.0, 1.5 Hz, 1H),2.69 (dd, J = 6.5, 1.2 Hz, 2H), 2.39 (dd, J = 4.6, 1.5 Hz, 3H), 2.27 (dd, J =12.5, 4.9 Hz, 1H), 1.93 (dd, J = 12.4, 2.3 Hz, 1H), 1.70 – 1.58 (m, 1H), 1.56– 1.43 (m, 2H), 1.19 (d, J = 6.1 Hz, 3H), 0.88 (dd, J= 6.6, 0.6 Hz, 6H). The chemical shifts δ = 8.14 – 8.08 (m, 2H), 7.42 – 7.36 (m, 2H) are the hydrogen absorption peaks on the benzene ring of the spacer arm, and 7.15 – 7.08 (m, 4H), 7.08 – 7.02 (m, 2H) are the double bond absorption peaks of the spacer arm. The presence of these peaks proves that the spacer arm coupling was successful, indicating that the hapten was successfully prepared.

[0029] Example 2: Preparation of vancomycin immunogen Take 50 mg of keyhole hemocyanin (KLH), dissolve it in 5 mL of PB buffer, add 1 mL of aqueous solution containing 1.9 mg of dithiothreitol (DDT), and react at room temperature for 30 min to obtain solution A. Take 21 mg of maleimide-vancomycin hapten prepared in Example 1, dissolve it in 0.5 mL of N,N-dimethylformamide, add it to solution A, and react at room temperature for 4 h. Purify by dialyzing with 0.02 M PB buffer for 3 days, changing the medium three times a day to obtain vancomycin-KLH conjugate, which is the vancomycin immunogen. Aliquot and store at -20 ℃ for later use.

[0030] Example 3: Preparation of vancomycin coating agent Take 50 mg of keyhole hemocyanin (KLH), dissolve it in 5 mL of PB buffer, add 1 mL of aqueous solution containing 1.9 mg of dithiothreitol (DDT), and react at room temperature for 30 min to obtain solution A. Take 10 mg of maleimide-vancomycin hapten prepared in Example 1, dissolve it in 0.5 mL of N,N-dimethylformamide, add it to solution A, and react at room temperature for 4 h. Purify by dialyzing with 0.02 M PB buffer for 3 days, changing the medium three times a day to obtain vancomycin-KLH conjugate, which is the vancomycin coating agent. Aliquot and store at -20 ℃ for later use.

[0031] Example 4: Preparation of vancomycin antibody and ovalbumin antibody (1) Animal immunization Vancomycin immunogen was injected into different Balb / c mice at a dose of 150 μg / mouse to induce the production of antiserum.

[0032] Ovalbumin was injected into different Balb / c mice at an immunization dose of 150 μg / mouse to induce the production of antiserum.

[0033] (2) Cell fusion and cloning Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 8:1 (number-matching). The cell supernatant was analyzed using an indirect competitive ELISA method to screen for positive wells. The positive wells were then cloned using a limiting dilution method until a hybridoma cell line stably secreting monoclonal antibodies was obtained.

[0034] (3) Cell cryopreservation and thawing Hybridoma cells were prepared into 1×10⁻⁶ cells using cryopreservation solution. 6 Cell suspensions of cells / mL were stored long-term in liquid nitrogen. Upon thawing, the cryovials were removed and immediately placed in a 37 °C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture flasks for incubation.

[0035] (4) Preparation and purification of monoclonal antibodies Incremental culture method: Hybridoma cells were placed in cell culture medium and cultured at 37 °C. The culture medium was purified using the octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, which were then stored at -20 °C.

[0036] The cell culture medium consisted of RPMI 1640 medium supplemented with fetal bovine serum and sodium bicarbonate, with the final concentration of fetal bovine serum in the cell culture medium being 20 wt% and the final concentration of sodium bicarbonate in the cell culture medium being 0.2 wt%; the pH of the cell culture medium was 7.4.

[0037] Example 5: Preparation of an enzyme-linked immunosorbent assay kit (1) Preparation of ELISA plates coated with coating antigen The vancomycin-coated progenitor and ovalbumin prepared in Example 3 were diluted to 20 μg / mL using coating buffer. 100 μL was added to each well and incubated at 25 °C in the dark for 2 h. The liquid in the well was discarded, and the well was washed twice with washing buffer for 30 s each time. The well was then patted dry. 200 μL of blocking buffer was added to each well and incubated at 25 °C in the dark for 2 h. The liquid in the well was discarded and the well was patted dry. After drying, the well was vacuum sealed with aluminum foil for storage. (2) Prepare 6 bottles of vancomycin standard solution with concentrations of 0, 0.075, 0.225, 0.675, 2.025, and 6.075 µg / L; and 6 bottles of ovalbumin standard solution with concentrations of 0, 0.05, 0.15, 0.45, 1.35, and 4.05 µg / L; (3) Prepare vancomycin antibody labeled with horseradish peroxidase and ovalbumin antibody labeled with horseradish peroxidase; (4) Prepare the substrate colorimetric solution, which consists of solution A and solution B. Solution A is urea peroxide, and solution B is tetramethylbenzidine. (5) Prepare a stop solution with a composition of 2 mol / L sulfuric acid; (6) Prepare a washing solution with a pH of 7.4, containing 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide preservative, and 0.1~0.3 mol / L phosphate buffer, wherein the percentages are weight-volume percentages; (7) Assemble the enzyme-linked immunosorbent assay kit.

