Method for detecting malachite green, crystal violet and methylene blue

Abstract

The invention discloses a method for detecting malachite green, crystal violet and methylene blue, which is characterized in that two different haptens are respectively used as an immunogen and a coating antigen to obtain a high-sensitivity monoclonal antibody for resisting malachite green, crystal violet and methylene blue. The invention further establishes a chemiluminescence immune method for detecting malachite green, crystal violet and methylene blue based on biotin-streptavidin, and the method has the advantages of rapidness, sensitivity, low cost and high throughput, and is suitable for simultaneously detecting the residual quantity of malachite green, crystal violet or methylene blue in aquatic products.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of aquatic drug residues, and more specifically, to a method for detecting malachite green, crystal violet and methylene blue. Background Art

[0002] Both malachite green and crystal violet are triphenylmethane compounds, which were once widely used as fungicides and parasiticides in aquaculture. They are easily metabolized into leucomalachite green and leucocrystal violet in aquatic organisms, and these substances accumulate in the liver, kidneys, muscles, etc. of aquatic organisms and are ingested by the human body, having side effects such as high toxicity, high residue, teratogenicity, carcinogenicity and mutagenicity. Therefore, they have been prohibited from use in many countries.

[0003] Methylene blue is an artificially synthesized thiazine dye. Due to its low toxicity, it is often used as a substitute for malachite green and crystal violet. In recent years, many studies have shown that methylene blue at a certain concentration can have toxic side effects on aquatic organisms, and being ingested by the human body through bioaccumulation will threaten physical health. Therefore, strengthening the management of aquaculture, standardizing the use of drugs in farms, and strengthening the residue detection of malachite green, crystal violet and methylene blue are crucial for ensuring food safety.

[0004] Currently, the main method for detecting malachite green, crystal violet and methylene blue dye compounds in aquatic products is high-performance liquid chromatography. Although this method is accurate and sensitive, it has high operating costs and requires professional personnel to operate, and cannot meet the needs of on-site rapid detection. Chemiluminescence immunoassay has high sensitivity, high specificity, stable chemical properties and low cost, and is suitable for various immunoassay scenarios. The biotin-streptavidin system has high specificity and high affinity, is not interfered by acids and bases, high temperature, proteolytic enzymes and organic solvents, and combined with chemiluminescence immunoassay, can amplify the reaction signal and improve the detection sensitivity.

[0005] Malachite green, crystal violet and methylene blue are dye drugs in aquaculture. Although they have been prohibited or restricted from use, due to their low price and easy availability, there are still illegal use phenomena. Currently, the immunoassay methods for the three drugs can only detect one of them, and there is no immunoassay method for simultaneously detecting malachite green, crystal violet and methylene blue. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for detecting malachite green, crystal violet and methylene blue.

[0007] The first purpose of the present invention is to provide a compound for use as a hapten for malachite green, crystal violet and methylene blue, or in the preparation of artificial antigens for malachite green, crystal violet and methylene blue.

[0008] The second object of the present invention is to provide another compound for use as a hapten for malachite green, crystal violet and methylene blue, or for use in the preparation of artificial antigens of malachite green, crystal violet and methylene blue.

[0009] The third object of the present invention is to provide a conjugate for use in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, or for use in the preparation of antibodies for detecting malachite green, crystal violet and methylene blue.

[0010] The fourth object of the present invention is to provide another conjugate for use in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, or for use in the preparation of antibodies for detecting malachite green, crystal violet and methylene blue.

[0011] The fifth object of the present invention is to provide a composition for detecting artificial antigens of malachite green, crystal violet and methylene blue.

[0012] The sixth object of the present invention is to provide a composition of an immunogen and a coating antigen.

[0013] The seventh object of the present invention is to provide a composition of an antibody and a coating antigen.

[0014] The eighth object of the present invention is to provide one or more of the above compositions for use in the preparation of a kit for detecting malachite green, crystal violet and methylene blue, or for use in detecting malachite green, crystal violet and methylene blue.

