Artificial complex antigens, broad-spectrum detection antibodies, and ELISA detection methods for sulfonamides and tetracycline antibiotics

Abstract

This invention provides an artificial complex antigen for sulfonamides and tetracyclines, a broad-spectrum detection antibody, and an ELISA detection method, belonging to the field of immunoassay technology. This invention constructs an artificial complex antigen for sulfonamides (SAs) and tetracyclines (TCs). A sulfonamide fragment and a tetracycline fragment are introduced into the artificial antigen, and the sulfonamide fragment and tetracycline fragment are linked to a carrier protein via a glutaraldehyde linker arm to obtain the artificial complex antigen for sulfonamides and tetracyclines. After immunization with animals, this artificial complex antigen yields a broad-spectrum antibody with high specificity and titer against SAs and TCs, allowing direct detection of the total amount of SAs and TCs in a sample. This invention also provides an ELISA detection method for the total amount of sulfonamides and tetracyclines, which exhibits good linearity for both SAs and TCs.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to artificial complex antigens, broad-spectrum detection antibodies, and ELISA detection methods for sulfonamides and tetracycline antibiotics. Background Technology

[0002] Antibiotics can enter the environment at any stage from production to consumption. Residual antibiotics in the environment can lead to the development of antibiotic resistance genes and alter the microbial ecosystem. Furthermore, due to accumulation in the food chain, antibiotics can cause unpredictable harm to humans. Currently, antibiotic pollution of water bodies is increasingly serious, with medical and health services, livestock farming, and aquaculture being the main sources of antibiotic pollution in environmental waters. Although the degree of pollution varies greatly from place to place, the detection frequency is high. In my country, the types of antibiotics with the highest detected concentrations are, in descending order: sulfonamides, tetracyclines, macrolides, quinolones, and chloramphenicol. Therefore, developing rapid and efficient detection methods for antibiotics is particularly important.

[0003] Currently, the main methods for detecting antibiotics in water bodies, both domestically and internationally, are instrumental analysis methods. These methods have high requirements for instrument conditions, and the pretreatment procedures are relatively complex and time-consuming. ELISA, utilizing antigen-antibody specific reactions, effectively compensates for the shortcomings of instrumental analysis methods. Because it does not require expensive equipment and offers high specificity, high sensitivity, and simple and rapid operation, it has shown unique advantages in on-site screening and rapid detection of large numbers of samples.

[0004] Current research has explored ELISA methods for detecting antibiotics in water. These methods all employ a carrier protein coupled with a hapten to prepare an artificial antigen, which is then used to immunize animals to prepare specific polyclonal / monoclonal antibodies, leading to the development of corresponding ELISA detection methods. However, given the wide variety of antibiotics, a single ELISA method can only measure the content of one type of antibiotic. If it is necessary to determine the total amount of a particular class of antibiotics or several classes of antibiotics in a sample, multiple measurements are required, followed by data aggregation, inevitably increasing workload and extending detection time. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an artificial complex antigen for sulfonamides and tetracyclines, a broad-spectrum detection antibody, and an ELISA detection method thereof. The artificial complex antigen provided by this invention can be used to prepare a broad-spectrum antibody that can simultaneously detect multiple antibiotics, enabling the simultaneous detection of sulfonamides and tetracyclines.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an artificial composite antigen having the structure shown in Formula I:

[0008]

[0009] In Formula I, “N-Protein-N” represents the carrier protein.

[0010] Preferably, the carrier protein is bovine serum albumin or ovalbumin.

[0011] This invention provides a method for preparing the above-mentioned artificial composite antigen, comprising the following steps:

[0012] Sulfonamide, glutaraldehyde, carrier protein, alcohol solvent and buffer solution were mixed and subjected to the first coupling reaction to obtain a compound having the structure shown in formula a;

[0013]

[0014] The compound having the structure shown in Formula a, tetracycline, and glutaraldehyde are mixed and subjected to a second coupling reaction to obtain an artificial complex antigen having the structure shown in Formula I.

[0015] Preferably, the temperature of the first coupling reaction is 22-26°C and the time is 4-5 hours;

[0016] The second coupling reaction is carried out at a temperature of 22–26°C for 4–5 hours.

[0017] This invention provides the application of the above-mentioned artificial composite antigen in the preparation of broad-spectrum detection antibodies for sulfonamides and tetracyclines.

[0018] This invention provides a broad-spectrum detection antibody for sulfonamides and tetracyclines, obtained by immunizing host animals with the aforementioned artificial composite antigen.

[0019] The present invention provides a kit for detecting sulfonamides and / or tetracyclines, comprising a coating antigen, a blocking agent, a broad-spectrum detection antibody for sulfonamides and tetracyclines, an enzyme-labeled secondary antibody, a chromogenic agent, and an enzyme-labeled plate;

[0020] The coating antigen is the aforementioned artificial composite antigen;

[0021] The carrier protein in the coated antigen structure is different from the carrier protein in the artificial composite antigen structure used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines.

[0022] Preferably, the carrier protein in the coated antigen structure is ovalbumin, and the carrier protein in the artificial composite antigen structure used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines is bovine serum albumin.