[0038] Experimental Example: Detection of Vancomycin and Ovalbumin in Test Biological Products I. Detection of Vancomycin 1. Sample preparation method for the test biological product: Dilute the test biological product with sample diluent to a vancomycin drug content range of 0.075~6.075µg / L, and set aside for use.

[0039] 2. Instructions for using the enzyme-linked immunosorbent assay (ELISA) kit Number the microwells corresponding to the test biopharmaceutical and standard in sequence, and perform two parallel wells for each test biopharmaceutical and standard, recording the positions of the standard wells and the test biopharmaceutical wells. Add 20-80 μL of the test biopharmaceutical and standard to the corresponding microwells, and then add 20-80 μL of enzyme conjugate working solution (the above enzyme conjugate concentrate is diluted with enzyme conjugate diluent at a volume ratio of 1:10 to obtain the enzyme conjugate working solution), gently shake to mix, cover with a cover plate membrane, and incubate at 25 ℃ in the dark for 30 min. Shake off the liquid in the wells, add 250 μL of washing working solution per well, wash thoroughly 4-5 times, with 10 s intervals between each wash, discard the washing solution in the wells, and pat dry with absorbent paper. Add 50 μL of substrate solution A per well, then add 50 μL of substrate solution B per well, gently shake to mix, cover with a cover plate membrane, and incubate at 25 ℃ in the dark for 15 min. Add 50 μL of stop solution per well, gently shake to mix, set the microplate reader to 450 nm, and measure the OD value of each well.

[0040] 3. Analysis of ELISA kit test results The percentage absorbance of the standard or test biological product is equal to the average absorbance value of the standard or test biological product (in a two-well sample) divided by the average absorbance value of the first standard (0 standard), and then multiplied by 100% to obtain the percentage absorbance value of the standard or test biological product. A standard curve is plotted with the percentage absorbance of the standard on the ordinate and the logarithm of the standard concentration (µg / L) on the abscissa. The percentage absorbance of the test biological product is substituted into the standard curve, and the corresponding concentration of the test biological product is read from the standard curve. Multiplying this concentration by the corresponding dilution factor gives the actual concentration of the analyte in the sample.

[0041] 4. Detection limit test According to the above method, 20 blank vaccines were used as samples of the biological products to be tested. The mean and standard deviation of the biological products to be tested were determined. The detection limit of the sample was calculated by adding three times the standard deviation to the mean of the biological products to be tested. The results are shown in Table 1.

[0042] Table 1: Detection limit determination results (unit: μg / L) As shown in Table 1, the detection limit for vancomycin in the vaccine is 0.329 μg / L.

[0043] 5. Accuracy and precision testing The accuracy of enzyme-linked immunosorbent assay (ELISA) is expressed as recovery rate, and the precision is expressed as coefficient of variation. Following the usage and result analysis methods of the ELISA kit described above, blank vaccine samples were added with vancomycin standard to concentrations of 0.5 μg / L, 1.0 μg / L, and 2.0 μg / L, with three replicates for each concentration for each sample. The results are shown in Table 2.

[0044] Table 2: Results of Accuracy and Precision Measurements As shown in Table 2, the average recovery rate of vancomycin in the vaccine by the enzyme-linked immunosorbent assay kit was 86.4% to 93.4%, and the intra-batch and inter-batch coefficients of variation were both 7.0% to 9.4%.

[0045] 6. Vancomycin monoclonal antibody cross-reactivity determination Antibody specificity is evaluated using cross-reactivity rate, which compares the ability of antibodies to bind to antigen analogs and specific antigens. The lower the cross-reactivity rate between an antibody and antigen analogs, and the higher the cross-reactivity rate with specific antigens, the better the antibody's specificity. Drugs such as polymyxin E, kanamycin, streptomycin, gentamicin, penicillin, tetracycline, chloramphenicol, neomycin, chlortetracycline, oxytetracycline, and doxycycline may also be found in vaccines; therefore, these drugs are selected for specificity comparison.

[0046] Table 3: Cross-reactivity rates of vancomycin antibodies As shown in Table 3, the vancomycin antibody is specific to vancomycin and has no cross-reactivity with its structural analogues.

