Nitrite artificial hapten as well as preparation method and application thereof

Abstract

The invention provides preparation and application of a nitrite hapten, an artificial antigen and an antibody. According to the design strategy of the hapten, an annular connecting arm of a benzene ring is introduced on the basis of retaining the characteristics of molecules to be detected to the greatest extent, the structural rigidity is kept, and the complexity and the stability of the molecules are enhanced, so that an organism can be stably stimulated to generate an antibody with strong specificity and high sensitivity in the immune process. The nitrite colloidal gold immunochromatography detection kit prepared from the antigen and the antibody has the advantages of strong specificity, high sensitivity, simple pretreatment method (the reaction time is 25-30 min), low cost, stable color, long quality guarantee period of the test strip, wide application range and the like, and can be used for rapidly detecting nitrite in samples such as sausages, luncheon meat, preserved szechuan pickles and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a nitrite artificial hapten and a preparation method and application thereof. Background Art

[0002] Nitrite (NIT) is an inorganic compound that is commonly found in the environment and biological organisms and can be used as a colorant and preservative in food. The most common is sodium nitrite, which is a white to light yellow powder or granule, slightly salty, and easily soluble in water. In the food industry, nitrite is an important food additive because of its unique antibacterial and color-developing properties, which can significantly improve the flavor and color retention of food. Although nitrite plays an indispensable role in the human body, such as assisting blood pressure regulation, preventing pathogenic bacteria from invading, and thus maintaining the normal functioning of the human physiological system, excessive intake may bring serious health risks. When the human body ingests excessive nitrite in a short period of time, it is easy to cause acute poisoning, which will cause irreversible oxidation of hemoglobin, converting it into high hemoglobin, and further leading to tissue hypoxia. In addition, nitrite will react with compounds such as secondary amines and tertiary amines to generate carcinogenic nitrosamines.

[0003] At present, instrumental methods are the main analytical methods for detecting nitrite. Including chromatographic methods such as high performance liquid chromatography, spectrophotometry, fluorescence and electrochemical sensors. Large-scale instrumental methods have high sensitivity and good selectivity, and can accurately detect nitrite residues, but they are expensive and require professional technicians to operate. In addition, the sample preparation and operation process also takes a certain amount of time, which is not suitable for rapid detection and analysis scenarios. Spectrophotometry is a chemical analysis method with high sensitivity, wide linear range, fast, efficient and high selectivity, but it also has some disadvantages, such as complex sample processing, many factors affecting accuracy and limited scope of application. Patent CN 113583110 A discloses a benzotriazole hapten, an artificial antigen and an antibody and a preparation method and application thereof. Benzotriazole is the product of the derivative reaction of nitrite and o-phenylenediamine. Nitrite can be completely reacted by adding an excess of o-phenylenediamine. The content of nitrite can be indirectly detected by detecting benzotriazole. In this patent, 5-aminobenzotriazole or 5-carboxylbenzotriazole is used as a hapten. The amino or carboxyl structure is introduced into the structure while retaining the characteristic structure of the nitrite derivative benzotriazole, which is conducive to stimulating the animal immune response to produce antibodies with stronger specificity and higher sensitivity, and is used to indirectly detect the content of nitrite in food. However, the hapten design strategy is to directly use the active group on the benzotriazole ring to couple the protein without introducing a connecting arm under the premise of retaining the molecular characteristics of the analyte, and the rigidity and stability are poor. Due to the complex three-dimensional structure of the carrier protein, it is easy to wrap the small molecule hapten inside, resulting in it being unable to be fully exposed to the antigen presenting cells during the immunization process, and the body will not be able to produce specific antibodies against the small molecule hapten. Therefore, there is still a need for a hapten with good rigidity and stability, high sensitivity and specificity for detecting nitrite, so as to achieve effective control and regulation of the nitrite content in food. Summary of the invention

[0004] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide a nitrite artificial hapten.

[0005] The second object of the present invention is to provide the use of the above-mentioned nitrite artificial hapten in the preparation of nitrite artificial antigen.

[0006] The third object of the present invention is to provide a nitrite artificial antigen and a nitrite artificial antibody.

[0007] The fourth object of the present invention is to provide an immunochromatographic detection kit for detecting nitrite, which indirectly detects the content of nitrite by adding an excess of o-phenylenediamine to completely derivatize nitrite to generate benzotriazole.

[0008] A fifth object of the present invention is to provide a method for detecting nitrite in a sample.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention provides a nitrite artificial hapten, the structural formula of which is shown in formula (I), .

[0010] The design strategy of small molecule haptens mainly considers retaining the molecular characteristics of the analyte, the introduction of active groups and the design of the connecting arm. The hapten design strategy of the present invention introduces the connecting arm on the basis of retaining the molecular characteristics of the analyte to the greatest extent, maintains the structural rigidity and enhances the complexity and stability of the molecule, so as to ensure that the body can be stably stimulated to produce specific antibodies during the immunization process. By introducing the cyclic connecting arm structure of the benzene ring to increase the rigidity and stability of the molecule and maintain the overall rigidity of the hapten, the improvement of the rigidity of the small molecule hapten can improve the antibody titer, affinity and sensitivity produced by the immune body; and the molecular characteristics of the analyte are fully exposed to improve the immunogenicity of the hapten, thereby preparing antibodies with good specificity.