[0015] The ninth object of the present invention is to provide a kit for detecting malachite green, crystal violet and methylene blue.

[0016] To achieve the above objects, the present invention is realized by the following technical solutions:

[0017] The present invention claims to protect a compound MCM-1 for use as a hapten for malachite green, crystal violet and methylene blue, or for use in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, and its structural formula is shown in I,

[0018]

[0019] and another compound MCM-2 for use as a hapten for malachite green, crystal violet and methylene blue, or for use in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, and its structural formula is shown in II,

[0020]

[0021] The present invention claims to protect the use of a conjugate MCM-1-pro in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, or in the preparation of antibodies against malachite green, crystal violet and methylene blue. The conjugate is the compound MCM-1 conjugated with a carrier protein, and its structural formula is as shown in III.

[0022]

[0023] And another conjugate MCM-2-pro, its use in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, or in the preparation of antibodies for detecting malachite green, crystal violet and methylene blue. The conjugate is the compound MCM-2 conjugated with a carrier protein, and its structural formula is as shown in IV.

[0024]

[0025] Preferably, the carrier protein is one or more of chicken ovalbumin (OVA), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or lactoferrin (LF).

[0026] The above conjugates are prepared by the active ester method.

[0027] The present invention claims to protect a composition of artificial antigens of malachite green, crystal violet and methylene blue, containing the conjugate shown in structural formula III and the conjugate shown in structural formula IV.

[0028] The present invention claims to protect a composition of an immunogen and a coating antigen. The immunogen is the conjugate MCM-2-KLH, and its structural formula is as shown in IV-1.

[0029]

[0030] The coating antigen is the conjugate MCM-1-OVA, and its structural formula is as shown in III-1.

[0031]

[0032] The present invention claims to protect a composition of an antibody and a coating antigen, containing an antibody and a coating antigen. The antibody is obtained by immunizing an animal with the conjugate MCM-2-KLH shown in structural formula IV-1.

[0033]

[0034] The coating antigen is the conjugate MCM-1-OVA shown in structural formula III-1.

[0035]

[0036] Preferably, the antibody is conjugated with biotin.

[0037] The present invention also claims one or several of the above-mentioned compositions for use in the preparation of a kit for detecting malachite green, crystal violet and methylene blue, or for detecting malachite green, crystal violet and methylene blue.

[0038] Preferably, the detection is qualitative detection and / or quantitative detection.

[0039] Preferably, the kit is a biotin-streptavidin chemiluminescent immunoassay kit.

[0040] The present invention also claims a kit for detecting malachite green, crystal violet and methylene blue, which contains any one of the above-mentioned compositions or is prepared by using any one of the above-mentioned compositions.

[0041] Preferably, the kit is a chemiluminescent immunoassay kit based on biotin-streptavidin. The conjugate shown in Structural Formula Ⅳ-1 is used to immunize an animal to obtain an antibody conjugated with biotin.

[0042] Preferably, the conjugate shown in Structural Formula Ⅲ-1 is coated on a solid-phase carrier.

[0043] Preferably, it also contains one or several of a washing solution, an enzyme-labeled plate, a diluent and a chemiluminescent solution.

[0044] Preferably, the detection is qualitative detection and / or quantitative detection.

[0045] As a specific embodiment, the washing solution: 8 g / L NaCl, 2.4 g / L Na2HPO4·12H2O, 0.6 mL / L Tween-20.