[0023] Alternatively, the carrier protein in the coated antigen structure may be bovine serum albumin, and the carrier protein in the artificial composite antigen structure used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines may be ovalbumin.

[0024] This invention provides an ELISA method for detecting the total amount of sulfonamides and tetracyclines, comprising the following steps:

[0025] Add the buffer solution containing the coating antigen to the ELISA plate and perform the first incubation to obtain the ELISA plate coated with the coating antigen.

[0026] The blocking agent is added to the enzyme-labeled plate coated with the coated antigen, a second incubation is performed, and the unbound material is washed away to obtain the blocked enzyme-labeled plate.

[0027] The test solution and broad-spectrum detection antibodies against sulfonamides and tetracyclines were added to the blocked ELISA plate, and a third incubation was performed. Unbound material was washed away to obtain the ELISA plate after competitive binding.

[0028] The enzyme-labeled secondary antibody was added to the enzyme-labeled plate after competitive binding, and a fourth incubation was performed. Unbound material was washed away, and then a colorimetric reagent was added to perform a colorimetric reaction. The absorbance of the resulting colorimetric reaction solution at 492 nm was measured.

[0029] The inhibition rate is calculated based on the absorbance value;

[0030] The total concentrations of sulfonamide and tetracycline antibiotics in the test solution were obtained based on the inhibition rate and the standard curve; the standard curve was a linear relationship curve between the natural logarithm of the total concentrations of sulfonamide and tetracycline antibiotics and the inhibition rate.

[0031] Preferably, the sulfonamide antibiotic is one or more of sulfonamides, sulfapyridine, sulfadiazine, and sulfathiazole;

[0032] The tetracycline antibiotics are one or more of tetracycline, chlortetracycline, oxytetracycline, and cyclocycline.

[0033] This invention provides an artificial composite antigen with the structure shown in Formula I. This invention constructs an artificial composite antigen for sulfonamides (SAs) and tetracyclines (TCs) antibiotics. A sulfonamide fragment and a tetracycline fragment are introduced into the artificial antigen, and the sulfonamide fragment and the tetracycline fragment are linked to a carrier protein via a glutaraldehyde linker arm to obtain the artificial composite antigen for sulfonamides and tetracyclines. After immunization with animals, this artificial composite antigen yields broad-spectrum antibodies with high specificity and titer against SAs and TCs, allowing direct detection of the total amount of SAs and TCs in a sample.

[0034] This invention provides an ELISA method for detecting the total amount of sulfonamides and tetracyclines, which exhibits good linearity for both SAs and TCs. Example results show that the ELISA method provided by this invention has good IC50 accuracy for sulfonamides (SA), sulfapyridine (SP), sulfadiazine (SD), sulfathiazole (ST), tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and cyclomycin (DC). 50 The concentrations were 16.3, 21.8, 18.3, 20.5, 17.3, 22.3, 17.9, and 20.2 ng / mL, respectively, with a detection limit IC50 of [missing value]. 10 The specific concentrations were 0.46, 0.54, 0.48, 0.53, 0.49, 0.51, 0.50, and 0.49 ng / mL, respectively, demonstrating high sensitivity and low detection limits. The recoveries of SAs and TCs in single-spike water samples ranged from 97.12% to 98.87%, effectively detecting all of these antibiotics in water. The recoveries of total SAs and TCs in mixed-spike water samples ranged from 96.25% to 98.56%, effectively detecting the total amount of SAs and TCs in water. Attached Figure Description

[0035] Figure 1 This is the synthetic route for the artificial composite antigen of the present invention;

[0036] Figure 2 The image shows the ultraviolet spectral scanning results of the artificial composite antigen obtained in this invention. Detailed Implementation

[0037] This invention provides an artificial composite antigen having the structure shown in Formula I:

[0038]

[0039] In this invention, "N-Protein-N" represents a carrier protein, and the "N" in "N-Protein-N" indicates that the carrier protein is linked to the glutaraldehyde structure through "N".

[0040] In this invention, the carrier protein is preferably bovine serum albumin (BSA) or ovalbumin (OVA).

[0041] In this invention, when the carrier protein is bovine serum albumin, the artificial complex antigen is designated as SA-BSA-TC; when the carrier protein is ovalbumin, the artificial complex antigen is designated as SA-OVA-TC.

[0042] This invention provides a method for preparing the above-mentioned artificial composite antigen, comprising the following steps:

[0043] Sulfonamide, glutaraldehyde, carrier protein, alcohol solvent and buffer solution were mixed and subjected to the first coupling reaction to obtain a compound having the structure shown in formula a;

[0044]

[0045] The compound having the structure shown in Formula a, tetracycline, and glutaraldehyde are mixed and subjected to a second coupling reaction to obtain an artificial complex antigen having the structure shown in Formula I.

[0046] This invention involves mixing sulfonamide, glutaraldehyde, a carrier protein, an alcohol solvent, and a buffer solution to perform a first coupling reaction, yielding a compound having the structure shown in formula a. In this invention, the alcohol solvent is preferably methanol. In this invention, the buffer solution is preferably a PBS buffer solution. In this invention, the pH value of the PBS buffer solution is preferably 7.2, and the concentration is preferably 0.01 mol / L. This invention does not have special requirements for the mixing method; any mixing method well known to those skilled in the art can be used, such as stirring.