[0011] The present invention also provides a method for preparing the nitrite artificial hapten. The method comprises the steps of mixing 5-hydroxybenzotriazole and methyl 4-bromomethylbenzoate to carry out a condensation reaction, and extracting to obtain the hapten.

[0012] Furthermore, the molar ratio of the 5-hydroxybenzotriazole to methyl 4-bromomethylbenzoate is 1:1 to 1.5.

[0013] Furthermore, the molar ratio of the 5-hydroxybenzotriazole to methyl 4-bromomethylbenzoate is 1:1.2.

[0014] The present invention also provides the use of the above-mentioned nitrite artificial hapten in the preparation of nitrite artificial antigen.

[0015] The present invention provides a nitrite artificial antigen, which is a combination of the above-mentioned hapten and a carrier protein, and has a structural formula as shown in formula (II): , The carrier protein is lactoferrin (LF) or chicken ovalbumin (OVA).

[0016] Furthermore, the preparation method of the nitrite artificial antigen is to mix the above hapten with N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride, and then add a carrier protein to obtain the nitrite artificial antigen.

[0017] Furthermore, the mass ratio of the hapten, N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride is 4-6:4:4-6.

[0018] Preferably, the mass ratio of the hapten, N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride is 5:4:5.

[0019] The present invention provides a nitrite artificial antibody, which is prepared by immunizing mice with the antigen.

[0020] Furthermore, the prepared antigen was diluted to 1 mg / mL with phosphate buffer solution and then mixed and emulsified with an equal volume of Freund's complete adjuvant; Balb / C mice were immunized by multiple subcutaneous injections on the back, with an immunization dose of 100 μg per mouse; booster immunizations were performed every two weeks, and mice with high titers and inhibition rates were screened for cell solution experiments, and after obtaining high-purity positive hybridoma cell lines, ascites was prepared and purified to obtain monoclonal antibodies.

[0021] The present invention also provides an immunochromatographic test kit for detecting nitrite, the kit comprising an immunochromatographic test paper and a microwell containing a gold-labeled antibody; the immunochromatographic test paper comprises a bottom plate and a sample pad, a reaction membrane and a water absorbent pad arranged in sequence on the bottom plate; non-overlapping detection lines and quality control lines are sequentially arranged on the reaction membrane from the sample pad to the water absorbent pad; the detection line is coated with 0.8-1 mg / mL of the above-mentioned nitrite artificial antigen as a T line; the quality control line is coated with 0.02-0.2 mg / mL of a sheep anti-mouse secondary antibody as a C line.

[0022] Furthermore, the detection line is coated with 0.8 mg / mL of artificial antigen as the T line; and the quality control line is coated with 0.05 mg / mL of goat anti-mouse secondary antibody as the C line.

[0023] Furthermore, the artificial antigen is a coating antigen, and the structural formula of the coating antigen is shown in formula (III), .

[0024] Furthermore, the preparation method of the gold-labeled antibody comprises preparing a colloidal gold solution with chloroauric acid and trisodium citrate, adjusting the pH value of the colloidal gold solution to 6-7, adding the above-mentioned nitrite artificial antibody for reaction, sealing, and solid-liquid separation to obtain the colloidal gold-labeled antibody.

[0025] Furthermore, the mass ratio of chloroauric acid to trisodium citrate is 1:0.8-1.2.

[0026] Preferably, the mass ratio of chloroauric acid to trisodium citrate is 1:1.

[0027] Furthermore, the reagent for adjusting pH is potassium carbonate.

[0028] Furthermore, the mass volume ratio of the antibody to the colloidal gold solution is 4-10 μg:1 mL.

[0029] Preferably, the mass volume ratio of the antibody to the colloidal gold solution is 7 μg:1 mL.

[0030] The invention provides a method for detecting nitrite in a sample, comprising mixing the sample to be tested with an extractant and a derivatization reagent, extracting and re-dissolving to obtain a pre-treated test liquid; and then using the immunochromatographic detection kit to detect nitrite in the pre-treated test liquid.

[0031] Further, the sample to be tested is mixed with the extractant, a derivatization reagent is added for derivatization reaction, a potassium hydrogen phosphate aqueous solution and an extractant are added, the supernatant is concentrated, and the pre-treated test solution is obtained; the immunochromatographic test paper is placed flat, 100 μL of the test solution is added to the microwell containing the gold-labeled antibody, blown and mixed, and then allowed to react for 3 minutes, and the test solution is added dropwise to the sample addition hole of the sample pad, and the result is observed after standing for 3 to 5 minutes.

