Thermal signal enhanced enzyme linked immunosorbent assay kit for detecting promethazine medicine

Abstract

The invention discloses a thermal signal enhanced enzyme linked immunosorbent assay (ELISA) kit for detecting promethazine drugs. Comprising a kit body, an elisa plate arranged in the kit body and a reagent arranged in the kit body, and each hole of the elisa plate is coated with a promethazine cloned antibody A; the reagent comprises a thermal signal latex microsphere coupled promethazine anti-clonal antibody B, a promethazine standard solution, a binding enhancing solution, a washing solution and an extracting agent. According to the invention, more than 40% of the polysucrose 400 can promote the combination of the promethazine monoclonal antibody B and the promethazine-antibody A compound, so that the sensitivity is obviously improved; the antibody B is coupled with thermal signal sensitive latex microspheres, detection is carried out through a heat-sensitive sensor, the method has the advantages of being high in sensitivity, simple, convenient, rapid and accurate, and compared with a traditional colorimetric ELISA method, the sensitivity can be improved by two orders of magnitude. The method is expected to play an important role in detection of promethazine drug residues in beef and mutton foods.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a thermal signal enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug residues in beef and mutton by a double antibody sandwich method. Background Art

[0002] Promethazine, belonging to the benzamide drugs, is often used as a sedative, an antihistamine, and a pre-anesthetic drug in veterinary practice. However, due to its low cost and ability to promote animal weight gain, some illegal traders may illegally use promethazine in economic animals such as beef and mutton to increase their weight and thus obtain higher economic benefits. This practice not only violates relevant regulations and livestock product safety standards but also may pose a serious threat to consumers' health. Long-term consumption of meat products containing promethazine residues may cause adverse reactions in humans, such as drowsiness, dizziness, dry mouth, blurred vision, and even heart problems.

[0003] Therefore, it is particularly important to detect promethazine drug residues in beef and mutton, which helps food safety regulatory authorities control and prevent the illegal use of this drug, protect consumers from its potential health risks, and ensure the quality and safety of meat products. In addition, reliable detection methods can also enhance consumers' confidence in meat quality, which is of great significance for maintaining the sustainable development of the meat industry.

[0004] Currently, commonly used detection techniques include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and enzyme-linked immunosorbent assay (ELISA). Although HPLC and GC-MS have high accuracy, they are expensive in equipment, complex in operation, require professional technicians, and have cumbersome sample pretreatment, which is not suitable for large-scale sample screening.

[0005] Traditional ELISA methods have obvious advantages in batch detection due to their rapidity, relatively low cost, and simplicity in operation. However, when it comes to small molecules such as promethazine, conventional ELISA usually uses the competitive method for detection. This method requires small molecule drugs to compete with enzyme-labeled drugs for binding sites, resulting in limited detection sensitivity. In addition, competitive ELISA is often greatly affected by matrix effects and is prone to false positive or false negative results.

[0006] To improve the sensitivity and specificity of detection, researchers have been working hard to develop new ELISA methods. The small molecule sandwich ELISA is a promising technique that can effectively enhance the sensitivity of detecting small molecule drugs. This method relies on two different antibodies - one that can bind to the target drug and another that can recognize the complex formed after the drug binds. However, to implement this technique, the key lies in screening suitable antibody pairs, which is quite difficult technically, especially for small molecules like promethazine.

[0007] Thermometric Lateral Flow Immunoassay (TLFIA) is a detection method that uses temperature changes as signal output to improve the detection sensitivity of Lateral Flow Immunoassay (LFIA). LFIA is a widely used technique for rapid on-site detection, and its basic principle is to use specific antigen-antibody reactions to detect target molecules in samples. However, traditional LFIA usually relies on visual observation of color changes for qualitative or semi-quantitative analysis, which has certain limitations in sensitivity and quantification ability. To overcome these limitations, researchers have developed the TLFIA technique to achieve higher sensitivity detection by measuring the temperature changes caused by reporter molecules.

