A nitrite artificial hapten, and a preparation method and application thereof

Abstract

The application provides preparation and application of nitrite hapten, artificial antigen and antibody. The hapten design strategy of the application is to introduce a benzene ring as a cyclic connecting arm on the basis of retaining the characteristics of a to-be-tested molecule to the greatest extent, and to keep structural rigidity and enhance the complexity and stability of the molecule, so as to ensure that the body can be stably stimulated to produce specific and high-sensitivity antibodies in the immune process. The nitrite colloidal gold immunochromatographic detection kit prepared by using the above-mentioned antigen and antibody has the advantages of high specificity, high sensitivity, simple pretreatment method (reaction time of 25-30 min), low cost, stable color, long shelf life of test strips and wide application range, and can be used for rapid detection of nitrite in sausage, luncheon meat and preserved vegetable and the like.

Description

TECHNICAL FIELD

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

[0002] Nitrite (NIT) is a kind of inorganic compound, which exists universally in the environment and biological organisms and can be applied to food as color fixative and preservative. The most common one is sodium nitrite, which is white to light yellow powder or granular, slightly salty, and easily soluble in water. In the food industry, nitrite is an important food additive, because of its unique performance of antibacterial and color development, which can significantly improve the flavor and color retention of food. Although nitrite plays an indispensable role in the human body, such as helping to regulate blood pressure and preventing pathogenic bacteria from invading, thereby maintaining the normal operation of the human physiological system, excessive intake may pose a serious health risk. When the human body ingests excessive nitrite in a short time, it is easy to cause acute poisoning, which can cause irreversible oxidation of hemoglobin, convert it into high hemoglobin, and further cause tissue hypoxia. In addition, nitrite can also react with secondary and tertiary amine compounds to generate nitrosamine with carcinogenicity.

[0003] Currently, instrumental methods are the main analysis methods for detecting nitrite, including chromatographic methods such as high performance liquid chromatography, spectrophotometry, fluorescence method and electrochemical sensor. Large instrument methods have high sensitivity and good selectivity, and can accurately detect nitrite residues, but the cost is relatively high, and professional technical personnel are needed to operate, and the sample preparation and operation process also need a certain time, which is not suitable for the scene needs of rapid detection and analysis. Spectrophotometry is a chemical analysis method with high sensitivity, wide linear range, rapid and efficient and high selectivity, but also has some shortcomings, such as complex sample processing, many influencing factors on precision and limited application range. Patent CN 113583110 A discloses a kind of benzotriazole hapten, artificial antigen and antibody and its preparation method and application, benzotriazole is the product of nitrite and o-phenylenediamine derivative reaction, by adding excess o-phenylenediamine, nitrite can be completely reacted, by detecting benzotriazole, the content of nitrite can be indirectly detected; In the patent, 5-amino benzotriazole or 5-carboxy benzotriazole is used as a hapten, the introduction of amino or carboxyl structure in the structure retains the characteristic structure of benzotriazole, the derivative of nitrite, which is conducive to stimulating the immune response of animals to produce antibodies with stronger specificity and higher sensitivity, and is used for indirect detection of the content of nitrite in food. However, the hapten design strategy is to retain the molecular characteristics of the measured substance without introducing a connecting arm and directly using the active group on the benzotriazole ring to couple the protein, which has poor rigidity and stability. Because of the complex three-dimensional structure of the carrier protein, small molecule haptens are easily wrapped inside, which makes them unable to fully expose to antigen presenting cells during the immune process, and the body cannot produce specific antibodies against small molecule haptens. Therefore, there is still a need for a hapten that can be used to detect nitrite, which has good rigidity and stability, high sensitivity and specificity, so as to effectively control and regulate the content of nitrite in food. SUMMARY

[0004] The purpose of the present application is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide a nitrite artificial hapten.

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

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

[0007] The fourth purpose of the present application is to provide an immunochromatographic test kit for detecting nitrite, which indirectly detects the content of nitrite by adding excess o-phenylenediamine to completely react with nitrite to generate benzotriazole.

[0008] The fifth purpose of the present application is to provide a method for detecting nitrite in a sample.

[0009] The above object of the present application is achieved by the following technical solution:

[0010] The present application provides a nitrite artificial hapten, the structural formula is shown as formula (I),

[0011] .