[0032] The results are interpreted as follows: Negative: The quality control line, i.e., the C line, is colored; the test line, i.e., the T line, is darker in red than or equal to the quality control line, indicating that the sample does not contain nitrite or its content is lower than 0.2 mg / kg, which is judged as negative.

[0033] Positive: The quality control line, i.e., the C line, shows color; the test line, i.e., the T line, does not appear red or appears but is lighter in color than the quality control line, indicating that the sample contains nitrite and its content is higher than 0.2 mg / kg, which is judged as positive.

[0034] Invalid: When the quality control line, i.e., the C line, does not show red, regardless of whether the test line, i.e., the T line, shows color, it means that the experimental results are invalid.

[0035] The nitrite colloidal gold immunochromatographic test paper of the present invention is designed based on the specific binding of antigen and antibody and the principle of chromatography. When testing, 100 μL of the test solution is added to the microwell containing the gold-labeled antibody, blown and mixed, and then allowed to react for 3 minutes. The test solution is added dropwise to the sample pad, and the liquid uses the capillary siphon effect to migrate upward for chromatography. When the sample does not contain the target analyte or the content is lower than the detection limit, the gold-labeled antibody migrates upward, and the artificial antigen captured on the detection line of the reaction membrane causes the detection line to develop color. The remaining gold-labeled antibody continues to migrate upward and combines with the goat anti-mouse IgG on the quality control line to cause the quality control line to develop color. When the color intensity of the test line is greater than or equal to the color intensity of the quality control line, the test result is determined to be negative. When the sample contains a certain amount of the analyte and the content is higher than the detection limit, the analyte will compete with the artificial antigen for the gold-labeled antibody. The gold-labeled antibody bound to the analyte will not bind to the artificial antigen on the test line, but will still bind to the goat anti-mouse IgG on the quality control line. Therefore, as the concentration of the analyte increases, the color of the test line will become lighter and lighter until it disappears. When the color intensity of the test line is lighter than that of the quality control line, the test result is judged as positive. When the quality control line does not show color, it is an invalid result. After the reaction is completed, the standard curve is established by naked eye observation and colloidal gold reader reading to achieve the determination of the nitrite content in the sample.

[0036] Furthermore, the extractant is hydrochloric acid, citric acid, p-toluenesulfonic acid or phosphate buffer.

[0037] Furthermore, the extractant is p-toluenesulfonic acid.

[0038] Preferably, the extractant is p-toluenesulfonic acid with a concentration of 5%.

[0039] Furthermore, the ratio of the sample to be tested to the extractant is 2.0-4.0 g: 3 mL.

[0040] Preferably, the ratio of the sample to be tested to the extractant is 3 g:3 mL.

[0041] Furthermore, the derivatization agent is o-phenylenediamine.

[0042] Preferably, the derivatization temperature is 80°C and the derivatization time is 10 min. Furthermore, the extractant is ethyl acetate.

[0043] Furthermore, the ratio of the sample to be tested to the reconstitution solution is 2.0-4.0 g: 500 μL.

[0044] Furthermore, the ratio of the sample to be tested to the reconstitution solution is 3 g:500 μL.

[0045] Preferably, the reconstitution solution is a 0.09-0.11 M PB solution.

[0046] More preferably, the reconstitution solution is a 0.1 M PB (pH 6.0) solution.

[0047] Therefore, the present invention also provides the use of the above kit in detecting nitrite.

[0048] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the preparation and application of a nitrite hapten, an artificial antigen and an antibody. The hapten design strategy of the present invention introduces a connecting arm on the basis of retaining the characteristics of the molecule to be detected to the greatest extent, maintains the structural rigidity and enhances the complexity and stability of the molecule, so as to ensure that the body can be stably stimulated to produce specific antibodies during the immunization process. The rigidity and stability of the molecule are increased by introducing a benzene ring, which is a cyclic connecting arm structure, and the molecular characteristics of the object to be detected are fully exposed to improve the immunogenicity of the hapten, so as to prepare an antibody with good specificity. The IC value of the antibody is 50 The concentration of nitrite was 209.75 ng / mL, the linear range was 60.39-728.53 ng / mL, and the sensitivity was high. The above antigen-antibody composition was used to prepare a nitrite colloidal gold immunochromatographic detection kit. Experiments verified that the kit had high detection sensitivity, good specificity, and no cross-reaction with common additives in food; the pretreatment method was simple (reaction time 25-30 min), and the results could be observed 5 minutes after the sample was added; the detection kit of the present invention has the advantages of strong specificity, high sensitivity, fast detection speed, simple operation, low cost, stable color and long shelf life of the test strip, and is used for semi-quantitative detection of nitrite. It is suitable for rapid detection of nitrite in samples such as sausages, luncheon meat and mustard tubers by units or individuals such as sanitation, quality inspection, and farmers' markets. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the mass spectrum of nitrite hapten.

[0050] Figure 2 This is the standard curve of ic-ELISA.