[0047] In this invention, the volume ratio of methanol to PBS is preferably 1:1.

[0048] In this invention, the molar ratio of sulfonamide, glutaraldehyde, and carrier protein is preferably 33:178:1 to 1.4; in this invention, when the carrier protein is bovine serum albumin, the molar ratio of sulfonamide, glutaraldehyde, and carrier protein is preferably 33:178:1.4, and when the carrier protein is ovalbumin, the molar ratio of sulfonamide, glutaraldehyde, and carrier protein is preferably 33:178:1.

[0049] In this invention, the temperature of the first coupling reaction is preferably 22-26°C, more preferably room temperature, and the time is preferably 4-5 hours.

[0050] This invention involves mixing the compound having the structure shown in Formula a, tetracycline, and glutaraldehyde, and performing a second coupling reaction to obtain an artificial complex antigen having the structure shown in Formula I. This invention does not have special requirements for the mixing method; any mixing method well known to those skilled in the art can be used, such as stirring.

[0051] In this invention, the molar ratio of the compound having the structure shown in Formula a, tetracycline, and glutaraldehyde is preferably 1:1:5 to 5.5.

[0052] In this invention, the temperature of the second coupling reaction is preferably 22-26°C, more preferably room temperature, and the time is preferably 4-5 hours.

[0053] After obtaining the second coupling reaction, the present invention preferably dialyzes the resulting second coupling reaction solution. In the present invention, the dialysis is preferably performed in PBS buffer solution, and the molecular weight cutoff of the dialysis bag used for the dialysis is preferably 8000-10000 Da; in the present invention, the dialysis time is preferably 72 h, and the temperature is preferably 4 °C; during the dialysis process, the solution is changed every 12 hours.

[0054] This invention provides the application of the above-mentioned artificial composite antigen in the preparation of broad-spectrum detection antibodies for sulfonamides and tetracyclines.

[0055] This invention provides a broad-spectrum detection antibody for sulfonamides and tetracyclines, obtained by immunizing host animals with the aforementioned artificial composite antigen. In this invention, the host animal is preferably a mouse or rabbit. This invention does not specify any particular method for immunizing the host animal; methods well-known to those skilled in the art can be used.

[0056] As a specific embodiment of the present invention, when the host animal is a mouse, the method of immunizing the host animal preferably includes the following steps:

[0057] Artificial composite antigen and adjuvant were injected into mice on days 0, 3, 28, and 49, respectively. After immunization, blood was collected from the tail of the mice to obtain antiserum, which contained broad-spectrum antibodies for detecting sulfonamides and tetracyclines.

[0058] This invention provides a kit for detecting sulfonamides and / or tetracycline antibiotics, comprising a coating antigen, a blocking agent, a broad-spectrum detection antibody for the above-mentioned sulfonamides and tetracycline antibiotics, an enzyme-labeled secondary antibody, a chromogenic agent, and an enzyme-labeled plate;

[0059] The coating antigen is the aforementioned artificial composite antigen;

[0060] The carrier protein in the coated antigen structure is different from the carrier protein in the artificial composite antigen structure used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines. In this invention, the artificial composite antigen used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines is an immunogenic antigen.

[0061] In this invention, the sulfonamide antibiotic is preferably one or more of sulfonamide (SA), sulfapyridine (SP), sulfadiazine (SD), and sulfathiazole (ST);

[0062] The tetracycline antibiotics mentioned are one or more of tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and cyclomycin (DC).

[0063] In this invention, the chemical formulas of sulfonamide (SA), sulfapyridine (SP), sulfadiazine (SD), sulfathiazole (ST), tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and cyclomycin (DC) are as follows:

[0064]

[0065] In this invention, the carrier protein in the coating antigen structure is preferably ovalbumin, and the carrier protein in the artificial composite antigen structure used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines is bovine serum albumin. That is, in this invention, the coating antigen is SA-OVA-TC, and the immunogenic antigen is SA-BSA-TC.

[0066] Alternatively, the carrier protein in the coating antigen structure may be bovine serum albumin, and the carrier protein in the artificial composite antigen structure used to prepare broad-spectrum detection antibodies against sulfonamides and tetracyclines may be ovalbumin. That is, in this invention, the coating antigen is SA-BSA-TC, and the immunogenic antigen is SA-BSA-TC.

[0067] In this invention, the sealing agent is preferably one or more of bovine serum albumin, ovalbumin, gelatin, and skim milk powder.

[0068] In this invention, the enzyme-labeled secondary antibody is preferably HRP-labeled goat anti-mouse IgG.

[0069] In this invention, the colorimetric agent is preferably a mixture of o-phenylenediamine, Na2HPO4, citric acid, H2O2 and water.

[0070] There are no special requirements in this field regarding the type of ELISA plate; any ELISA plate familiar to those skilled in the art can be used.

[0071] This invention provides an ELISA method for detecting the total amount of sulfonamides and tetracyclines, comprising the following steps:

[0072] Add the buffer solution containing the coating antigen to the ELISA plate and perform the first incubation to obtain the ELISA plate coated with the coating antigen.