[0046] Based on the above-mentioned kit, a method for detecting malachite green, crystal violet and / or methylene blue in aquatic products for non-diagnostic purposes is provided:

[0047] S1. Coating the artificial antigen MCM-1-OVA conjugated with chicken ovalbumin as a coating antigen in different micro-wells of an enzyme-labeled plate, incubating, washing the plate and blocking;

[0048] S2. Respectively adding standard solutions of malachite green, crystal violet or methylene blue or treated sample solutions to be detected into the corresponding micro-wells of the enzyme-labeled plate;

[0049] S3. Adding a solution of an antibody conjugated with biotin into the micro-wells of the enzyme-labeled plate, incubating and washing the plate;

[0050] S4. Adding a solution of streptavidin labeled with horseradish peroxidase into all micro-wells of the enzyme-labeled plate, incubating and washing the plate;

[0051] S5. Add equal volumes of commercially available chemiluminescence solution A and solution B, and immediately read the absorbance value of each well using a multifunctional microplate reader;

[0052] S6. Use the four-parameter fitting module of Origin9.0 to plot the standard curve and calculate the corresponding detection parameters of the inhibition curve;

[0053] S7. Obtain the test results of the sample to be tested according to the curve.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The present invention provides a method for detecting malachite green, crystal violet and methylene blue. The method uses two different artificial antigens as immunogen and coating antigen respectively to obtain monoclonal antibodies against malachite green, crystal violet and methylene blue with high sensitivity. Further, a chemiluminescence immunoassay based on biotin-streptavidin is established for detecting malachite green, crystal violet and methylene blue. The method is rapid, sensitive, low-cost and high-throughput, and is suitable for simultaneously detecting the residues of malachite green, crystal violet or methylene blue in aquatic products. Description of the Drawings

[0056] Figure 1 It is the mass spectrum of hapten MCM-1.

[0057] Figure 2 It is the mass spectrum of hapten MCM-2.

[0058] Figure 3 It is the UV scanning chart of artificial antigen MCM-1-OVA.

[0059] Figure 4 It is the UV scanning chart of artificial antigen MCM-2-KLH.

[0060] Figure 5 It is the standard curve of malachite green.

[0061] Figure 6 It is the standard curve of crystal violet.

[0062] Figure 7 It is the standard curve of methylene blue. Detailed Embodiments

[0063] The present invention will be further described in detail below with reference to the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0064] Example 1 Synthesis of Hapten

[0065] I. Experimental Methods

[0066] 3.05 g of hydroxybenzaldehyde was mixed with 5 mL of 5 mol / L NaOH solution and stirred well until dissolved. After adding 2.9 g of sodium chloroacetate and 20 mL of distilled water, the mixture was stirred and reacted at 60 °C for 2 - 3 h. After the reaction, the pH of the solution was adjusted to 2 with concentrated hydrochloric acid, and then adjusted to alkaline with NaHCO₃. Dichloromethane was used for extraction to remove the incompletely reacted raw materials. The aqueous phase was reserved, and 4 - formylphenoxyacetic acid was precipitated by adjusting with concentrated HCl.

[0067] 0.585 g of 4 - formylphenoxyacetic acid and 1.6 g of anhydrous zinc chloride were added to a two - necked flask. Under N₂ protection, 20 mL of absolute ethanol and 0.725 mL of N,N - dimethylaniline were successively added to the two - necked flask. The mixture was heated to 90 °C, stirred and reacted. Samples were taken for dilution at intervals, and the progress of the reaction was judged by TLC. After 24 h, the reaction ended. After rotary evaporation and drying, an appropriate amount of dichloromethane was added for dissolution, and then petroleum ether was added and stirred until precipitation occurred. After filtration and drying, the target hapten (MCM - 1) was obtained, which was sub - packed and stored at 4 °C, with attention paid to light - proof treatment.

[0068] MCM - 1 was dissolved in 90 mL of chloroform, and then 4.90 mg of p - benzoquinone tetrachloride and 1.5 mL of glacial acetic acid were successively added. The mixture was stirred and reacted at room temperature for 2 h. The precipitate of the reaction product was filtered and washed twice with an equal - volume chloroform - carbon tetrachloride solution (100 mL), and then dried under vacuum at 40 °C overnight to obtain MCM - 2.

[0069] The products MCM - 1 and MCM - 2 were subjected to mass spectrometry detection.