[0051] Figure 3 The structure diagram and top view of the nitrite colloidal gold immunochromatographic test paper are shown in FIG. Figure 3 A is a schematic diagram of the structure, and B is a top view. Among them, 1 is a sample pad; 2 is a reaction membrane; 3 is a detection line; 4 is a quality control line; 5 is a water absorption pad; and 6 is a bottom plate.

[0052] Figure 4 This is the result analysis diagram of the nitrite colloidal gold immunochromatography detection kit.

[0053] Figure 5This is the effect of potassium carbonate volume on nitrite colloidal gold immunochromatographic detection kit.

[0054] Figure 6 This is the effect of the antibody volume on the nitrite colloidal gold immunochromatographic detection kit.

[0055] Figure 7 The effect of antigen concentration on nitrite colloidal gold immunochromatographic detection kit.

[0056] Figure 8 This is the effect of the concentration of goat anti-mouse secondary antibody on the nitrite colloidal gold immunochromatographic detection kit.

[0057] Fig. 9 To study the effect of derivatization temperature on nitrite colloidal gold immunochromatographic detection kit.

[0058] Fig.10 To study the effect of derivatization time on nitrite colloidal gold immunochromatographic detection kit.

[0059] Fig.11 The influence of the type of extractant on the pretreatment extraction effect.

[0060] Fig.12 The effect of extractant concentration on the pretreatment extraction effect.

[0061] Fig.13 This is the colloidal gold standard curve.

[0062] Fig.14 This is a graph showing the results of a specific experiment.

[0063] Fig.15 This is the stability test result diagram. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0065] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0066] Example 1 Preparation of nitrite hapten and monoclonal antibody (1) The synthesis process of nitrite hapten is shown in the following reaction equation:

[0067] Preparation of hapten: Add 1.35 g (10 mmol) 5-hydroxybenzotriazole and 2.76 g (20 mmol) potassium carbonate to N,N-dimethylformamide (DMF), stir evenly, add 4.58 g (12 mmol) methyl 4-bromomethylbenzoate, react at 80°C for 5h, add water-ethyl acetate for extraction and then evaporate. The product is dissolved in 3 mL methanol, and 1 mL of 10% lithium hydroxide aqueous solution is added dropwise and reacted at 50°C until completely hydrolyzed, and concentrated hydrochloric acid is added to adjust the pH to acidic, and ethyl acetate is added for extraction and then evaporated to obtain the hapten.

[0068] The mass spectrum of the prepared nitrite hapten is shown in Figure 1 As shown, it has a structure shown in formula (I): .

[0069] (2) Preparation of immunogen and coating agent: 10 mg of hapten was dissolved in 200 μL DMF, 8 mg of N-hydroxysuccinimide (NHS) and 10 mg of 1-ethylcarbodiimide hydrochloride (EDC) were added thereto to dissolve them completely, and then stirred at room temperature in the dark for 4 h to obtain an activation solution. The carrier proteins LF and OVA were dissolved in alkaline CB buffer at a concentration of 10 mg / mL, and the activation solution was added dropwise in a stirring ice bath. After coupling at room temperature overnight, the immunogen (NIT-LF) and coating agent (NIT-OVA) were obtained, which have the structure shown in formula (II): , The carrier protein is lactoferrin (LF) or chicken ovalbumin (OVA).

[0070] (3) Preparation of monoclonal antibodies: The prepared immunogen (NIT-LF) was diluted to 1 mg / mL with phosphate buffer solution, and then mixed with an equal volume of Freund's complete adjuvant for emulsification; Balb / C mice were immunized by multiple subcutaneous injections on the back, with an immunization dose of 100 μg per mouse; booster immunizations were performed every two weeks, and mice with high titers and inhibition rates were screened for cell solution experiments. After obtaining high-purity positive hybridoma cell lines, ascites was prepared and purified to obtain monoclonal antibodies.

[0071] (4) Establishment of ic-ELISA curve: Dilute the coated original NIT-OVA to 1 μg / mL with carbonate buffer (CB, 0.1M pH=9.8), coat the plate with 96-well ELISA plate, add 100 μL to each well, incubate at 37°C for 4 h, pour off the liquid in the wells, wash twice with washing solution, 30 s each time, pat dry, then add 120 μL of blocking solution (i.e., 5% skim milk powder by weight) to each well, incubate at 37°C for 2 h, pour off the liquid in the wells, pat dry. The monoclonal antibody prepared was diluted 1:8000 times with phosphate buffer (PBS, 0.01M, pH=7.4), and the drug to be detected, benzotriazole, was diluted 2-fold to 5000 ng / mL, 2500 ng / mL, 1250 ng / mL, 625 ng / mL, 312.5 ng / mL, 156.25 ng / mL, 78.12 ng / mL, 39.06 ng / mL, 19.53 ng / mL, and 9.76 ng / mL, and added to the enzyme-labeled plate coated with NIT-OVA according to the general indirect competitive ELISA method, incubated at 37°C for 40 min, washed five times, and patted dry; sheep anti-mouse secondary antibody-HRP diluted 5000 times with phosphate-tween buffer (PBST, 0.01M) was added at 100 μL / well, incubated at 37°C for 30 min, washed five times, and patted dry; color development solution was added, 100 μL / well per well μL, color development for 10 min; add 50 μL 10% H 2 SO 4 The reaction was terminated by adding the solution and the OD value was read at 450 nm. 0 The ordinate is the logarithmic value of the corresponding standard concentration and the abscissa is the nitrite content. The molar ratio of benzotriazole to nitrite is 1:1. The standard curve is drawn by normalization method. The standard curve is as follows: Figure 2 , the relevant standard curve parameters are shown in Table 1.