[0073] The blocking agent is added to the enzyme-labeled plate coated with the coated antigen, a second incubation is performed, and the unbound material is washed away to obtain the blocked enzyme-labeled plate.

[0074] The test solution and broad-spectrum detection antibodies against sulfonamides and tetracyclines were added to the blocked ELISA plate, and a third incubation was performed. Unbound material was washed away to obtain the ELISA plate after competitive binding.

[0075] The enzyme-labeled secondary antibody was added to the enzyme-labeled plate after competitive binding, and a fourth incubation was performed. Unbound material was washed away, and then a colorimetric reagent was added to perform a colorimetric reaction. The absorbance of the resulting colorimetric reaction solution at 492 nm was measured.

[0076] The inhibition rate of the antibiotic against the antibody was calculated based on the absorbance value.

[0077] The total concentrations of sulfonamide antibiotics and tetracycline antibiotics in the test solution were obtained based on the inhibition rate and the standard curve.

[0078] The standard curve is a linear relationship between the natural logarithm of the total concentration of sulfonamide antibiotics and tetracycline antibiotics and the inhibition rate.

[0079] This invention involves adding a buffer solution containing the coated antigen to an ELISA plate and performing a first incubation to obtain an ELISA plate coated with the coated antigen. In this invention, the concentration of the buffer solution containing the coated antigen is preferably 0.125–2 μg / mL, specifically preferably 2, 1, 0.5, 0.25, or 0.125 μg / mL, and most preferably 0.5 μg / mL. In this invention, the preferred amount of buffer solution containing the coated antigen added is 100 μL / well.

[0080] In this invention, the temperature of the first incubation is preferably 4°C, and the incubation time is preferably overnight.

[0081] This invention involves adding a blocking agent to an ELISA plate coated with the antigen, performing a second incubation, and washing away unbound material to obtain a blocked ELISA plate. In this invention, the blocking agent is preferably one or more of bovine serum albumin, ovalbumin, gelatin, and skim milk powder, with bovine serum albumin being the most preferred. In this invention, the concentration of the blocking agent is preferably 1 wt%, and the addition amount is preferably 250 μL / well.

[0082] In this invention, the second incubation temperature is preferably 37°C, and the incubation time is preferably 1 hour.

[0083] In this invention, the method of washing away unbound material is preferably washing with a buffer solution.

[0084] In this invention, the test solution and broad-spectrum detection antibodies against sulfonamides and tetracyclines are added to the blocked ELISA plate, followed by a third incubation. Unbound material is then washed away to obtain a competitively bound ELISA plate. In this invention, the test solution is preferably water, serum, or skim milk.

[0085] In this invention, the dilution ratio of the broad-spectrum detection antibodies against sulfonamides and tetracyclines is preferably 1:16000 to 1:512000, specifically preferably 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, and most preferably 1:128000.

[0086] In this invention, the amount of the test solution added is preferably 50 μL / well, and the amount of the broad-spectrum detection antibody for sulfonamides and tetracyclines added is preferably 50 μL / well.

[0087] In this invention, the temperature of the third incubation is preferably 4–40°C, more preferably 25–35°C, and most preferably 30°C. In this invention, the time of the third incubation is preferably 1 hour.

[0088] In this invention, the method of washing away unbound material is preferably washing with a buffer solution.

[0089] In this invention, the enzyme-labeled secondary antibody is added to the competitively bound ELISA plate, incubated for a fourth time, and unbound material is washed away. Then, a chromogenic reagent is added to initiate a colorimetric reaction, and the absorbance of the resulting chromogenic reaction solution is measured at 492 nm. In this invention, the enzyme-labeled secondary antibody is selected as HRP-labeled goat anti-mouse IgG. Preferably, the dilution factor of the enzyme-labeled secondary antibody is 1:64000, and the preferred addition amount is 50 μL / well.

[0090] In this invention, the fourth incubation temperature is preferably 37°C, and the incubation time is preferably 1 hour.

[0091] In this invention, the method of washing away unbound material is preferably washing with a buffer solution.

[0092] In this invention, the amount of the colorimetric reagent added is preferably 100 μL / well. In this invention, the temperature of the colorimetric reaction is preferably room temperature, and the time is preferably 15 min. In this invention, the colorimetric reaction is preferably carried out under light-protected conditions.

[0093] This invention calculates the inhibition rate based on the absorbance value. In this invention, the method for calculating the inhibition rate is as follows:

[0094] Inhibition rate (%) = (AA) i ) / (A-A0)

[0095] A: Absorbance of positive control well (50 μL antibody dilution buffer + 50 μL broad-spectrum antibody at 2x concentration)

[0096] A i : Absorbance of the well containing antibiotic concentration i

[0097] A0: Absorbance of blank control well (100 μL antibody dilution).

[0098] The present invention obtains the total concentration of sulfonamide antibiotics and tetracycline antibiotics in the test solution based on the inhibition rate and the standard curve; the standard curve is a linear relationship curve between the natural logarithm of the total concentration of sulfonamide antibiotics and tetracycline antibiotics and the inhibition rate.