[0070] II. Experimental Results

[0071] The mass spectrometry results are shown in Figure 1 and Figure 2 :

[0072] The relative molecular mass of MCM - 1 is 404.5, and there is an ultra - strong signal peak at 403.4 in the mass spectrometry diagram of MCM - 1. The results are all in line with the expectations, indicating that the synthesis of the hapten is successful, and its structural formula is as shown in Formula I

[0073]

[0074] The relative molecular mass of MCM - 2 is 403.5, and MCM - 2 has an ultra - strong signal peak at 405.5. The results are all in line with the expectations, indicating that the synthesis of the hapten is successful, and its structural formula is as shown in Formula II

[0075]

[0076] Example 2 Synthesis of Artificial Antigen

[0077] I. Experimental Methods

[0078] Using the active ester method, haptens MCM-1 and MCM-2 were respectively conjugated with chicken ovalbumin (OVA) and keyhole limpet hemocyanin (KLH) to prepare immunogens and coating antigens. Specifically, the method is as follows:

[0079] (1) Dissolve 0.02 mmol of hapten in 200 μL of DMF, add 0.02 mmol of EDC and 0.02 mmol of NHS, and react overnight at 4 °C in the dark with magnetic stirring. Take the supernatant as solution A.

[0080] (2) Dissolve 10 mg of carrier protein in 2 mL of 0.1 mol / L PBS buffer solution and stir to dissolve. This is solution B.

[0081] (3) Under magnetic stirring, slowly drop solution A into solution B, react overnight at 4 °C in the dark, and dialyze with 0.01 mol / L PBS solution (pH 7.4) for 3 days, changing the dialysis solution twice a day, to obtain artificial antigens MCM-2-KLH and MCM-1-OVA. After aliquoting, store at -20 °C.

[0082] Perform ultraviolet scanning determination (200 - 400 nm) on the prepared artificial antigens, carrier proteins, and haptens, and obtain the absorption curves.

[0083] II. Experimental Results

[0084] The ultraviolet scanning diagrams are shown in Figure 3 and Figure 4 . The results show that the ultraviolet scanning curves of the hapten, carrier protein, and artificial antigen do not overlap with each other and the characteristic absorption peaks have corresponding offsets, indicating that both artificial antigens MCM-2-KLH and MCM-1-OVA are successfully prepared.

[0085] The structure of the prepared artificial antigen MCM-1-OVA is shown in Formula III-1:

[0086]

[0087] The structure of the prepared artificial antigen MCM-2-KLH is shown in Formula IV-1:

[0088]

[0089] Example 3 Preparation of Hybridoma Cell Lines and Monoclonal Antibodies Against Malachite Green, Crystal Violet, and Methylene Blue

[0090] I. Immunization of Mice

[0091] Female Balb / c mice were immunized with the artificial antigen MCM-2-KLH prepared in Example 2. For the first immunization, 500 μL (0.5 μg) of 1 mg / mL MCM-2-KLH artificial antigen was emulsified with an equal volume of Freund's complete adjuvant, with 200 μL administered to each mouse. Multiple immunizations were performed subcutaneously.

[0092] After the initial immunization, booster immunizations were performed every two weeks. The booster immunizations were emulsified with an equal volume of Freund's incomplete adjuvant. Other procedures were the same as the initial immunization. One week after the two booster immunizations, blood was collected from the tail to determine the antiserum titer. When the titer remained stable, the mice with the best immune effect (the best inhibitory effect) were selected for a booster immunization. Splenocytes were collected 3 days later for subsequent experiments. The titer and inhibition of the antiserum were tested using icELISA. The specific detection method is as follows:

[0093] (1) Coating: Dilute the coating agent (MCM-1-OVA) to 1 μg / mL with 0.01 mol / L pH 9.0 carbonate buffer, add 100 μL / well to the wells of the ELISA plate, and incubate in a 4°C water bath for 12 h.