[0072] Table 1 Detection parameters of monoclonal antibodies

[0073] Example 2 Preparation of nitrite colloidal gold immunochromatographic detection kit 1. Main Materials The sample pad, nitrocellulose membrane (NC membrane), absorbent pad and polyvinyl chloride base were from Shanghai Liangxin Technology Co., Ltd.; chloroauric acid and trisodium citrate were from Sinopharm Chemical Reagent Co., Ltd.; goat anti-mouse IgG was from Beijing Quanshijin Biological Co., Ltd.; nitrite standard was from Tanmo Quality Inspection Technology Co., Ltd.; sample pad treatment solution and gold label rehydration solution were from Guangzhou Wanlian Biotechnology Co., Ltd.; samples (sausage, luncheon meat, mustard tuber) were commercially available; the remaining reagents were of analytical grade.

[0074] 2. Preparation of Nitrite Colloidal Gold Immunochromatographic Detection Kit (1) Preparation of colloidal gold solution: colloidal gold particles were prepared by the trisodium citrate reduction method. 100.0 mL of ultrapure water was weighed into a 500 mL round-bottom flask, and the flask was placed in a magnetic stirring oil bath. The mixture was stirred and heated under reflux until boiling. Then 4 mL of 10 g / L chloroauric acid aqueous solution was added. After boiling again, 4 mL of 10 g / L trisodium citrate aqueous solution was quickly added. The mixture was heated for 10 min and then stopped. The mixture was stirred under residual heat and cooled to room temperature to obtain a colloidal gold solution with a mass concentration of 0.02%.

[0075] (2) Preparation of gold-labeled antibody microwells: Pipette 1 mL of colloidal gold solution, adjust the pH, add 8 μL of 0.1 mol / L potassium carbonate aqueous solution to the colloidal gold solution, shake and mix, then add 5 μg of antibody, shake and mix, and react at room temperature for 10 min for labeling. Add 10 μL of 100 g / L BSA solution for blocking, shake and mix, and react at room temperature for 5 min to block the sites on the colloidal gold that are not bound to the antibody. After blocking, centrifuge for 12 min (4°C, 12000 rpm), discard the supernatant, add 500 μL of gold diluent to dissolve, and dispense the dissolved product into microwells in a certain volume, and dry in a 45°C oven overnight to obtain the gold-labeled antibody.

[0076] The gold diluent is a 0.1 mol / L PB solution containing 0.1% BSA, 1% sucrose, 0.3% PVP-40 and 0.01% preservative PC300.

[0077] (3) Preparation of reaction membrane: Dilute the artificial antigen to 0.8 mg / mL with 0.01 mol / L PB solution, and dilute the goat anti-mouse secondary antibody to 0.05 mg / mL with 0.01 mol / L PB solution. Use a membrane scribing instrument to spray the diluted artificial antigen and goat anti-mouse secondary antibody onto the NC membrane, respectively, as the detection T line and quality control C line, and place them in a 45°C drying oven overnight for use.

[0078] (4) Preparation of sample pad: Soak the glass cellulose membrane in the sample pad treatment solution for 10 min, take it out and dry it at 45°C overnight. The sample pad treatment solution is a 0.1 mol / L PB solution containing 1% Tween-20 and 0.2% sucrose.

[0079] (5) Assemble the test strip card: Figure 3 As shown, it is a schematic diagram of the structure of the nitrite colloidal gold immunochromatographic test paper of the present invention ( Figure 3 A) and top view ( Figure 3B), including a bottom plate 6 and a sample pad 1, a reaction membrane 2 and a water absorbent pad 5 arranged in sequence on the bottom plate 6; the reaction membrane 2 is provided with a detection line 3 and a quality control line 4 which do not overlap each other, and the two ends of the reaction membrane 2 are respectively connected to the water absorbent pad 5 and the sample pad 1, and the connecting parts of the sample pad 1, the reaction membrane 2 and the water absorbent pad 5 overlap by 1 to 2 mm and are attached to the bottom plate 6. The bottom plate is cut into 3 mm wide using a strip cutter and then loaded into a plastic card.