[0008] In TLFIA, the reporter molecules are deposited on the nitrocellulose membrane. When light shines on these reporter molecules, they absorb light energy and convert it into heat energy, resulting in a local temperature increase. This temperature change can be measured by temperature detection devices such as infrared cameras. Since the temperature increase is proportional to the number of captured reporter molecules, the concentration of target molecules in the sample can be quantitatively analyzed by measuring the temperature change.

[0009] The key advantage of TLFIA lies in its high sensitivity and quantification ability. By using latex beads (LBs) containing endothermic substances as reporter molecules, researchers have found that under 520-nanometer light irradiation, the light absorption of LBs can be enhanced by 1.3 - 3.2 times. This enhancement effect is attributed to the multiple scattering of light in this porous medium, and this mechanism has an important impact on the improvement of LFIA sensitivity.

[0010] Another important feature of TLFIA is its applicability to a variety of detection tools. Compared with traditional gold nanoparticle-based LFIA, CLB-based TLFIA provides a 10-fold increase in sensitivity. In addition, since CLBs allow different endothermic substances to be uniformly doped into the polystyrene matrix, they perform better than gold nanoparticles in multiplex detection.

[0011] Although TLFIA has many advantages, it also faces some challenges in practical applications. First, the high light power used in TLFIA may cause safety problems. Second, an infrared camera is a precision and expensive device, which may limit the application of TLFIA in environments with limited resources. In addition, TLFIA is most suitable for dry samples, and the sample drying process may slow down the detection speed, which goes against the original intention of rapid detection.

[0012] To overcome these challenges, research needs to focus on improving TLFIA technology to enhance its feasibility in practical applications. For example, by selecting a more suitable light source wavelength for excitation, the detection sensitivity can be further improved. In addition, the development of a more compact, portable and cost-effective TLFIA device will help apply this high-sensitivity detection technology to clinical and on-site detections.

[0013] In summary, TLFIA is a new detection technology with high sensitivity and quantitative ability. It achieves precise detection of target molecules in samples by measuring the temperature changes caused by reporter molecules. With the continuous progress and optimization of technology, TLFIA is expected to become an important tool for clinical and on-site detections, providing more accurate and sensitive detection means for disease prevention and control. Summary of the Invention

[0014] The purpose of the present invention is to provide a heat signal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug residues in beef and mutton and its usage method.

[0015] A heat signal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug residues in beef and mutton, comprising a box body, an enzyme-labeled plate arranged in the box body, and reagents arranged in the box body; each well of the enzyme-labeled plate is coated with promethazine monoclonal antibody A; the reagents include: promethazine anti-monoclonal antibody B conjugated with heat signal latex microspheres, promethazine standard solution, binding enhancement solution, washing solution, extraction agent.

[0016] The enzyme-labeled plate is preferably a milky white opaque polystyrene 96-well non-heat-source enzyme-labeled plate.

[0017] Each well of the enzyme-labeled plate is coated with promethazine monoclonal antibody A; it is a monoclonal antibody prepared by immunizing an animal with an artificial immunogen made by coupling the promethazine drug nucleus with bovine serum albumin; wherein the concentration of the coated antibody is preferably 0.1 - 10 μg / mL.

[0018] The promethazine standard solution is diluted from promethazine pure product. The diluent is 0.05 mmol / L PBS containing 10% methanol (the percentage is by volume) with pH = 7.4. The concentrations of promethazine standards are 0 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 25.0 ng / mL, 125.0 ng / mL and 625.0 ng / mL respectively.

[0019] The monoclonal antibody B of promethazine is a monoclonal antibody prepared by immunizing an animal with an artificial immunogen made from the promethazine anti-clonal antibody A and the promethazine conjugate. Its working concentration is preferably 1:16000.

[0020] The inclusions of the thermal signal latex microspheres are one or several of polyethylene glycol (PEG), polyacrylamide (PAM), polyacrylic acid (PAA), polysaccharides, polyvinylpyrrolidone (PVP), hyaluronic acid (HA), carboxymethyl starch, hydroxymethyl cellulose, carboxymethyl cellulose, hydrolyzed polyacrylamide (HPAM).

[0021] The binding enhancement solution is an aqueous solution containing 0.574 g of NaH2PO4·2H2O, 3.26 g of Na2HPO4·12H2O and 40 - 400 g of Ficoll 400 per liter.