[0094] (2) Blocking: Pour off the liquid in the ELISA plate, wash twice with a plate washer, and pat dry. Add 120 μL of blocking solution (2% bovine serum albumin solution) to each well, block in a 37°C water bath for 3 hours, spin dry the blocking solution, dry in a 37°C oven, and store at 4°C until ready for use.

[0095] (3) Add antiserum: Antiserum was serially diluted with PBS (0.01 M, pH 7.4). Malachite green was diluted to 1 μg / mL with PBS (0.01 M, pH 7.4). 50 μL of PBS and 50 μL of serially diluted antiserum were added to the titer wells. 50 μL of serially diluted antiserum and 50 μL of drug diluted with PBS were added to the inhibition wells. Incubate at 37°C for 30 min. Wash the plate 5 times with plate washing buffer and pat dry.

[0096] (4) Add secondary antibody: Add goat anti-mouse IgG-HRP diluted 5000-fold in PBS (100 μL / well) and incubate at 37°C for 30 min. Wash the plate 5 times and pat dry.

[0097] (5) Color development: Mix TMB color development solution in equal proportions, add 100 μL to each well, and incubate at 37°C for 10 min.

[0098] (6) Termination: Add 10% H2SO4 stop solution to terminate the reaction, 50 μL / well.

[0099] (7) Reading: Measure the absorbance (OD) of each well at a wavelength of 450 nm using a microplate reader. The antiserum titer is the antiserum titer corresponding to the antiserum dilution factor corresponding to an absorbance value of approximately 1.0.

[0100] According to the determination by the microplate reader, select the ones with higher OD value and dilution factor, which are recorded as having higher titer. According to the formula: Inhibition rate (%) = (A 效价 - A 抑制 ) / A 效价 × 100%, calculate the inhibition rate. The greater the inhibition rate, the more obvious the inhibition effect.

[0101] II. Preparation of hybridoma cell lines

[0102] Mix sp2 / 0 myeloma cells and spleen cells at a ratio of 1:5 - 1:10, and fuse them under the action of polyethylene glycol (PEG 2000). After suspending the fused cells, plate them in a 96-well plate containing feeder cells and HAT medium (final concentration: 80% RPMI1640 basal medium, 20% fetal bovine serum, 1% HAT), and culture them in an incubator at 37°C and 5% CO2. After fusion, when the cell colonies grow to cover 1 / 4 of the bottom of the well, take the supernatant and screen the cell wells secreting antibodies against malachite green, crystal violet, and methylene blue using icELISA (the method is the same as above). Select the monoclonal cell wells with the highest positive value (OD value detected by the microplate reader) and obvious competitive inhibition (larger inhibition rate value), and clone and expand the cells using the limited dilution method to obtain hybridoma cell lines.

[0103] III. Preparation of monoclonal antibodies

[0104] Inject 0.5 mL of liquid paraffin into the abdominal cavity of Balb / c mice. After 1 - 2 weeks, inject about 2 × 10 6 hybridoma cells into the abdominal cavity, 0.5 mL for each mouse. About 10 days after inoculation, it can be seen that the abdomen of the mice swells. Sacrifice the mice before they die, collect the ascites, centrifuge at 12000 r / min at 4°C for 15 min, remove the upper fat and the lower fibrin and cells, aliquot the middle slightly yellow liquid, and store it at -20°C for later use. The antibody is purified by the conventional caprylic acid - ammonium sulfate method to obtain monoclonal antibodies against malachite green, crystal violet, and methylene blue.