[0080] 3. Detection principle of nitrite colloidal gold immunochromatographic detection kit The nitrite colloidal gold immunochromatographic test paper of the present invention is designed based on the specific binding of antigen and antibody and the chromatography principle. Figure 4 As shown, when testing, 100 µL of the test solution is added to the microwell containing the gold-labeled antibody, blown and mixed, and then allowed to react for 3 min. The test solution is added dropwise to the sample pad 1, and the liquid migrates upward for chromatography using the capillary siphon effect. When the sample does not contain the target analyte or the content is lower than the detection limit, the gold-labeled antibody migrates upward and is captured by the artificial antigen fixed on the detection line 3 of the reaction membrane 2 to color the detection line. The remaining gold-labeled antibody continues to migrate upward and binds to the goat anti-mouse IgG on the quality control line 4 to color the quality control line. When the color intensity of the detection line 3 is greater than or equal to the color intensity of the quality control line 4, the test result is judged to be negative. When the sample contains a certain amount of the analyte and the content is higher than the detection limit, the analyte will compete with the artificial antigen for the gold-labeled antibody. The gold-labeled antibody bound to the analyte will not bind to the artificial antigen on the test line 3, but will still bind to the goat anti-mouse IgG on the quality control line 4. Therefore, as the concentration of the analyte increases, the color of the test line 3 will become lighter and lighter until it disappears. When the color intensity of the test line 3 is lighter than that of the quality control line 4, the test result is judged to be positive. When the quality control line 4 does not show color, it is an invalid result. After the reaction is completed, the standard curve is established by naked eye observation and colloidal gold reader reading to achieve the determination of the nitrite content in the sample.

[0081] Method of using the nitrite colloidal gold immunochromatographic detection kit (1) Sample pretreatment: Grind sausage, luncheon meat and mustard tuber (or other samples to be tested) separately. Weigh 3 g of the sample to be tested and add it to a clean centrifuge tube, then add 3 mL of extractant (5% p-toluenesulfonic acid) and 100 µL of o-phenylenediamine derivatization reagent (3 mg / mL) and shake evenly. After reacting at 80°C for 10 min, add 500 µL of potassium dihydrogen phosphate aqueous solution (4 mol / L), add 2 mL of ethyl acetate and shake evenly, then centrifuge at 3000 r for 3 min, take the supernatant and blow dry, finally re-dissolve with 500 μL of reconstitution solution (0.1M PB pH6.0 solution), shake evenly again, and obtain the pre-treated test solution.

[0082] (2) Lay the nitrite colloidal gold immunochromatographic test paper prepared in this example flat, take 100 μL of the test solution and add it to the microwell containing the gold-labeled antibody, pipette and mix well, and then let it stand for 3 min. Add the test solution dropwise to the sample well of the sample pad, let it stand for 3 to 5 min, and then observe the result.

[0083] The results are analyzed as Figure 4 The results are interpreted as follows: Negative (-): The quality control line (C line) is colored, and the test line (T line) is darker than or equal to the quality control line (C line), indicating that the sample does not contain nitrite or its content is lower than 0.2 mg / kg, which is judged as negative.

[0084] Positive (+): The quality control line (C line) is colored, and the test line (T line) does not appear red or appears but the color is lighter than the quality control line (C line), indicating that the sample contains nitrite and its content is higher than 0.2 mg / kg, which is judged as positive.

[0085] Invalid: When the quality control line (C line) does not show red, regardless of whether the test line (T line) shows color, it means that the experimental results are invalid.

[0086] Example 3 Effects of different preparation conditions on the effect of nitrite colloidal gold immunochromatographic detection kit 1. Effect of nitrite colloidal gold immunochromatographic detection kit under different preparation conditions (1) Colloidal gold labeled antibodies are bound and coated on the surface of colloidal gold particles by electrostatic adsorption. The pH value of the labeling system will affect the adsorption efficiency of the antibody and the colloidal gold particles and the biological activity of the antibody. The volumes of potassium carbonate are 4, 6, 8 and 10 μL, and the corresponding pH values ​​are 6.2, 6.6, 7.0 and 7.4, respectively. Other preparation conditions and parameters are the same as those in Example 2.

[0087] (2) The volumes of the antibodies were adjusted to 3, 5, 7 and 9 μg / mL, respectively. Other preparation conditions and parameters were the same as those in Example 2.

[0088] (3) The concentrations of the antigen were set to 0.4, 0.6, 0.8 and 1 mg / mL, respectively. Other preparation conditions and parameters were the same as those in Example 2.

[0089] (4) The concentrations of goat anti-mouse secondary antibody were 0.02, 0.05, 0.1 and 0.2 mg / mL, respectively. Other preparation conditions and parameters were the same as those in Example 2.

[0090] (5) The derivatization temperatures were set to room temperature, 40°C, 60°C, 80°C and 90°C, respectively. Other preparation conditions and parameters were the same as those in Example 2.

[0091] (6) The derivatization time was set to 5, 10, 15 and 20 min respectively. The other preparation conditions and parameters were the same as those in Example 2.

[0092] (7) 1 mol / L hydrochloric acid, citric acid, p-toluenesulfonic acid and 0.1 M PB (pH 6.0) solution were used as extractants to pre-treat sausage, luncheon meat and mustard tuber.