[0099] In this invention, the method for plotting the standard curve preferably includes the following steps:

[0100] A series of standard solutions of sulfonamide and / or tetracycline antibiotics with known concentrations are provided. When the standard solutions include both sulfonamide and tetracycline antibiotics, the concentration of the standard solutions is the total concentration of the sulfonamide and tetracycline antibiotics. In this invention, the concentrations of the standard solutions of sulfonamide and / or tetracycline antibiotics with known concentrations are preferably 1, 2.5, 5, 10, 25, 50, or 100 ng / mL.

[0101] Using standard solutions of sulfonamides and / or tetracyclines with known gradient concentrations as test solutions, the inhibition rates corresponding to the standard solutions of sulfonamides and / or tetracyclines with known gradient concentrations were obtained according to the above method. A standard curve was plotted with the inhibition rate on the ordinate and the natural logarithm of the antibiotic concentration on the abscissa.

[0102] The artificial complex antigens, broad-spectrum detection antibodies, and ELISA detection methods for sulfonamide and tetracycline antibiotics provided by the present invention will be described in detail below with reference to the embodiments. However, these should not be construed as limiting the scope of protection of the present invention.

[0103] Example 1: Synthesis of Artificial Complex Antigen

[0104] Prepare a methanol / PBS (pH = 7.2, 0.01 mol / L) mixed solution at a volume ratio of 1:1. Add 80 mg SA to 20 mL of the methanol / PBS mixed solution and mix thoroughly. Add 1.4 g BSA or 0.6 g OVA to 50 mL of PBS solution and mix thoroughly. Add 1 mL of 25% GA and stir at room temperature for 4 h to prepare the artificial antigen SA-BSA / SA-OVA. Then add 200 mg TC, mix thoroughly, and add another 1 mL of 25% GA. Stir at room temperature for 4 h to prepare the composite artificial antigen SA-BSA-TC / SA-OVA-TC. Place the solution in a dialysis bag and dialyze with PBS (pH = 7.2, 0.01 mol / L) at 4 °C for 72 h, changing the solution every 12 hours. After dialysis, determine the protein content of the dialysis product and store it at -20 °C for later use.

[0105] The synthetic route of the artificial composite antigen of this invention is as follows: Figure 1 As shown.

[0106] After diluting the composite artificial antigen to 1 mg / mL (based on protein concentration), ultraviolet spectroscopy was performed, and the results are as follows. Figure 2 As shown. By Figure 2 It can be seen that SA-BSA-TC and SA-OVA-TC have the light absorption characteristics of BSA, OVA, TC and SA. At the same time, the maximum absorption peak has shifted to a certain extent, which shows its own characteristics, indicating that SA and TC are successfully coupled with BSA and OVA.

[0107] Example 2: Preparation of broad-spectrum detection antibodies

[0108] BALB / c female mice were immunized using the immunization protocol shown in Table 1. Each mouse was immunized four times on days 0, 3, 28, and 49. For each immunization, 100 μg, 50 μg, 50 μg, and 50 μg of artificial complex antigen were mixed with equal volumes of FCA, FCA, and FICA to prepare immunizing agents. Mice were immunized by subcutaneous injection at multiple sites on the back of the neck.

[0109] Table 1 Immunization regimen

[0110]

[0111] Blood was collected from mouse tails to obtain antiserum. The titer of the antiserum against SAs and TCs antibiotics was determined using an indirect noncompetitive ELISA (inELISA), and the sensitivity to SAs and TCs antibiotics was determined using an indirect competitive ELISA (icELISA). This method was used to detect the specificity of the composite antibody against SAs and TCs antibiotics. Antibody IgG was separated using a saturated ammonium sulfate precipitation method.

[0112] The specific steps for determining antiserum titer are as follows (in ELISA):

[0113] ① Coating: Dilute the coating antigen to 1 μg / mL with coating buffer, add 100 μL / well to the microplate, and coat overnight at 4℃;

[0114] ② Blocking: Pour out the liquid from the wells of the microplate and blot dry. Add 250 μL of blocking buffer to each well, incubate at 37°C for 1 hour, pour out the liquid from the wells, and blot dry. Add 200 μL of washing buffer to each well, gently shake for 90 seconds, pour out the liquid from the wells, and blot dry. Repeat washing 3 times.

[0115] ③ Add the serum to be tested: Dilute the antiserum serially from 1:4000 to 1:256000 using antibody dilution buffer, 100 μL / well. Incubate at 37℃ for 1 h, pour out the liquid from the wells, and blot dry. Wash 3 times.

[0116] ④ Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse IgG1 at a ratio of 1:10000 with antibody dilution buffer, 100 μL / well. Incubate at 37℃ for 1 h, pour out the liquid from the wells, and pat dry. Wash 3 times.

[0117] ⑤ Color development: Add 100 μL of freshly prepared substrate color development solution to each well and react at room temperature in the dark for 15 min.

[0118] ⑥ Termination: Add 50 μL of termination solution to each well.

[0119] ⑦ Result determination: The absorbance at 492 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0120] The titer of the antiserum is calculated using the midpoint titer, which is the serum dilution at which the absorbance value is closest to 1.0. This dilution is then used as the working concentration of the antibody to determine its sensitivity.

[0121] The specific steps for determining the sensitivity of antiserum are as follows (icELISA):

[0122] ① Wrapped: Same as above.