[0047] II. Detection of ovalbumin 1. Sample preparation method for the biological product to be tested: Dilute the biological product to be tested with sample diluent to a concentration of ovalbumin of 0.05~4.05 µg / L, and set aside for use.

[0048] 2. Detection limit test The test was conducted according to the method of the first detection limit, and the results are shown in Table 4.

[0049] Table 4: Detection limit determination results (unit: μg / L) As shown in Table 4, the detection limit for vancomycin in the vaccine is 167.0 μg / L.

[0050] 3. Accuracy and precision testing The accuracy of enzyme-linked immunosorbent assay (ELISA) is expressed as recovery rate, and the precision is expressed as coefficient of variation. Following the usage and result analysis methods of the ELISA kit described above, blank vaccine samples were added with ovalbumin standards to concentrations of 200 μg / L, 400 μg / L, and 800 μg / L, with three replicates for each concentration for each sample. The results are shown in Table 5.

[0051] Table 5: Results of Accuracy and Precision Measurements As shown in Table 5, the average recovery rate of ovalbumin in the vaccine by the enzyme-linked immunosorbent assay kit was 85.0% to 93.4%, and the intra-batch and inter-batch coefficients of variation were both 6.2% to 8.8%.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing antibodies to detect ovalbumin and vancomycin in biological products, characterized in that, The ovalbumin antibody was obtained by direct immunization of animals, and the vancomycin antibody was obtained by preparing a vancomycin immunogen and then immunizing animals. The vancomycin immunogen was obtained by conjugating a vancomycin hapten with a carrier protein. The structure of the vancomycin hapten is shown in formula (I). Equation (Ⅰ).

2. The method for preparing antibodies for detecting ovalbumin and vancomycin in biological products according to claim 1, characterized in that, The method for preparing the vancomycin hapten includes: preparing it by nucleophilic substitution reaction of vancomycin with N-(P-maleimide phenyl) isocyanate.

3. The method for preparing antibodies to detect ovalbumin and vancomycin in biological products according to claim 1, characterized in that, The carrier protein is bovine serum albumin, ovalbumin, human serum albumin, or keyhole hemocyanin.

4. An enzyme-linked immunosorbent assay (ELISA) kit, comprising: The enzyme-labeled plate is coated with a coating agent, a standard solution, an antibody, an enzyme conjugate concentrate, an enzyme conjugate dilution, a substrate chromogenic solution, a stop solution, and a washing solution. The coating agents are vancomycin coating agent and ovalbumin, respectively. The antibodies are vancomycin antibody and ovalbumin antibody prepared by any of the preparation methods described in claims 1 to 3, respectively. The enzyme conjugates are enzyme-labeled vancomycin antibody and ovalbumin antibody, respectively.

5. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 4, characterized in that, The labeled enzyme of the enzyme conjugate is horseradish peroxidase or bacterial-extracted alkaline phosphatase.

6. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 5, characterized in that, The enzyme conjugate is labeled with horseradish peroxidase; the enzyme conjugate is obtained by conjugating the labeled enzyme with the aforementioned vancomycin antibody and ovalbumin antibody.

7. The enzyme-linked immunosorbent assay kit according to claim 6, characterized in that, When the labeled enzyme is horseradish peroxidase, the substrate colorimetric solution consists of substrate solution A and substrate solution B, where substrate solution A is urea peroxide and substrate solution B is tetramethylbenzidine; the stop solution is a 1-2 mol / L sulfuric acid solution.

8. The enzyme-linked immunosorbent assay kit according to claim 4, characterized in that, The standard solutions are vancomycin standard solution and ovalbumin standard solution, respectively. The concentrations of the vancomycin standard solution are 0, 0.075, 0.225, 0.675, 2.025, and 6.075 μg / L, respectively, and the concentrations of the ovalbumin standard solution are 0, 0.05, 0.15, 0.45, 1.35, and 4.05 μg / L, respectively.

9. The enzyme-linked immunosorbent assay kit according to any one of claims 4 to 8, characterized in that, The enzyme-linked immunosorbent assay kit described above has a detection limit of 0.5 μg / L for vancomycin and a detection limit of 200 μg / L for ovalbumin.

10. The enzyme-linked immunosorbent assay kit according to any one of claims 4 to 8, characterized in that, The enzyme-linked immunosorbent assay (ELISA) kit showed an average recovery rate of 85%–100% for vancomycin and ovalbumin in biological products, with intra-assay and inter-assay coefficients of variation both less than 10%. The cross-reactivity rate for vancomycin was 100%, and the cross-reactivity rates for polymyxin E, kanamycin, streptomycin, gentamicin, penicillin, tetracycline, chloramphenicol, neomycin, chlortetracycline, oxytetracycline, and doxycycline were all less than or equal to 1%.

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