[0093] (8) Sausage, luncheon meat and pickled mustard tuber were pretreated with aqueous p-toluenesulfonic acid solutions at concentrations of 2.5%, 5%, 10% and 15%, respectively.

[0094] 2. Experimental results (1) If Figure 5 As shown, in 1 mL of colloidal gold solution, when 0.1MK 2 CO 3 When the solution volume was 4 µL, the color of the colloidal gold solution was purple, the color of the test strip was uneven, and the inhibition rate was poor. 2 CO 3 When the solution volume increased from 6 μL to 10 μL, the color development and T / C value of the test strip increased slightly and then did not change significantly. 2 CO 3 The inhibition rate was highest when the solution volume was 8 µL, so 0.1 MK was selected. 2 CO 3 The optimal amount of potassium carbonate added is 8 µL.

[0095] (2) If Figure 6 As shown in the figure, in 1 mL of colloidal gold solution, when the antibody labeling concentration is lower than 5 µg / mL, the color intensity of the test strip is too low and the inhibition rate is poor. As the antibody labeling concentration increases from 5 µg / mL to 9 µg / mL, the T / C value of the test strip does not change significantly, and the inhibition rate reaches the highest value at the antibody labeling concentration of 7 µg / mL and then decreases. Therefore, the antibody labeling concentration of 7 µg / mL, which has the best T / C value and inhibition rate, is selected as the optimal antibody labeling concentration.

[0096] (3) If Figure 7 As shown in the figure, with the increase of antigen concentration, the T / C value of the test strip gradually increased. When the antigen concentration was greater than 0.8 mg / mL, the T / C value and inhibition rate of the test strip tended to be stable. Therefore, 0.8 mg / mL was selected as the optimal T line coating concentration.

[0097] (4) If Figure 8As shown in the figure, as the concentration of goat anti-mouse secondary antibody increased from 0.02 mg / mL to 0.2 mg / mL, the T / C value and inhibition rate of the test strip reached the highest value when the concentration of goat anti-mouse secondary antibody reached 0.05 mg / mL and then decreased. When the concentration of goat anti-mouse secondary antibody was 0.05 mg / mL, the T / C value of the test strip was greater than 1 and the sensitivity was good. Therefore, 0.05 mg / mL was selected as the optimal C line coating concentration.

[0098] (5) If Fig. 9 As shown in the figure, with the increase of the derivatization temperature, the T / C value of the test strip has no obvious change and the color intensity meets the requirements. The inhibition rate of the test strip shows an overall upward trend. The inhibition rate reaches the highest when the derivatization temperature is 80℃. Therefore, 80℃ is selected as the optimal derivatization temperature.

[0099] (6) If Fig.10 As shown in the figure, as the derivatization time increased from 5 min to 20 min, the T / C value of the test strip did not change significantly and the color intensity met the requirements. The derivatization efficiency reached the highest after the derivatization time was 10 min and there was no significant change. Therefore, 10 min was selected as the optimal derivatization time.

[0100] (7) Fig.11 As shown in the figure, when citric acid and 0.1M PB buffer (pH 6.0) were used as pretreatment extractants, nitrite in the sample could not be extracted, and the sample solution matrix had large interference, resulting in poor derivatization efficiency. 1 M hydrochloric acid has the oxidizing property of an inorganic acid, and the sample solution matrix directly extracted also has certain matrix interference, resulting in low derivatization efficiency. In contrast, the electron absorption of the aromatic ring of p-toluenesulfonic acid, a non-oxidizing inorganic acid, was used to enhance the ionization ability of hydrogen ions and improve the extraction effect. The matrix interference was small and the inhibition rate was the highest. Therefore, p-toluenesulfonic acid was selected as the pretreatment extractant.

[0101] (8) Fig.12 As shown in the figure, when 2.5% p-toluenesulfonic acid is used as the pretreatment extractant, the extractant cannot completely acidify the impurity protein, resulting in a decrease in the derivatization efficiency. When the p-toluenesulfonic acid content reaches 5%, it can acidify the impurity protein to a certain extent, reduce the interference of the sample matrix, and increase the derivatization efficiency. When the p-toluenesulfonic acid content is greater than 10%, the solution is too acidic, resulting in false positives in negative samples (T / C value <1.0). Therefore, 5% p-toluenesulfonic acid is selected as the optimal pretreatment extractant.

[0102] Example 4 Establishment of a standard curve for nitrite detection using a nitrite colloidal gold immunochromatographic detection kit A series of nitrite standard solutions (0, 0.125, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 8.0, 16.0, 32.0 μg / mL) were prepared with 0.1 mol / L PB (pH 6.0) solution, and a derivatization reagent was added. The mixture was reacted at 80°C for 10 min. 100 μL of the nitrite colloidal gold immunochromatographic detection kit prepared in Example 2 was taken to detect the standard solution and a standard curve was established.

[0103] like Fig.13 The standard curve is shown in Figure 1. 20 ~IC 80 0.4~3.9 μg / mL, IC 50 The detection limit was 1.3 μg / mL and 0.2 μg / mL.