[0123] ② Closed: Same as above.

[0124] ③ Competition: Various antibiotic standard solutions were serially diluted 2-fold from 4000 μg / L to 7.812 μg / L using antibody dilution buffer. The solutions were added to 96-well plates at 50 μL / well. Then, 50 μL of antiserum at twice the working concentration was added to each well, and the mixture was thoroughly mixed. A positive control well was included (50 μL of pure water + 50 μL of antiserum at twice the working concentration). The plates were incubated at 37°C for 1 hour. The liquid in the wells was poured out and patted dry. The plates were washed 3 times.

[0125] ④ The subsequent steps of adding enzyme-labeled secondary antibody, color development, termination, and result determination are the same as above.

[0126] A regression analysis was performed with the inhibition rate on the ordinate and the natural logarithm of the antibiotic concentration on the x-axis to calculate the IC50 concentration of the antibiotic that produces 50% inhibition. 50 IC 50 The lower the value, the higher the sensitivity.

[0127] The titer and sensitivity of mouse antiserum are shown in Table 2.

[0128] Table 2. Titer and Sensitivity of Mouse Antiserum

[0129]

[0130] As shown in Table 2, when SA-BSA-TC is used as the immunogen and SA-OVA-TC is used as the coating antigen, the resulting broad-spectrum detection antibody has good specificity and high sensitivity.

[0131] Example 3: Establishment and Specificity Detection of ELISA Detection Method

[0132] (1) Determination of coating agent concentration and antibody dilution ratio

[0133] Dilute the coating stock (SA-OVA-TC) to concentrations of 2, 1, 0.5, 0.25, and 0.125 μg / mL, and coat the ELISA plates overnight at 4°C. Dilute the broad-spectrum antibody to 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000, and perform checkerboard titration. Take the OD... 450 Combinations with IC50 values ​​around 1 were used for icELISA assays. The optimal combination was the one with the highest IC50 values ​​among all combinations. 50 The combination with the smallest value.

[0134] The specific steps of square titration are as follows:

[0135] The hapten was coated at concentrations of 2, 1, 0.5, 0.25, and 0.125 μg / mL and incubated overnight at 4°C. The broad-spectrum antibody was diluted at 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000 and then subjected to checkerboard titration.

[0136] ① Wrapped: Same as above.

[0137] ② Closed: Same as above.

[0138] ③ Add broad-spectrum antibody: Add broad-spectrum antibody at different dilution ratios, 100 μL / well, and incubate at 37℃ for 1 h. Pour out the liquid from the wells and pat dry. Wash 3 times.

[0139] ④ Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse IgG1 at a ratio of 1:10000 with antibody dilution buffer, 100 μL / well. Incubate at 37℃ for 1 h, pour out the liquid from the wells, and pat dry. Wash 3 times.

[0140] ⑥ Color development: Same as above.

[0141] ⑦ Termination: Same as above.

[0142] ⑧ Result determination: Same as above.

[0143] A combination of hapten concentration and monoclonal antibody dilution with absorbance values ​​close to 1.0 was selected for indirect competitive ELISA with hapten coating. The results are shown in Table 3.

[0144] Table 3 Screening based on original coating concentration and antibody dilution factor

[0145]

[0146] Table 4 shows that when the original coating concentration is 0.5 μg / mL and the antibody dilution ratio is 128000, the IC50 against most SA and TC antibiotics is [data missing]. 50 The optimal combination is chosen to have the lowest possible concentration and highest sensitivity, with an original coating concentration of 0.5 μg / mL and an antibody dilution ratio of 128,000.

[0147] (2) Determination of reaction temperature for ELISA competitive reaction system

[0148] Under the conditions of a coating concentration of 0.5 μg / mL and an antibody dilution ratio of 128000, the same icELISA method as in 3(1) was used to determine the IC50 using different reaction temperatures (4, 25, 30, 35, 40℃). 50 Select IC 50 The temperature at which the minimum temperature is reached is determined as the optimal reaction temperature, and the results are shown in Table 4.

[0149] Table 4 Screening of Optimal Reaction Temperatures

[0150]

[0151] Table 4 shows that at a reaction temperature of 30℃, the IC50 for most SAs and TCs antibiotics is... 50 The values ​​are all the lowest and the sensitivity is the highest, therefore 30℃ is chosen as the optimal reaction temperature for the competitive reaction.

[0152] (3) Determination of pH of ELISA competitive reaction system

[0153] Under the conditions of a coating agent concentration of 0.5 μg / mL, an antibody dilution ratio of 128000, and a competitive reaction temperature of 30℃, the same icELISA method as in step (1) of Example 3 was used to determine the IC50 using different reaction pH values ​​(6.4, 7.4, 8.4, 9.4). 50 The pH at which the IC50 is minimized is selected as the optimal reaction pH, and the results are shown in Table 5.

[0154] Table 5. Screening for optimal reaction pH

[0155]

[0156] As shown in Table 5, the IC50 for most SAs and TCs antibiotics is the lowest and the sensitivity is the highest when the reaction pH is 7.4. Therefore, pH 7.4 is selected as the optimal pH for the competitive reaction.