[0012] The design strategy of the small molecule hapten mainly considers the reservation of the molecular characteristics of the tested substance, the introduction of active groups and the design of the connecting arm. The hapten design strategy of the present application introduces the connecting arm on the basis of reserving the molecular characteristics of the tested substance to the greatest extent, maintains the structural rigidity and enhances the complexity and stability of the molecule, so as to ensure that the specific antibodies of the body can be stably stimulated in the immune process. The rigidity of the small molecule hapten is improved by introducing the cyclic connecting arm structure of the benzene ring to increase the rigidity and stability of the molecule, maintain the overall rigidity of the hapten, and improve the antibody titer, affinity and sensitivity produced by the immune body. And the molecular characteristics of the tested substance are fully exposed to improve the immunogenicity of the hapten, so as to prepare the antibody with good specificity.

[0013] The present application also provides a preparation method of the nitrite artificial hapten, which comprises mixing 5-hydroxybenzotriazole and 4-bromomethyl benzoic acid methyl ester to perform condensation reaction, and extracting to obtain the hapten.

[0014] Further, the molar ratio of the 5-hydroxybenzotriazole to the 4-bromomethyl benzoic acid methyl ester is 1:1-1.5.

[0015] Further, the molar ratio of the 5-hydroxybenzotriazole to the 4-bromomethyl benzoic acid methyl ester is 1:1.2.

[0016] The present application also provides the application of the above-mentioned nitrite artificial hapten in the preparation of a nitrite artificial antigen.

[0017] The present application provides a nitrite artificial antigen, which is the combination of the above-mentioned hapten and a carrier protein, and the structural formula is shown as formula (II):

[0018] ,

[0019] The carrier protein is lactoferrin (LF) or chicken egg white albumin (OVA).

[0020] Further, the preparation method of the nitrite artificial antigen comprises mixing the above-mentioned hapten with N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride, and then adding the carrier protein to obtain the nitrite artificial antigen.

[0021] Further, the mass ratio of the hapten, N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride is 4-6:4:4-6.

[0022] Preferably, the mass ratio of the hapten, N-hydroxysuccinimide and 1-ethylcarbodiimide hydrochloride is 5:4:5.

[0023] The application provides a nitrite artificial antibody, which is prepared by immunizing a mouse with the antigen.

[0024] Further, the prepared antigen is diluted to 1 mg / mL with a phosphate buffer solution, and then mixed and emulsified with an equal volume of Freund's complete adjuvant; Balb / C mice are immunized by subcutaneous injection at multiple points on the back, and the immunization amount of each mouse is 100 μg; the immunization is performed every two weeks, and a cell solution experiment is performed on mice with high titer and inhibition rate to obtain a positive hybridoma cell strain with high purity, and then an ascites is prepared and a monoclonal antibody is purified.

[0025] The application further provides an immunochromatographic detection kit for detecting nitrite, which comprises an immunochromatographic detection test paper and a micropore containing a gold-labeled antibody.

[0026] Further, the detection line is coated with 0.8 mg / mL of the artificial antigen as a T line, and the quality control line is coated with 0.05 mg / mL of a goat anti-mouse secondary antibody as a C line.

[0027] Further, the artificial antigen is a coating agent, and the structural formula of the coating agent is shown in formula (III).

[0028] .

[0029] Further, the preparation method of the gold-labeled antibody comprises the following steps: preparing a colloidal gold solution by using chloroauric acid and trisodium citrate, adjusting the pH value of the colloidal gold solution to 6-7, adding the nitrite artificial antibody to the colloidal gold solution to react, blocking, and performing solid-liquid separation to obtain the colloidal gold-labeled antibody.

[0030] Further, the mass ratio of the chloroauric acid and the trisodium citrate is 1:0.8-1.2.

[0031] Preferably, the mass ratio of the chloroauric acid and the trisodium citrate is 1:1.

[0032] Further, the pH adjusting agent is potassium carbonate.

[0033] Further, the mass-volume ratio of the antibody to the colloidal gold solution is 4-10 μg: 1 mL.

[0034] Preferably, the mass-volume ratio of the antibody to the colloidal gold solution is 7 μg: 1 mL.

[0035] The present application provides a method for detecting nitrite in a sample, which comprises mixing the sample to be tested with an extraction agent and a derivatization reagent, extracting, redissolving, and obtaining a pretreated sample solution; and then detecting the nitrite in the pretreated sample solution by using the above-mentioned immunochromatographic detection kit.

[0036] Further, the sample to be tested is mixed with an extraction agent, a derivatization reagent is added for derivatization reaction, and then potassium phosphate dibasic aqueous solution and an extraction agent are added, and the supernatant is concentrated and redissolved to obtain a pretreated sample solution; the immunochromatographic test paper is placed flat, 100 μL of the sample solution is added to the microwells containing the gold-labeled antibody, and then mixed and blown, and then left to react for 3 min; the sample solution is added dropwise to the sample addition hole of the sample pad, and then left to stand for 3-5 min to observe the results.