[0022] The washing solution is a pH = 7.4, 0.1 mol / L phosphate buffer solution containing 0.05% (by volume) Tween - 20.

[0023] The extractant is an aqueous solution containing 0.5 - 5% ammonium chloride.

[0024] Preparation of the solution of the present invention:

[0025] The promethazine standard solution, the promethazine anti - clonal antibody B conjugated with thermal signal latex microspheres, the binding enhancement solution and the washing solution involved in the kit of the present invention and their formulations have a great influence on the detection sensitivity of the kit of the present invention. The main components of each solution and their preparation methods are as follows:

[0026] 1. Promethazine standard solution: By a conventional method, promethazine pure drug is formulated into promethazine standard solutions with concentrations of 0 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 25.0 ng / mL, 125.0 ng / mL and 625.0 ng / mL respectively using 0.05 mmol / L PBS containing 10% methanol (the percentage is by volume) with pH = 7.4.

[0027] 2. Promethazine anti-clonal antibody B conjugated with thermal signal latex microspheres: First, dissolve one or several of polyethylene glycol (PEG), polyacrylamide (PAM), polyacrylic acid (PAA), polysaccharides, polyvinylpyrrolidone (PVP), hyaluronic acid (HA), carboxymethyl starch, hydroxymethyl cellulose, carboxymethyl cellulose, hydrolyzed polyacrylamide (HPAM) in deionized water to prepare a solution with a concentration of 1%. At room temperature, drop this solution into an emulsifier containing acrylate monomers to form an emulsion. Under nitrogen protection, add an initiator to initiate the polymerization reaction to generate latex microspheres with a diameter of about 300 nm. Then, treat the microspheres with succinic anhydride to convert the surface hydroxyl groups into carboxyl groups. After the reaction, wash the microspheres with a large amount of deionized water to remove the unreacted succinic anhydride. Finally, suspend the microspheres in a buffer solution containing EDC and NHS to activate the carboxyl groups. Then add the diluted antibody solution and conduct a coupling reaction at room temperature. After reacting for a certain time, add glycine to terminate the reaction and remove the uncoupled antibody by centrifugal washing.

[0028] 3. Binding enhancement solution: Add 0.574 g of NaH2PO4·2H2O and 3.26 g of Na2HPO4·12H2O to a 1 L container, and then add 40 - 400 g of Ficoll 400, and make up the volume to 1 L with water.

[0029] 4. Washing solution: Add Tween-20 to a pH = 7.4, 0.1 mol / L phosphate buffer solution at a volume fraction of 0.05%.

[0030] Coating of the enzyme-labeled plate of the present invention:

[0031] In the present invention, the enzyme-labeled plate is coated by placing promethazine clonal antibody A in a set coating solution, reacting and coating at a set concentration in an incubator at 37°C.

[0032] The coating solution used in the present invention is a sodium carbonate-sodium bicarbonate buffer solution with a pH of 9.5. Promethazine clonal antibody A coated on the microplate in the present invention can bind well to the plastic surface of the microplate in an alkaline environment and can withstand multiple washings of the plate. The concentration of the coated antigen used is 0.1 - 10 μg / mL.

[0033] The coated microplate can be blocked with a blocking solution. Ovalbumin (OVA) is preferably used as the inert protein in the blocking solution, and NaN3 needs to be added to prevent deterioration.

[0034] Use of the kit:

[0035] 1. Take about 0.5 mg of muscle tissue, add 500 μL of extraction solution, and extract at 75°C for 30 min;

[0036] 2. Centrifuge, take 50 μL of the supernatant, add it to the ELISA plate, add 50 μL of promethazine anti-clone antibody B conjugated with heat signal latex microspheres, and then add 50 μL of binding enhancement solution, and incubate at 37 °C for 30 min;

[0037] 3. Pour out the solution and add 250 μL of washing solution;

[0038] 4. Pour out the washing solution and repeat step 3;

[0039] 5. Pour out the washing solution and use a thermal sensing device to detect the ELISA plate.