[0157] (4) Determination of blocking agent for ELISA competitive reaction system

[0158] Under the conditions of a coating agent concentration of 0.5 μg / mL, an antibody dilution ratio of 128000, a competitive reaction temperature of 30℃, and pH = 7.4, the same icELISA method as in 3(1) was used, with different blocking agents (1% BSA, 1% OVA, 1% gelatin, 3% skim milk powder) selected to determine the IC50. 50 Select IC 50 The pH value at which the reaction is minimized was determined to be the optimal blocking agent, and the results are shown in Table 6.

[0159] Table 6 Screening of Optimal Reaction Blocking Agents

[0160]

[0161] As shown in Tables 3-6, the optimized ELISA detection method based on broad-spectrum antibodies against sulfonamides and tetracyclines is as follows: using 0.5 μg / mL SA-OVA-TC as the coating agent, the broad-spectrum antibody dilution ratio is 1:128000, the competitive reaction system temperature is 30℃, pH is 7.4, and 1% BSA is used as the blocking agent.

[0162] (5) Establishment of the standard curve

[0163] Using the established ELISA detection method, antibiotic standard solutions of different concentrations (0.5, 1, 2.5, 5, 10, 25, 50 ng / mL) were used to competitively react with broad-spectrum antibodies. The inhibition rate was plotted on the ordinate, and lg(10×C) was plotted on the other. 标准品 (C) 标准品 Establish a standard curve with the concentration of the standard on the x-axis and calculate the IC50. 50 and detection lower limit IC 10 .

[0164] Specific steps for establishing a standard curve (icELISA):

[0165] ① Coating: Dilute the coating antigen to 0.5 μg / mL with coating buffer, add 100 μL / well to the microplate, and coat overnight at 4℃.

[0166] ② Blocking: Pour out the liquid from the wells of the microplate and blot dry. Add 250 μL of 1% BSA to each well, incubate at 37°C for 1 h, pour out the liquid from the wells, and blot dry. Add 200 μL of washing buffer to each well, gently shake for 90 s, pour out the liquid from the wells, and blot dry. Repeat washing 3 times.

[0167] ③ Competition: Antibiotic standard solutions (SAs) and TCs, as well as randomly mixed SAs and TCs samples, were serially diluted to 1, 2.5, 5, 10, 25, 50, and 100 ng / mL and added to 96-well plates (50 μL / well). Then, 50 μL of a 1:64000 diluted composite antibody was added to each well and mixed thoroughly, resulting in antibiotic standard solution concentrations of 1, 2.5, 5, 10, 25, 50, and 100 ng / mL, and a final composite antibody dilution ratio of 1:128000. A positive control well was also included (50 μL of pure water + 50 μL of a 1:64000 diluted composite antibody). The plates were incubated at 37°C for 1 hour. The liquid in the wells was poured out and patted dry. The plates were washed three times.

[0168] ④ Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse IgG1 at a ratio of 1:10000 with antibody dilution buffer, 100 μL / well. Incubate at 37℃ for 1 h, pour out the liquid from the wells, and pat dry. Wash 3 times.

[0169] ⑤ Color development: Add 100 μL of freshly prepared substrate color development solution to each well and react at room temperature in the dark for 15 min.

[0170] ⑥ Termination: Add 50 μL of termination solution to each well.

[0171] ⑦ Result determination: The absorbance at 492 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0172] A regression analysis was performed with the inhibition rate on the ordinate and the natural logarithm of the antibiotic concentration on the x-axis to calculate the IC50 concentration of the antibiotic that produces 50% inhibition. 50 IC 50 The lower the value, the higher the sensitivity; the detection limit is expressed in IC50. 10 count.

[0173] Standard curve and IC 50 IC 10 The values ​​are shown in Table 7.

[0174] Table 7 Standard Curve, IC 50 IC 10

[0175]

[0176]

[0177] Table 7 shows that the constructed ELISA detection method exhibits good linearity for both SA and TC antibiotics. For SA, SP, SD, ST, TC, CTC, OTC, DC, and total IC50, the method also shows good linearity. 50 The concentrations were: 16.3, 21.8, 18.3, 20.5, 17.3, 22.3, 17.9, 20.2, and 17.6 ng / mL, respectively, with a detection limit IC50 of 17.6.10 The values ​​were 0.46, 0.54, 0.48, 0.53, 0.49, 0.51, 0.50, 0.49, and 0.50 ng / mL, respectively.

[0178] Example 4 Spike Recovery Experiment

[0179] (1) Single-spiked water recovery experiment

[0180] Spiked water samples with a concentration of 100 ng / mL were prepared using SAs and TCs antibiotic standards, and the amounts of SAs and TCs in the spiked water samples were determined using a constructed ELISA method.

[0181] Specific steps for water spike recovery experiment:

[0182] ① Coating: Dilute the coating antigen to 0.5 μg / mL with coating buffer, add 100 μL / well to the microplate, and coat overnight at 4℃.

[0183] ② Blocking: Pour out the liquid from the wells of the microplate and blot dry. Add 250 μL of 1% BSA to each well, incubate at 37°C for 1 h, pour out the liquid from the wells, and blot dry. Add 200 μL of washing buffer to each well, gently shake for 90 s, pour out the liquid from the wells, and blot dry. Repeat washing 3 times.