[0037] The results are interpreted as follows:

[0038] Negative: the quality control line, i.e. the C line, shows color; the detection line, i.e. the T line, shows red color which is deeper than or equal to the quality control line, indicating that the sample does not contain nitrite or the content of nitrite is lower than 0.2 mg / kg, and is judged as negative.

[0039] Positive: the quality control line, i.e. the C line, shows color; the detection line, i.e. the T line, does not show red color or shows red color which is shallower than the quality control line, indicating that the sample contains nitrite and the content of nitrite is higher than 0.2 mg / kg, and is judged as positive.

[0040] Invalid: when the quality control line, i.e. the C line, does not show red color, no matter whether the detection line, i.e. the T line, shows color or not, it indicates that the experimental results are invalid.

[0041] The nitrite colloidal gold immunochromatographic test paper of the application is designed based on the specific binding of antigen and antibody and the chromatography principle. When detecting, 100 muL of the sample to be detected is added to the micropore containing the gold-labeled antibody, and after mixing by blowing, the sample is left to react for 3 min. The sample to be detected is added dropwise to the sample pad, and the liquid moves upward by capillary siphon effect. When the sample does not contain the target to be detected or the content is lower than the detection limit, the gold-labeled antibody moves upward, the artificial antigen on the detection line of the reaction membrane is captured to make the detection line color, and the remaining gold-labeled antibody continues to move upward and is combined with the goat anti-mouse IgG on the quality control line to make the quality control line color. When the color intensity of the detection line is greater than or equal to that of the quality control line, the detection result is determined to be negative. When the sample contains a certain amount of the to-be-detected substance and the content is higher than the detection limit, the to-be-detected substance competes with the artificial antigen for the gold-labeled antibody, the gold-labeled antibody combined with the to-be-detected substance does not combine with the artificial antigen on the detection line, but still combines with the goat anti-mouse IgG on the quality control line. Therefore, as the concentration of the to-be-detected substance increases, the color of the detection line becomes lighter and lighter until the line disappears, and when the color intensity of the detection line is lighter than that of the quality control line, the detection result is determined to be positive. When the quality control line does not color, it is an invalid result. After the reaction is completed, the naked eye observation and colloidal gold reading instrument reading are performed to establish a standard curve, so that the content of nitrite in the sample is determined.

[0042] Further, the extracting agent is hydrochloric acid, citric acid, p-toluenesulfonic acid or phosphate buffer.

[0043] Still further, the extracting agent is p-toluenesulfonic acid.

[0044] Preferably, the extracting agent is 5% p-toluenesulfonic acid.

[0045] Further, the ratio of the sample to be detected to the extracting agent is 2.0-4.0 g:3 mL.

[0046] Preferably, the ratio of the sample to be detected to the extracting agent is 3 g:3 mL.

[0047] Further, the derivative reagent is o-phenylenediamine.

[0048] Preferably, the derivative temperature is 80 DEG C, and the derivative time is 10 min.

[0049] Further, the extracting agent is ethyl acetate.

[0050] Further, the ratio of the sample to be detected to the reconstituting solution is 2.0-4.0 g:500 muL.

[0051] Still further, the ratio of the sample to be detected to the reconstituting solution is 3 g:500 muL.

[0052] Preferably, the reconstituted solution is a 0.09–0.11 M PB solution.

[0053] More preferably, the reconstituted solution is a 0.1 M PB (pH 6.0) solution.

[0054] Therefore, the present invention also provides the application of the above-mentioned kit in the detection of nitrite.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] This invention provides a method for preparing and applying nitrite hapten, artificial antigen, and antibody. The hapten design strategy of this invention introduces a linker arm while preserving the characteristics of the analyte molecule to the greatest extent possible, maintaining structural rigidity, and enhancing the complexity and stability of the molecule. This ensures that the hapten can stably stimulate the body to produce specific antibodies during immunization. Introducing a benzene ring as a cyclic linker arm increases the rigidity and stability of the molecule, and fully exposes the characteristics of the analyte molecule, thereby improving the immunogenicity of the hapten. This results in the preparation of an antibody with good specificity. The IC50 of this antibody is... 50 The detection limit was 209.75 ng / mL, with a linear range of 60.39–728.53 ng / mL, exhibiting high sensitivity. A colloidal gold immunochromatographic assay kit for nitrite was prepared using the above antigen-antibody composition. Experiments verified that the kit has high detection sensitivity, good specificity, and no cross-reactivity with common food additives. The pretreatment method is simple (reaction time 25–30 min), and results can be observed 5 minutes after sample addition. The assay kit of this invention has advantages such as high specificity, high sensitivity, fast detection speed, simple operation, low cost, stable color, and long shelf life of the test strips, 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 pickled vegetables by units or individuals in health, quality inspection, and agricultural markets. Attached Figure Description

[0057] Figure 1 This is the mass spectrum of the nitrite hapten.