[0105] Example 4 Detection of malachite green, crystal violet, and methylene blue by biotin - streptavidin chemiluminescence immunoassay

[0106] I. Experimental method

[0107] Using artificial antigen MCM - 2 - KLH as the immunogen and artificial antigen MCM - 1 - OVA as the coating antigen, establish a method for detecting malachite green, crystal violet, and methylene blue based on biotin - streptavidin chemiluminescence immunoassay, including the following steps:

[0108] S1. Dilute the coated original MCM-1-OVA with coating buffer (0.01 mol / L carbonate buffer) to 125 ng / mL, and coat 100 μL per well in different micro-wells of the ELISA plate. After incubating at 4°C for 16 hours, wash the plate twice, then block with 2% bovine serum albumin solution for 3 h, discard the blocking solution, and dry it in an oven at 37°C;

[0109] S2. Add standard solutions of malachite green, crystal violet or methylene blue with gradient concentrations to the corresponding micro-wells of the ELISA plate. The concentrations of malachite green are: 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.048828125, 0.0244140625, 0.01221 ng / mL

[0110] The concentrations of crystal violet are: 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625 ng / mL

[0111] Methylene blue: 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625 ng / mL;

[0112] S3. Dilute the monoclonal antibodies against malachite green, crystal violet and methylene blue prepared in Example 3 with 0.01 mol / L carbonate buffer to 1.5 mg / mL. Take 1 mL and mix it with 340 μL of activated biotin solution dissolved in 1 mg / mL dimethyl sulfoxide at 4°C for 12 h. Ultrafilter with a 30 kDa ultrafiltration tube to obtain a biotinylated antibody solution. Dilute the biotinylated antibody solution 1000 times with 0.01 mol / L phosphate solution and add it to the micro-wells of the ELISA plate, incubate and wash the plate;

[0113] S4. Add horseradish peroxidase-labeled streptavidin solution to all micro-wells of the ELISA plate. After incubating for 30 min, wash the plate with the washing solution;

[0114] S5. Add an equal volume mixture of chemiluminescence Solution A and Solution B, and immediately read the absorbance value of each well with a multi-functional microplate reader;

[0115] S6. Use the concentration of the standard solution as the abscissa and B / B0 as the ordinate, and draw a standard curve using the four-parameter fitting module of Origin9.0. The absorbance value of each standard solution concentration well is B, and the absorbance value of the zero standard well is B0; Calculate the limit of detection LOD (drug concentration at 10% inhibition rate, IC10 ) and the half-maximal inhibitory concentration (the drug concentration at 50% inhibition rate, IC 50 ).

[0116] II. Experimental Results

[0117] The standard curves of malachite green, crystal violet and methylene blue are shown in Figure 5 , Figure 6 and Figure 7 ,

[0118] Among them, the standard curve established for malachite green is: y = 0.108 + 0.888 / (1+(x / 0.667) 0.855 ) R 2 = 0.9996

[0119] The standard curve established for crystal violet is: y = 0.146 + 0.725 / (1+(x / 0.897) 1.577 ) R 2 = 0.9995

[0120] The standard curve established for methylene blue is: y = 0.201 + 0.811 / (1+(x / 0.463) 1.198 ) R 2 = 0.9969

[0121] The half-inhibitory concentrations (IC 50 ) of this detection method for malachite green, crystal violet and methylene blue are 0.66, 0.9 and 0.46 ng / mL respectively, the detection limits are 0.04, 0.22 and 0.08 ng / mL respectively, and the detection ranges are 0.12 - 3.75, 0.37 - 2.16 and 0.12 - 1.81 ng / mL respectively. It can meet the detection requirements for the maximum residue limits of malachite green, crystal violet and methylene blue in aquatic products.

[0122] Example 5 Detection of Malachite Green, Crystal Violet and Methylene Blue in Aquatic Products by Biotin-Streptavidin Chemiluminescent Immunoassay

[0123] 1. Sample Pretreatment

[0124] Homogenization: For fish samples, take the dorsal muscle part; for shrimp samples, take the muscle part and homogenize at 4°C.