[0040] The beneficial effects of the present invention: The double antibody sandwich method of the present invention is used to detect promethazine, and it is found that more than 40% of ficoll 400 can promote the binding of promethazine monoclonal antibody B to the promethazine-antibody A complex, significantly improving the sensitivity; the antibody B of the present invention is conjugated with heat signal-sensitive latex microspheres and detected by a thermal sensor, which has the characteristics of high sensitivity, simplicity, rapidity and accuracy. Compared with the traditional colorimetric ELISA method, the sensitivity can be increased by two orders of magnitude. It is expected to play an important role in the detection of promethazine drug residues in beef and mutton foods. Brief Description of the Drawings

[0041] Figure 1 is the working principle diagram of the present invention.

[0042] Figure 2 is the working curve of the promethazine drug antibody of the present invention.

[0043] Figure 3 is the effect diagram of using ficoll 400 at different concentrations. Detailed Embodiments

[0044] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0045] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are all conventional means well known to those skilled in the art.

[0046] The working principle of the present invention is as Figure 1 shown.

[0047] Example 1: Preparation of immunogen, coating antigen and monoclonal antibody

[0048] (1) Synthesis of immunogen

[0049] Take 12 mg of promethazine and dissolve it in 1 mL of DMF. Take 15 mg of EDC and dissolve it thoroughly in 0.2 mL of water, then add it thereto. Stir at room temperature for 24 h to obtain reaction solution A.

[0050] Weigh 40 mg of BSA and dissolve it thoroughly in 3 mL of PBS (pH 7.2). Slowly add reaction solution A drop by drop to the protein solution and stir at room temperature for 24 h. Dialyze with 0.01 mol / L PBS at 4 °C for 3 d, changing the dialysis solution 3 times a day to remove unreacted small molecule substances. Aliquot and store at -20 °C for later use.

[0051] Weigh 30 mg of OVA and 12 mg of promethazine, react according to the above steps to synthesize SAs-OVA for coating.

[0052] (2) Preparation of promethazine monoclonal antibody

[0053] Animal immunization: Use the immunogen (SAs-BSA) prepared above at 100 μg / animal, dissolve the immunogen in physiological saline and mix it evenly with an equal volume of Freund's complete adjuvant, and inject subcutaneously into the neck and back of 6-8-week-old Balb / c female mice for immunization. On the 7th, 14th, and 28th days after the primary immunization, mix the immunogen with an equal volume of Freund's incomplete adjuvant and boost the immunization once each. Three days before fusion, boost the immunization once more with 100 μg / animal of the immune complex without Freund's adjuvant.

[0054] Cell fusion: Perform according to the conventional method. Mix the spleen cells of the immunized mice with mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, then slowly add the preheated fusogen (PEG4000) within 45 seconds for fusion. Suspend it evenly with HAT medium, add an appropriate amount of feeder cells, culture in a 96-well culture plate, and culture in an incubator at 37 °C and 5% CO2. Replace half of the medium with HT medium after 5 days and replace all the medium on the 9th day.

[0055] Screening of hybridoma cells: After cell fusion, when the cells grow to 1 / 2 of the culture well area, use the step-by-step screening method to screen hybridoma cells. The primary screening uses the indirect ELISA method. Coat the enzyme-labeled plate with the coated antigen (previously titrate its optimal coating concentration and positive serum dilution by the checkerboard method), add the culture supernatant of the tested wells, incubate, wash, and then add goat anti-mouse IgG-HRP and IgM-HRP, and use o-phenylenediamine (OPD) for color reaction. The positive wells screened out are further screened by the indirect competitive ELISA method. First, mix the cell supernatant with 100 μg / mL of promethazine in an equal volume, incubate at 37 °C in a water bath for 30 min, and then add it to the well-coated enzyme-labeled plate. At the same time, use PBS to replace promethazine as a control, and the other steps are the same as above. If the OD450nm value drops to less than 50% of the control well, it is judged as positive. The wells that are positive in 2-3 detections are immediately subcloned by the limiting dilution method.