[0058] Figure 2 This is the standard curve for ic-ELISA.

[0059] Figure 3 This is a schematic diagram and top view of the structure of a colloidal gold nitrite immunochromatographic test strip. Figure 3 In the diagram, A is a schematic diagram and B is a top view. 1 is the sample pad; 2 is the reaction membrane; 3 is the detection line; 4 is the quality control line; 5 is the absorbent pad; and 6 is the base plate.

[0060] Figure 4 This is a result analysis diagram of the nitrite colloidal gold immunochromatographic assay kit.

[0061] Figure 5 The effect of potassium carbonate volume on the nitrite colloidal gold immunochromatographic assay kit.

[0062] Figure 6 The effect of antibody volume on the nitrite colloidal gold immunochromatographic assay kit.

[0063] Figure 7 The effect of antigen concentration on the nitrite colloidal gold immunochromatographic assay kit.

[0064] Figure 8 The effect of goat anti-mouse secondary antibody concentration on the nitrite colloidal gold immunochromatographic assay kit.

[0065] Figure 9 To investigate the effect of temperature on the nitrite colloidal gold immunochromatographic assay kit.

[0066] Figure 10 To investigate the effect of derivatization time on the nitrite colloidal gold immunochromatographic assay kit.

[0067] Figure 11 The effect of the type of extractant on the extraction effect of pretreatment.

[0068] Figure 12 The effect of extractant concentration on the extraction efficiency of pretreatment.

[0069] Figure 13 This is a standard curve diagram of colloidal gold.

[0070] Figure 14 The image shows the results of a specific experiment.

[0071] Figure 15 The figure shows the results of the stability experiment. Detailed Implementation

[0072] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0073] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0074] Example 1: Preparation of nitrite hapten and monoclonal antibody

[0075] (1) The synthesis process of nitrite hapten is shown in the following reaction equation:

[0076]

[0077] Preparation of the hapten: 1.35 g (10 mmol) of 5-hydroxybenzotriazole and 2.76 g (20 mmol) of potassium carbonate were added to N,N-dimethylformamide (DMF) and stirred until homogeneous. 4.58 g (12 mmol) of methyl 4-bromomethylbenzoate was added, and the mixture was reacted at 80 °C for 5 h. The mixture was extracted with water-ethyl acetate and then rotary evaporated. The product was dissolved in 3 mL of methanol, and 1 mL of 10% lithium hydroxide aqueous solution was added dropwise. The mixture was reacted at 50 °C until complete hydrolysis. The pH was adjusted to acidic with concentrated hydrochloric acid, and the mixture was extracted with ethyl acetate and then rotary evaporated to obtain the hapten.

[0078] The mass spectrum of the prepared nitrite hapten is shown below. Figure 1 As shown, it has the structure shown in equation (I):

[0079] .

[0080] (2) Preparation of immunogen and coating agent: 10 mg of hapten was dissolved in 200 μL of DMF, and 8 mg of N-hydroxysuccinimide (NHS) and 10 mg of 1-ethylcarbodiimide hydrochloride (EDC) were added to dissolve them completely. The mixture was then stirred at room temperature in the dark for 4 h to obtain the activation solution. The carrier proteins LF and OVA were dissolved in alkaline CB buffer to a concentration of 10 mg / mL. The activation solution was added dropwise in an ice bath with stirring. After coupling overnight at room temperature, the immunogen (NIT-LF) and coating agent (NIT-OVA) were obtained, which have the structure shown in formula (II):

[0081] ,

[0082] The carrier protein is lactoferrin (LF) or ovalbumin (OVA).

[0083] (3) Preparation of monoclonal antibodies: The prepared immunogen (NIT-LF) was diluted to 1 mg / mL with phosphate buffer solution and then emulsified with an equal volume of Freund's complete adjuvant; Balb / C mice were immunized by multiple subcutaneous injections on the back, with each mouse receiving 100 μg of immunization; booster immunization was performed every two weeks, and mice with high titers and inhibition rates were selected for cell solution experiments. After obtaining a high purity positive hybridoma cell line, ascites was prepared and purified to obtain monoclonal antibodies.