[0125] Weigh 5.0 g (±0.1 g) of the homogenized sample into a centrifuge tube, add 10 mL of acetonitrile, and shake vigorously for 3 min. Subsequently, add 2 g of neutral alumina, shake vigorously for 3 min, and centrifuge at 4000 r / min for 5 min. Pipette 5 mL of the supernatant into another centrifuge tube, add 1 mL of n-hexane, shake vigorously, and centrifuge at 4000 r / min for 1 min. Pipette 4 mL of the lower layer solution into a 5 mL centrifuge tube, add 0.1 mL of 0.1% tetrachlorobenzoquinone solution, shake well, and dry under nitrogen or air at 65 °C. Dissolve the residue with 0.4 mL of PBS buffer and let it stand for 2 min.

[0126] 2. Detection of malachite green, crystal violet and methylene blue

[0127] According to the method of Example 4, change the sentence in S2. "Add standard solutions of gradient concentrations of malachite green, crystal violet or methylene blue to the corresponding microwells of the microplate respectively" to "Add the pretreated sample to be tested to the corresponding microwells of the microplate", and detect malachite green, crystal violet and methylene blue in the sample to be tested.

[0128] Based on the B / B0 of the test sample and the standard curve obtained in Example 4, obtain the contents of malachite green, crystal violet and methylene blue in the sample to be tested.

[0129] Example 6 Biotin-streptavidin chemiluminescence immunoassay for the addition recovery experiment of malachite green, crystal violet and methylene blue in aquatic products

[0130] I. Experimental method

[0131] Use fish meat and shrimp meat as matrices, set the addition levels of malachite green to 4.8, 24, 48 ng / g, and the addition levels of crystal violet and methylene blue to 8 ng / g, 16 ng / g, 24 ng / g. According to the method of Example 5, detect malachite green, crystal violet and methylene blue.

[0132] II. Experimental results

[0133] The results are shown in Table 1. The average addition recoveries of malachite green in fish meat and shrimp meat samples are between 78.7% and 88%, the recovery rate of crystal violet is 85.96 - 90.43%, and the recovery rate of methylene blue is 83.3 - 89.6%. The coefficients of variation are all less than 12.49%. The results prove that the method has good accuracy and can be used for the detection of malachite green, crystal violet and methylene blue drug residues in aquatic products.

[0134] Table 1 Sample addition recovery results of malachite green, crystal violet and methylene blue (n = 3)

[0135]

[0136]

[0137] Example 6 Cross - reaction Rate of Detection Methods for Malachite Green, Crystal Violet and Methylene Blue

[0138] I. Experimental Method

[0139] According to the method of Example 4, change "Add the standard solutions of malachite green, crystal violet or methylene blue with gradient concentrations into the corresponding micro - wells of the enzyme - linked immunosorbent assay (ELISA) plate respectively" in S2 to "Add the analogues of malachite green, crystal violet and methylene blue with gradient concentrations into the corresponding micro - wells of the ELISA plate respectively", and detect the contents of malachite green, crystal violet and methylene blue in the sample to be tested.

[0140] Among them, the analogues of malachite green, crystal violet and methylene blue are: brilliant green, leucomalachite green, leucomethylene blue, pararosaniline, N, N - dimethylaniline and toluene.

[0141] Calculate the IC 50 of each analogue through the curve respectively, and calculate the cross - reaction rate of the established biotin - streptavidin chemiluminescence immunoassay for the analogue.

[0142] Cross - reaction rate = Reactant (malachite green) IC 50 / Analogue IC 50 .

[0143] II. Experimental Results

[0144] The results are as shown in Table 2. The results show that the cross - reaction rates of malachite green, crystal violet and methylene blue with brilliant green are 26%, and the cross - reaction rates with other structural analogues are all less than 2.05%.

[0145] Table 2

[0146]

[0147]

[0148] Comparative Example 1

[0149] I. Experimental Method

[0150] According to the method of Example 2, using the active ester method, the haptens MCM - 1 and MCM - 2 are respectively conjugated with chicken ovalbumin (OVA) to prepare the coating antigens MCM - 1 - OVA and MCM - 2 - OVA; the hapten MCM - 2 is respectively conjugated with bovine serum albumin (BSA), lactoferrin (LF) and keyhole limpet hemocyanin (KLH) to prepare the immunogens MCM - 2 - BSA, MCM - 2 - LF and MCM - 2 - KLH.