[0056] Preparation of monoclonal antibody: The hybridoma cells after 2 - 3 subclonings and establishment of strains were cultured on a large scale. The supernatant was collected and its titer was determined by indirect ELISA, and then stored frozen. 8 - 10 - week - old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. 7 - 10 days later, the mice were intraperitoneally injected with 1 - 2×10 6 / mouse. 7 - 10 days later, the ascites of the mice were extracted, centrifuged, and the supernatant was taken to determine the titer and stored frozen for later use.

[0057] Example 2: Establishment of ELISA detection method

[0058] (1) Optimization of the concentrations of antibody A and antibody B (matrix)

[0059] Vertically, each well of the ELISA plate was coated with antibody A at a series of dilution concentrations of 160.0 μg / mL, 80.0 μg / mL, 40.0 μg / mL, 20.0 μg / mL, 10.0 μg / mL, 5.0 μg / mL, 0.5 μg / mL, 0.1 μg / mL, 100 μL / well. After being placed in an incubator at 37°C for 2 h, it was patted dry. It was blocked with 150 μL / well of blocking solution, placed in an incubator at 37°C for 2 h, washed once, and patted dry. 50 μL / well of promethazine antigen and 50 μL / well of a series of diluted monoclonal antibody B (1:1000 to 1:512000) were added. Incubated at room temperature (20 - 25°C) for 15 min, washed five times, and patted dry for the last time. The heat value was measured. The coating antibody A concentration and antibody B dilution with an obvious gradient change of the heat value with the concentration of the coating antibody A were used as the optimal concentrations for specificity determination.

[0060] (2) Determination of antibody sensitivity

[0061] According to the above optimization experiment of the concentrations of antibody A and antibody B, the applicant selected and determined that the concentration of antibody B was 1:64000 and the concentration of antibody A was 10 μg / mL for the determination of antibody sensitivity:

[0062] Coating: Antibody A was prepared into a 10 μg / mL solution with 0.05 M carbonate coating solution at pH = 9.6. 100 μL was added to each reaction well of the polystyrene plate and placed in an incubator at 37°C for 2 h. The solution in the well was discarded and patted dry.

[0063] Blocking: The above - coated ELISA plate was blocked with blocking solution, 150 μL / well, placed in an incubator at 37°C for 2 h, then washed once and patted dry.

[0064] Sample addition: Add 50 μL / well of promethazine solutions with different concentrations, and then add 50 μL / well of diluted promethazine monoclonal antibody B ((1:64000) and binding enhancement solution in a volume ratio of 1:1 to the above-mentioned blocked reaction wells. Incubate in the dark at room temperature (20 - 25 °C) for 15 min, then wash the plate five times and pat dry for the last time.

[0065] Measurement: Detect using a thermal sensing device.

[0066] Example 3: Chemiluminescent enzyme-linked immunosorbent assay kit for detecting promethazine thermal sensing signal

[0067] (1) Composition of the chemiluminescent enzyme-linked immunosorbent assay kit for detecting promethazine thermal sensing signal

[0068] Solid phase carrier (ELISA plate) coated with promethazine monoclonal antibody A.

[0069] Promethazine standard solutions: 0 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 25.0 ng / mL, 125.0 ng / mL, and 625.0 ng / mL.

[0070] Promethazine antibody B solution: Promethazine anti-clone antibody B conjugated with thermal signal latex microspheres, a monoclonal antibody prepared by immunizing animals with an artificial immunogen made from promethazine anti-clone antibody A and promethazine conjugate, and its working concentration is preferably 1:16000.

[0071] Binding enhancement solution: An aqueous solution containing 0.574 g of NaH2PO4·2H2O, 3.26 g of Na2HPO4·12H2O, and 40 - 400 g of Ficoll 400 per liter.

[0072] Washing solution: Phosphate buffer solution with pH = 7.4, 0.1 mol / L containing 0.05% (v / v) Tween-20.

[0073] Extraction agent: An aqueous solution containing 0.5 - 5% ammonium chloride.

[0074] (2) Preparation of ELISA plate

[0075] Dilute the coating antibody A to 10 μg / mL with the coating solution, add 100 μL to each well, place in an incubator at 37 °C for 2 h, pour out the coating solution, pat dry, then add 150 μL of blocking solution to each well, place in an incubator at 37 °C for 2 h, pour out the liquid in the wells, wash once with the washing solution, pat dry, and store in a vacuum-sealed manner with tin foil.