[0084] (4) Establishment of ic-ELISA curve: The original NIT-OVA coating was diluted to 1 μg / mL with carbonate buffer (CB, 0.1M pH=9.8), coated with 96-well microplate, 100 μL was added to each well, incubated at 37℃ for 4 h, the liquid in the well was poured off, and the plate was washed twice with washing buffer for 30 s each time, patted dry, and then 120 μL of blocking buffer (i.e., 5% skim milk powder by mass) was added to each well, incubated at 37℃ for 2 h, the liquid in the well was poured off and patted dry. Monoclonal antibodies prepared by 1:8000 dilution with phosphate-buffered saline (PBS, 0.01M, pH=7.4) were serially 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. These were added to NIT-OVA-coated microplates using a standard indirect competitive ELISA method. The plates were incubated at 37°C for 40 min, washed five times, and blotted dry. Goat anti-mouse secondary antibody-HRP (5000-fold dilution with PBST, 0.01M), 100 μL / well, was added and incubated at 37°C for 30 min. The plates were washed five times and blotted dry. Finally, 100 μL of chromogenic solution was added to each well. μL, color development for 10 min; add 50 μL of 10% H2SO4 solution to terminate the reaction, and read the OD value at 450 nm. Plot B / B0 as the ordinate and the logarithm of the corresponding standard concentration as the abscissa, and calculate the nitrite content. The molar ratio of benzotriazole to nitrite is 1:1. Use the normalization method to plot the standard curve, as shown in the figure. Figure 2 The relevant standard curve parameters are shown in Table 1.

[0085] Table 1 Detection parameters for monoclonal antibodies

[0086]

[0087] Example 2: Preparation of Nitrite Colloidal Gold Immunochromatographic Detection Kit

[0088] 1. Main materials

[0089] Sample pads, nitrocellulose membranes (NC membranes), absorbent pads, and polyvinyl chloride base plates were sourced from Shanghai Liangxin Technology Co., Ltd.; chloroauric acid and trisodium citrate were sourced from Sinopharm Chemical Reagent Co., Ltd.; goat anti-mouse IgG was sourced from Beijing TransGen Biotech Co., Ltd.; nitrite standards were sourced from Tanmo Quality Inspection Technology Co., Ltd.; sample pad treatment solution and gold standard reconstitution solution were sourced from Guangzhou Wanlian Biotechnology Co., Ltd.; samples (sausage, luncheon meat, and pickled mustard greens) were commercially available; all other reagents were of analytical grade.

[0090] 2. Preparation of Nitrite Colloidal Gold Immunochromatographic Detection Kit

[0091] (1) Preparation of colloidal gold solution: Colloidal gold particles were prepared by the reduction method of trisodium citrate. 100.0 mL of ultrapure water was weighed into a 500 mL round bottom flask and placed in a magnetically stirred oil bath. After stirring and heating to boiling, 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. Heating was continued for 10 min and then stopped. Stirring was continued under residual heat and cooled to room temperature to obtain a colloidal gold solution with a mass concentration of 0.02%.

[0092] (2) Preparation of gold-labeled antibody microwells: Take 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 to mix, add 5 μg of antibody, shake to mix, and react at room temperature for 10 min for labeling. Add 10 μL of 100 g / L BSA solution for blocking, shake to mix, and react at room temperature for 5 min to block the sites on the colloidal gold that have not bound to the antibody. After blocking, centrifuge for 12 min (4℃, 12000 rpm), discard the supernatant, add 500 μL of gold diluent to reconstitute, aliquot the reconstituted product into microwells at a certain volume, and dry overnight in a 45℃ oven to obtain gold-labeled antibody.

[0093] The gold diluent is a PB solution with a concentration of 0.1 mol / L, containing 0.1% BSA, 1% sucrose, 0.3% PVP-40, and 0.01% preservative PC300.

[0094] (3) Preparation of reaction membrane: The artificial antigen was diluted to 0.8 mg / mL with 0.01 mol / L PB solution, and the goat anti-mouse secondary antibody was diluted to 0.05 mg / mL with 0.01 mol / L PB solution. The diluted artificial antigen and goat anti-mouse secondary antibody were sprayed onto the NC membrane by a membrane scrubbing instrument as the detection T line and the quality control C line, respectively. The membrane was then left to stand overnight in a 45℃ drying oven for later use.