[0104] Example 5 Specificity and stability test of nitrite colloidal gold immunochromatographic detection kit 1. Experimental Methods (1) Specificity: o-phenylenediamine, o-phthalaldehyde, sodium sulfite, benzoyl peroxide, sodium benzoate, and potassium sorbate were selected and prepared into 50 μg / mL test samples respectively with 0.1 mol / L PB (pH 6.0) solution, and the 4 μg / mL benzotriazole standard solution of Example 4 was used as a control, and the nitrite colloidal gold immunochromatographic detection kit prepared in Example 2 was used for detection.

[0105] (2) Stability: The nitrite colloidal gold test paper prepared in Example 2 was sealed and placed in a 45°C oven for an accelerated stability test. The performance of the test paper was tested at 1, 7, 14, 21 and 28 days.

[0106] 2. Experimental results (1) If Fig.14 As shown, low concentration (4 μg / mL) of benzotriazole showed strong positive, while the other 6 high concentration (50 μg / mL) structural analogs were all negative. This shows that the nitrite colloidal gold immunochromatographic test paper prepared in Example 2 has strong specificity and no cross reaction with common structural analogs.

[0107] (2) If Fig.15 As shown, the T / C value and inhibition rate of the nitrite colloidal gold immunochromatographic test paper at different time periods are recorded. The T / C value and inhibition rate of the test strips at different time periods remain basically stable, indicating that the negative color development and positive inhibition of the nitrite colloidal gold immunochromatographic test paper prepared in Example 2 maintain good stability.

[0108] Since different artificial antigens stimulate the body to produce antibodies with different titers and different compatibility with the coating source, insufficient antibody labeling during the labeling process will affect the stability of the prepared detection kit. Therefore, the nitrite colloidal gold immunochromatography detection kit prepared by the above method of the present invention has good stability.

[0109] Example 6 Addition and recovery experiment of nitrite colloidal gold immunochromatographic detection kit Three samples of sausage, luncheon meat and mustard tuber were selected for the addition recovery experiment. 3 g of each sample was weighed, and the gradient concentration of nitrite added to each sample was 2.0, 10.0 and 50.0 mg / kg, with 3 replicates for each concentration, for a total of 27 groups. The pretreatment method of Example 2 and the nitrite colloidal gold immunochromatographic detection kit were used for detection.

[0110] As shown in Table 2 , the recovery rates of nitrite addition in the three samples ranged from 74.9% to 108.2%, with RSD < 14.4%, indicating that the immunochromatographic detection method established in this study has good accuracy.

[0111] Table 2 Nitrite addition recovery experiment in samples

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A nitrite artificial hapten, characterized in that: The structural formula is shown in formula (I), 。 2. The method for preparing the nitrite artificial hapten according to claim 1, characterized in that: The preparation method comprises the following steps: mixing 5-hydroxybenzotriazole and methyl 4-bromomethylbenzoate to carry out condensation reaction, and extracting to obtain the hapten.

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

4. A nitrite artificial antigen, characterized in that: The nitrite artificial antigen is the combination of the hapten described in claim 1 and a carrier protein, and its structural formula is shown in formula (II): ， The carrier protein is lactoferrin or chicken ovalbumin.

5. A nitrite artificial antibody, characterized in that: The artificial antibody is prepared by immunizing mice with the antigen according to claim 4.

6. An immunochromatographic detection kit for detecting nitrite, characterized in that: The kit comprises an immunochromatographic test strip and a microwell containing a gold-labeled antibody; the immunochromatographic test strip comprises a bottom plate (6) and a sample pad (1), a reaction membrane (2) and a water-absorbing pad (5) arranged in sequence on the bottom plate (6); a non-overlapping detection line (3) and a quality control line (4) are arranged in sequence on the reaction membrane (2) from the sample pad (1) to the water-absorbing pad (5); the detection line (3) is coated with 0.8-1 mg / mL of the nitrite artificial antigen according to claim 4 as a T line; and the quality control line (4) is coated with 0.02-0.2 mg / mL of a sheep anti-mouse secondary antibody as a C line.

7. The kit according to claim 6, characterized in that The preparation method of the gold-labeled antibody comprises preparing a colloidal gold solution with chloroauric acid and trisodium citrate, adjusting the pH value of the colloidal gold solution to 6-7, adding the nitrite artificial antibody of claim 5 for reaction, sealing, and solid-liquid separation to obtain the colloidal gold-labeled antibody.

8. Use of the immunochromatographic detection kit according to claim 6 or 7 in detecting nitrite.

9. A method for detecting nitrite in a sample, characterized in that: The sample to be tested is mixed with the extractant and the derivatization reagent, extracted, and redissolved to obtain a pre-treated test solution; and the nitrite in the pre-treated test solution is detected using the immunochromatographic detection kit according to claim 6 or 7.

10. The method according to claim 9, characterized in that: The extractant is hydrochloric acid, citric acid, p-toluenesulfonic acid or phosphate buffer; the derivatization reagent is o-phenylenediamine derivatization reagent.

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