[0151] Then, prepare malachite green, crystal violet, and methylene blue hybridoma cell lines and monoclonal antibodies according to the method of Example 3, and use icELISA to detect the titer and inhibition of the antiserum.

[0152] II. Experimental Results

[0153] The measurement results of the mouse antiserum by icELISA are shown in Table 3.

[0154] Table 3 Titers and inhibition rates of mouse sera against different coating antigens and immunogens

[0155]

[0156] The results showed that the titer of the antibody was higher when MCM-2-OVA (homologous coating) was used. The reason is that in the homologous mode, the antibody and the coating antigen have high affinity. The reason for the better inhibition rate of the antiserum against the drug under the condition of MCM-1-OVA (heterologous coating) may be that the recognition ability of the antiserum to the coating antigen is weakened, indirectly increasing the drug competitiveness and improving the inhibition rate. At the same time, the titer may be reduced due to the decrease in the recognition ability of the antibody to the coating antigen.

[0157] The best immune effect was achieved with the immunogen MCM-2-KLH. When the coating antigen was MCM-1-OVA and the malachite green drug concentration was 1000 ng / mL, the titer and inhibition rate were 32000 and 80%, respectively.

Claims

1. A compound, as a hapten of malachite green, crystal violet and methylene blue, or its application in the preparation of artificial antigens of malachite green, crystal violet and methylene blue, characterized in that, The structural formula of the said compound is as shown in Ⅰ, 2. A compound, used as a hapten for malachite green, crystal violet and methylene blue, or in the preparation of artificial antigens for malachite green, crystal violet and methylene blue, characterized in that, The structural formula of the said compound is as shown in Ⅱ, 3. An application of a conjugate in artificial antigens of malachite green, crystal violet and methylene blue, or in the preparation of antibodies against malachite green, crystal violet and methylene blue, characterized in that, The said conjugate is the compound described in claim 1 conjugated with a carrier protein, and its structural formula is as shown in Ⅲ, 4. An application of a conjugate in an artificial antigen of malachite green, crystal violet and methylene blue, or in the preparation of antibodies against malachite green, crystal violet and methylene blue, characterized in that, The said conjugate is the compound described in claim 2 conjugated with a carrier protein, and is characterized in that its structural formula is as shown in Ⅳ, 5. A composition of artificial antigens of malachite green, crystal violet and methylene blue, characterized in that, Contains the conjugate with the structural formula as shown in Ⅲ and the conjugate with the structural formula as shown in Ⅳ.

6. A composition of an immunogen and a coating antigen, characterized in that, The said immunogen is the conjugate with the structural formula as shown in Ⅳ-1, The said coating antigen is the conjugate with the structural formula as shown in Ⅲ-1, 7. A composition of an antibody and a coating antigen, characterized in that, Contains an antibody and a coating antigen, and the antibody is obtained by immunizing an animal with the conjugate with the structural formula as shown in Ⅳ-1, The said coating antigen is the conjugate with the structural formula as shown in Ⅲ-1 8. One or several of the compositions described in claim 5, the compositions described in claim 6, and the compositions described in claim 7 are used in the preparation of a kit for detecting malachite green, crystal violet, and methylene blue, or in the detection of malachite green, crystal violet, and methylene blue.

9. A kit for detecting malachite green, crystal violet and methylene blue, characterized in that, Contains any one of the compositions described in claims 5 to 7, or is prepared using any one of the compositions described in claims 5 to 7.

10. The kit according to claim 9, characterized in that, The said kit is a chemiluminescent immunoassay kit based on biotin-streptavidin, and the antibody obtained by immunizing an animal with the conjugate with the structural formula as shown in Ⅳ-1 is conjugated with biotin.

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