[0095] (4) Preparation of sample pads: The glass cellulose membrane was immersed in the sample pad treatment solution for 10 min, and then dried overnight at 45°C. The sample pad treatment solution was a 0.1 mol / L PB solution containing 1% Tween-20 and 0.2% sucrose.

[0096] (5) Assemble the test strip cards: such as Figure 3 The diagram shown is a schematic representation of the structure of the nitrite colloidal gold immunochromatographic test strip of the present invention. Figure 3 A) and top view ( Figure 3B) includes a base plate 6 and a sample pad 1, a reaction membrane 2, and an absorbent pad 5 arranged sequentially on the base plate 6. The reaction membrane 2 has non-overlapping detection lines 3 and control lines 4. The two ends of the reaction membrane 2 are connected to the absorbent pad 5 and the sample pad 1, respectively. The connecting portions of the sample pad 1, reaction membrane 2, and absorbent pad 5 overlap by 1–2 mm and are attached to the base plate 6. The base plate is cut into 3 mm wide strips using a strip cutter and then placed into a plastic card.

[0097] 3. Detection principle of the nitrite colloidal gold immunochromatographic assay kit

[0098] The nitrite colloidal gold immunochromatographic test strip of the present invention is designed based on the specific binding of antigen and antibody and the principle of chromatography. Figure 4 As shown, during the test, 100 µL of the test solution is added to the microwell containing the gold-labeled antibody, mixed thoroughly by pipetting, and allowed to stand for 3 min. The test solution is then dropped onto sample pad 1, and the liquid migrates upwards for chromatography using capillary action. When the sample does not contain the target analyte or the concentration is below the detection limit, the gold-labeled antibody migrates upwards and is captured by the artificial antigen immobilized on the detection line 3 of the reaction membrane 2, causing the detection line to develop color. The remaining gold-labeled antibody continues to migrate upwards and binds to goat anti-mouse IgG on the control line 4, causing the control line to develop color. When the color intensity of the detection line 3 is greater than or equal to the color intensity of the control line 4, the test result is considered negative. When a sample contains a certain amount of the analyte above 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 test line 3, but will still bind to goat anti-mouse IgG on control line 4. Therefore, as the concentration of the analyte increases, the color of test line 3 will become lighter and lighter until it disappears. When the color intensity of test line 3 is lighter than that of control line 4, the test result is considered positive. When control line 4 does not develop color, the result is invalid. After the reaction is complete, a standard curve is established by observing with the naked eye and reading with a colloidal gold reader to determine the nitrite content in the sample.

[0099] Instructions for use of the Nitrite Colloidal Gold Immunochromatographic Detection Kit (1) Sample pretreatment: Crush sausage, luncheon meat, and pickled mustard greens (or other samples to be tested) separately. Weigh 3 g of the sample to be tested into a clean centrifuge tube, add 3 mL of extraction reagent (5% p-toluenesulfonic acid) and 100 µL of o-phenylenediamine derivatizing reagent (3 mg / mL), shake well, react at 80℃ for 10 min, add 500 µL of dipotassium hydrogen phosphate aqueous solution (4 mol / L), add 2 mL of ethyl acetate, shake well, centrifuge at 3000 r for 3 min, take the supernatant and blow dry, finally reconstitute with 500 μL of reconstitution solution (0.1M PB pH6.0 solution), shake well again to obtain the pretreatment test solution.

[0100] (2) Place the nitrite colloidal gold immunochromatographic test paper prepared in this embodiment flat, add 100 µL of the test solution to the microwell containing the gold-labeled antibody, mix by blowing and letting stand for 3 min, add the test solution to the sample well of the sample pad, and observe the results after standing for 3-5 min.

[0101] Results analysis is as follows: Figure 4 As shown, the results are interpreted as follows:

[0102] Negative (-): The control line (C line) shows color development, and the test line (T line) is redder than or equal to the control line (C line), indicating that the sample does not contain nitrite or its content is less than 0.2 mg / kg, and is judged as negative.

[0103] Positive (+): The control line (C line) shows color, and the test line (T line) does not show red or shows red but is lighter in color than the control line (C line), indicating that the sample contains nitrite and its content is higher than 0.2 mg / kg, and is judged as positive.

[0104] Invalid: If the control line (C line) does not show red, the experimental result is invalid regardless of whether the test line (T line) shows color.