[0076] Example 4: Application of chemiluminescent enzyme-linked immunosorbent assay kit for detecting sulfonamides

[0077] (1) Sample pretreatment

[0078] Weigh about 0.5 mg of muscle tissue and transfer it to a 2 mL polystyrene centrifuge tube. Add 500 μL of 0.1 M sample extraction solution and immediately shake for 30 s.

[0079] Extract at 75 °C for 30 min.

[0080] Centrifuge at 3500 rpm for 1 min and take 50 μL of the supernatant for analysis.

[0081] (2) Detection steps

[0082] Sample addition: Add 50 μL / well of promethazine solutions with different concentrations, then add 50 μL of promethazine anti-clone antibody B conjugated with heat signal latex microspheres, and 50 μL of binding enhancement solution to the above-mentioned blocked reaction wells. Incubate at 37 °C in the dark for 15 min.

[0083] Washing: Pour out the liquid in the wells, add 250 μL of washing solution to each well, wash 5 times, and pat dry.

[0084] Detection: Measure the thermal value intensity of each well using a thermosensitive sensor device.

[0085] (3) Result judgment

[0086] Use the average value of the thermal values of the standard products and samples as the ordinate and the logarithm of the promethazine concentration as the abscissa to plot a standard curve ( Figure 2 ), and the concentration of each sample can be read from the standard curve.

[0087] Example 5: Kit specificity test

[0088] The drugs used for antibody cross-reactivity study are all drugs similar to promethazine in structure or function: Chlorpromazine: Similar to promethazine in structure, with one more chlorine atom and small changes in the side chain.

[0089] Cyproheptadine: Has strong antihistamine effect, and also has anti-5-hydroxytryptamine and anticholinergic effects.

[0090] Loratadine: A structural analogue of cyproheptadine.

[0091] Ketotifen: A structural analogue of cyproheptadine.

[0092] Diphenhydramine: Has antihistamine effect, but the action time is shorter.

[0093] Difenoxin: Another type of drug with sedative and hypnotic effects.

[0094] Tripelennamine: Belongs to phenothiazine antihistamines and is used to treat allergic reactions.

[0095] Metoclopramide: A drug with antiemetic effect.

[0096] Metformin: Although not belonging to the phenothiazine class, it is often used in combination with promethazine to treat mental illness.

[0097] Olanzapine: An antipsychotic drug with sedative effects.

[0098] Quetiapine: An antipsychotic drug used to treat schizophrenia.

[0099] Risperidone: An antipsychotic drug used to treat various mental disorders.

[0100] Aripiprazole: An antipsychotic drug used to treat schizophrenia.

[0101] Haloperidol: An antipsychotic drug with strong sedative effects.

[0102] Clozapine: An antipsychotic drug used to treat various mental disorders.

[0103] Sulpiride: An antipsychotic drug with sedative and antiemetic effects.

[0104] Ziprasidone: An antipsychotic drug used to treat schizophrenia.

[0105] Zolmitriptan: A drug used to treat migraine.

[0106] Sertraline: An antipsychotic drug used to treat depression.

[0107] Venlafaxine: An antidepressant drug that also has antihistamine effects.

[0108] The cross - reaction rate (%CR) is the percentage of the ratio of the detected concentration value of the antibody to the competitor to the actual concentration value of the competitor. The results are listed in Table 1:

[0109] Table 1 Specificity test of promethazine kit

[0110] Number Competitor Cross - reaction rate 1 Chlorpromazine 2.84％ 2 Cyproheptadine 1.01％ 3 Loratadine 4.96％ 4 Ketotifen 0.09％ 5 Diphenhydramine 0.70％ 6 Difenoxin 2.23％ 7 Tripelennamine 3.82％ 8 Metoclopramide 1.28％ 9 Metformin 0.54％ 10 Olanzapine 2.31％ 11 Quetiapine 1.79％ 12 Risperidone 4.19％ 13 Aripiprazole 2.32％ 14 Haloperidol 1.09％ 15 Clozapine 0.33％ 16 Sulpiride 3.79％ 17 Ziprasidone 2.92％ 18 Zolmitriptan 0.24％ 19 Sertraline 0.66％ 20 Venlafaxine 2.50％

[0111] Example 6: Accuracy test of the kit

[0112] Beef and mutton samples were spiked with different promethazine drugs respectively for the spike - recovery test. The recoveries of different drugs in different samples were calculated to determine the accuracy of the kit. One concentration was added to each sample, six samples were added for each concentration, and three batches of kits were selected for the test.