[0184] ③ Competition: Various antibiotic standard solutions were serially diluted to 1, 2.5, 5, 10, 25, 50, and 100 ng / mL, and added to 96-well plates at 50 μL / well. Then, 50 μL of a 1:64000 diluted composite antibody was added to each well and mixed thoroughly, so that the final concentrations of the antibiotic standard solutions were 1, 2.5, 5, 10, 25, 50, and 100 ng / mL, and the final dilution ratio of the composite antibody was 1:128000 (a standard curve was prepared for each well). One positive control well was set up (50 μL of pure water + 50 μL of a 1:64000 diluted composite antibody); the test wells (50 μL of spiked water sample + 50 μL of a 1:64000 diluted composite antibody) were incubated at 37℃ for 1 h. The liquid in the wells was poured out and patted dry. Wash 3 times.

[0185] ④ Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse IgG1 at a ratio of 1:10000 with antibody dilution buffer, 100 μL / well. Incubate at 37℃ for 1 h, pour out the liquid from the wells, and pat dry. Wash 3 times.

[0186] ⑤ Color development: Add 100 μL of freshly prepared substrate color development solution to each well and react at room temperature in the dark for 15 min.

[0187] ⑥ Termination: Add 50 μL of termination solution to each well.

[0188] ⑦ Result determination: The absorbance at 492 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0189] Regression analysis was performed with the inhibition rate on the ordinate and the natural logarithm of the antibiotic concentration on the abscissa to generate a standard curve and calculate the concentration of antibiotics in the spiked water sample to be tested.

[0190] The results of the single-spiking recovery rate determination in water are shown in Table 8.

[0191] Table 8 Results of Single Spike Recovery in Water

[0192]

[0193] As shown in Table 8, the constructed ELISA detection method has a recovery rate of 97.12% to 98.87% for SAs and TCs in single-spike water samples, and can effectively detect the above-mentioned antibiotics in water.

[0194] (2) Water Mixing Spike Recovery Experiment

[0195] Various antibiotics, including SAs and TCs, were randomly mixed to prepare mixed samples of SAs and TCs. Spiked water samples with a concentration of 100 ng / mL were prepared using the mixed samples. The total amount of SAs and TCs antibiotics in the spiked water samples was determined using the constructed ELISA detection method (method as above). The results are shown in Table 9.

[0196] Table 9 Results of the recovery rate determination of mixed spiked water

[0197]

[0198] As shown in Table 9, the recovery rate of the constructed ELISA detection method for the total amount of SAs and TCs in mixed standard water samples ranges from 96.25% to 98.56%, which can effectively detect the total amount of SAs and TCs in water.

[0199] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An artificial composite antigen, the structure of which is shown in Formula I: Equation I; In Formula I, "N-Protein-N" represents the carrier protein; The method for preparing the artificial composite antigen includes the following steps: Sulfonamide, glutaraldehyde, carrier protein, alcohol solvent and buffer solution were mixed and subjected to the first coupling reaction to obtain the compound with the structure shown in formula a; Formula a; The compound with the structure shown in Formula a, tetracycline and glutaraldehyde are mixed and subjected to a second coupling reaction to obtain the artificial complex antigen with the structure shown in Formula I. The molar ratio of sulfonamide, glutaraldehyde, and carrier protein is 33:178:1~1.4; The molar ratio of the compound with the structure shown in Formula a, tetracycline, and glutaraldehyde is 1:1:5~5.5; The alcohol solvent is methanol, and the buffer solution is PBS buffer solution; The temperature of the first coupling reaction is 22~26℃, and the time is 4~5h; The second coupling reaction is carried out at a temperature of 22-26°C for 4-5 hours.

2. The artificial composite antigen according to claim 1, characterized in that, The carrier protein is bovine serum albumin or ovalbumin.

3. The method for preparing the artificial composite antigen according to claim 1 or 2, comprising the following steps: Sulfonamide, glutaraldehyde, carrier protein, alcohol solvent and buffer solution were mixed and subjected to the first coupling reaction to obtain the compound with the structure shown in formula a; Formula a; The compound with the structure shown in Formula a, tetracycline and glutaraldehyde are mixed and subjected to a second coupling reaction to obtain the artificial complex antigen with the structure shown in Formula I. The molar ratio of sulfonamide, glutaraldehyde, and carrier protein is 33:178:1~1.4; The molar ratio of the compound with the structure shown in Formula a, tetracycline, and glutaraldehyde is 1:1:5~5.5; The alcohol solvent is methanol, and the buffer solution is PBS buffer solution; The temperature of the first coupling reaction is 22~26℃, and the time is 4~5h; The second coupling reaction is carried out at a temperature of 22-26°C for 4-5 hours.

4. The application of the artificial composite antigen according to claim 1 or 2 or the artificial composite antigen prepared by the preparation method according to claim 3 in the preparation of broad-spectrum detection antibodies for sulfonamides and tetracyclines; The sulfonamide antibiotics are one or more of sulfonamides, sulfapyridine, sulfadiazine, and sulfathiazole; The tetracycline antibiotics are one or more of tetracycline, chlortetracycline, oxytetracycline, and cyclocycline.

Images