[0105] Example 3: Effect of different preparation conditions on the efficacy of the nitrite colloidal gold immunochromatographic assay kit

[0106] 1. The effect of the nitrite colloidal gold immunochromatographic assay kit under different preparation conditions

[0107] (1) Colloidal gold-labeled antibodies bind to and encapsulate on the surface of colloidal gold particles through electrostatic adsorption. The pH value of the labeling system affects the adsorption efficiency of the antibody and the colloidal gold particles, as well as the bioactivity of the antibody. The volumes of potassium carbonate were 4, 6, 8, and 10 μL, corresponding to pH values ​​of 6.2, 6.6, 7.0, and 7.4, respectively. Other preparation conditions and parameters were the same as in Example 2.

[0108] (2) The antibody volumes were 3, 5, 7 and 9 μg / mL, respectively, and other preparation conditions and parameters were the same as in Example 2.

[0109] (3) The concentrations of the antigen were 0.4, 0.6, 0.8 and 1 mg / mL, respectively, and the other preparation conditions and parameters were the same as in Example 2.

[0110] (4) The concentrations of sheep anti-mouse secondary antibody were 0.02, 0.05, 0.1 and 0.2 mg / mL, respectively, and other preparation conditions and parameters were the same as in Example 2.

[0111] (5) The derivatization temperatures were set to room temperature, 40°C, 60°C, 80°C and 90°C respectively, and other preparation conditions and parameters were the same as in Example 2.

[0112] (6) The derivatization times were 5, 10, 15 and 20 min respectively, and the other preparation conditions and parameters were the same as in Example 2.

[0113] (7) Sausage, luncheon meat and pickled mustard tuber were pretreated using 1 mol / L hydrochloric acid, citric acid, p-toluenesulfonic acid and 0.1 M PB (pH 6.0) solution as extraction solvents respectively.

[0114] (8) Prepare sausages, luncheon meat and pickled mustard tubers with aqueous solutions containing 2.5%, 5%, 10% and 15% p-toluenesulfonic acid, respectively.

[0115] 2. Experimental Results

[0116] (1) such as Figure 5 As shown, in 1 mL of colloidal gold solution, when the volume of 0.1 M K₂CO₃ solution is 4 µL, the colloidal gold solution turns purple, the test strip shows uneven color development, and the inhibition rate is poor. As the volume of K₂CO₃ solution increases from 6 µL to 10 µL, the color development and T / C value of the test strip show a slight increase followed by no significant change. The inhibition rate is highest when the volume of K₂CO₃ solution is 8 µL. Therefore, 8 µL of 0.1 M K₂CO₃ solution is selected as the optimal amount of potassium carbonate to add.

[0117] (2) For example Figure 6 As shown, in 1 mL of colloidal gold solution, when the antibody labeling concentration was below 5 µg / mL, the color intensity of the test strip was too low and the inhibition rate was poor. As the antibody labeling concentration increased from 5 µg / mL to 9 µg / mL, the T / C value of the test strip did not change significantly, and the inhibition rate reached its highest value at an antibody labeling concentration of 7 µg / mL before decreasing. Therefore, the optimal antibody labeling concentration of 7 µg / mL, which yielded the best T / C value and inhibition rate, was selected as the optimal antibody labeling concentration.

[0118] (3) such as Figure 7 As shown, the T / C value of the test strip gradually increases with the increase of antigen concentration. When the antigen concentration is greater than 0.8 mg / mL, the T / C value and inhibition rate of the test strip tend to stabilize. Therefore, 0.8 mg / mL is selected as the optimal T-line coating concentration.

[0119] (4) such as Figure 8 As shown, 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 their highest values ​​at a concentration of 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.

[0120] (5) such as Figure 9 As shown, with the increase of derivatization temperature, the T / C value of the test strip did not change significantly and the color intensity met the requirements. The inhibition rate of the test strip showed an overall upward trend, and the inhibition rate reached the highest at a derivatization temperature of 80℃. Therefore, 80℃ was selected as the optimal derivatization temperature.

[0121] (6) For example Figure 10 As shown, 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 its highest point at a derivatization time of 10 min and then did not change significantly. Therefore, 10 min was selected as the optimal derivatization time.

[0122] (7) For example Figure 11 As shown, when citric acid and 0.1M PB buffer (pH 6.0) were used as pretreatment extraction agents, nitrite in the sample could not be extracted due to significant matrix interference in the sample solution, resulting in poor derivatization efficiency. 1M hydrochloric acid, due to its oxidizing properties as an inorganic acid, also caused matrix interference in the directly extracted sample solution, leading to low derivatization efficiency. In contrast, utilizing the electron absorption of the aromatic ring of toluenesulfonic acid by a non-oxidizing inorganic acid enhanced the ionization ability of hydrogen ions, improving the extraction effect. It exhibited minimal matrix interference and the highest inhibition rate. Therefore, p-toluenesulfonic acid was chosen as the pretreatment extraction agent.