[0113] Quantitative calculation of the average recovery rate was carried out according to the linear equation of the established standard curve. The results are shown in Table 2 below.

[0114] Table 2 Accuracy test of promethazine kit

[0115]

[0116] Judging from the above measurement results, the recovery rates of beef and mutton samples are between 90.0% and 110%, indicating that this kit has good accuracy.

[0117] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A heat signal enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drugs, comprising a box body, an enzyme-labeled plate arranged in the box body, and reagents arranged in the box body, characterized in that, Each well of the enzyme-linked immunosorbent assay (ELISA) plate is coated with promethazine monoclonal antibody A; the reagents include: thermosignal latex microspheres conjugated with promethazine anti-clonal antibody B, promethazine standard solution, binding enhancement solution, washing solution, and extractant.

2. The thermosignal enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein The ELISA plate is an opalescent opaque polystyrene 96-well pyrogen-free ELISA plate.

3. The thermosignal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein Each well of the ELISA plate is coated with promethazine monoclonal antibody A; it is a monoclonal antibody obtained by immunizing an animal with an artificial immunogen prepared by coupling the promethazine drug nucleus with bovine serum albumin; the coating antibody concentration is 0.1 - 10 μg / mL.

4. The thermosignal enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein, The promethazine standard solution is diluted from promethazine pure product, and the diluent is PBS with 10% methanol by volume, 0.05 mmol / L, and pH = 7.

4. The concentrations of the promethazine standards are 0 ng / mL, 1.0 ng / mL, 5.0 ng / mL, 25.0 ng / mL, 125.0 ng / mL, and 625.0 ng / mL respectively, and the percentage is by volume.

5. The thermosignal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, characterized in that, The promethazine monoclonal antibody B is a monoclonal antibody obtained by immunizing an animal with an artificial immunogen prepared by combining promethazine anti-clonal antibody A with a promethazine conjugate, and its working concentration is 1:16000.

6. The thermosignal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein The content of the thermosignal latex microspheres is one or more of polyethylene glycol, polyacrylamide, polyacrylic acid, polysaccharides, polyvinylpyrrolidone, hyaluronic acid, carboxymethyl starch, hydroxymethyl cellulose, carboxymethyl cellulose, and hydrolyzed polyacrylamide.

7. The thermosignal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein, The binding enhancement solution is an aqueous solution containing 0.574 g of NaH2PO4·2H2O, 3.26 g of Na2HPO4·12H2O, and 40 - 400 g of Ficoll 400 per liter.

8. The thermo-signal enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein The washing solution is a 0.1 mol / L phosphate buffer with a pH of 7.4 containing 0.05% Tween-20 by volume fraction.

9. The thermosignal-enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, wherein, The extractant is an aqueous solution containing 0.5 - 5% ammonium chloride.

10. The method of using the thermal signal enhanced enzyme-linked immunosorbent assay kit for detecting promethazine drug according to claim 1, characterized in that, Proceed as follows: (1) Take 0.3 - 0.7 mg of muscle tissue, add 500 μL of extractant, and extract at 75°C for 30 min. (2) Centrifuge, take 50 μL of the supernatant, add it to the ELISA plate, add 50 μL of thermosignal latex microspheres conjugated with promethazine anti-clonal antibody B, and then add 50 μL of binding enhancement solution, and incubate at 37°C for 30 min. (3) Pour out the solution and add 250 μL of washing solution. (4) Pour out the washing solution and repeat step 3. (5) Pour out the washing solution and use a thermal sensing device to detect the ELISA plate.

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