[0123] (8) such as Figure 12 As shown, when 2.5% p-toluenesulfonic acid is used as the pretreatment extractant, the extractant cannot completely acidify the impurities, leading to a decrease in derivatization efficiency. When the p-toluenesulfonic acid content reaches 5%, it can acidify the impurities to a certain extent, reduce sample matrix interference, and increase derivatization efficiency. However, when the p-toluenesulfonic acid content is greater than 10%, the solution becomes too acidic, causing false positives in negative samples (T / C value < 1.0). Therefore, 5% p-toluenesulfonic acid is selected as the optimal pretreatment extractant.

[0124] Example 4: Establishment of a standard curve for nitrite detection using a colloidal gold immunochromatographic assay kit.

[0125] 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 using 0.1 mol / L PB (pH 6.0) solution. Derivatizing reagents were added, and the mixture was reacted at 80℃ for 10 min. 100 μL of the standard solution was then tested using the nitrite colloidal gold immunochromatographic assay kit prepared in Example 2 to establish a standard curve.

[0126] like Figure 13 The figure shown is a standard curve graph, IC. 20 ~IC 80The concentration ranges from 0.4 to 3.9 μg / mL, and the IC50 concentration is [missing information]. 50 The concentration was 1.3 μg / mL, and the detection limit was 0.2 μg / mL.

[0127] Example 5: Specificity and stability testing of the nitrite colloidal gold immunochromatographic assay kit.

[0128] 1. Experimental Methods

[0129] (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 with 0.1 mol / L PB (pH 6.0) solution. The 4 μg / mL benzotriazole standard solution in Example 4 was used as a control and the nitrite colloidal gold immunochromatographic assay kit prepared in Example 2 was used for detection.

[0130] (2) Stability: The nitrite colloidal gold test paper prepared in Example 2 was sealed and placed in an oven at 45°C for accelerated stability testing. The performance of the test paper strips was tested on days 1, 7, 14, 21 and 28.

[0131] 2. Experimental Results

[0132] (1) such as Figure 14 As shown, low concentration (4 μg / mL) of benzotriazole showed a strong positive result, while the other six high concentrations (50 μg / mL) of structural analogs were all negative. This indicates that the nitrite colloidal gold immunochromatographic test strip prepared in Example 2 has high specificity and no cross-reactivity with common structural analogs.

[0133] (2) For example Figure 15 As shown, the T / C value and inhibition rate of the nitrite colloidal gold immunochromatographic test strip were recorded at different time periods. The T / C value and inhibition rate of the test strip remained basically stable at different time periods, indicating that the nitrite colloidal gold immunochromatographic test strip prepared in Example 2 maintained good stability in both negative color development and positive inhibition.

[0134] Since different artificial antigens stimulate the body to produce antibodies with different titers and different compatibility with the coating antigen, insufficient antibody labeling during the labeling process will affect the stability of the prepared detection kit. Therefore, the nitrite colloidal gold immunochromatographic detection kit prepared by the above method of the present invention has good stability.

[0135] Example 6: Addition and Recovery Experiment of Nitrite Colloidal Gold Immunochromatographic Detection Kit

[0136] Three samples—sausage, luncheon meat, and pickled mustard greens—were selected for nitrite recovery experiments. 3 g of each sample was weighed, and nitrite was added to each sample at gradient concentrations of 2.0, 10.0, and 50.0 mg / kg, with three replicates for each concentration, for a total of 27 groups. The pretreatment method described in Example 2 and a nitrite colloidal gold immunochromatographic assay kit were used for detection.

[0137] As shown in Table 2, the recovery rates of nitrite in the three samples ranged from 74.9% to 108.2%, with an RSD of <14.4%, indicating that the immunochromatographic detection method established in this study has good accuracy.

[0138] Table 2. Nitrite recovery experiment in samples

[0139]

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A nitrite artificial hapten, characterized in that, The structural formula of the nitrite artificial hapten is shown as formula (I), 。 2. The use of the nitrite artificial hapten in the preparation of a nitrite artificial antigen according to claim 1.

3. A nitrite artificial antigen, characterized in that, The nitrite artificial antigen is the hapten according to claim 1 combined with a carrier protein, and the structural formula is shown as formula (II): ， The carrier protein is lactoferrin or chicken egg white albumin.

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