Tolazoline antigen, synthetic method and application in preparation of immunological detection kit

Abstract

The present application relates to a kind of toluene thiazide antigens, its general structure is as follows: and the preparation method of toluene thiazide antigen, carboxyl is derived from succinic anhydride by toluene thiazide hapten, and is coupled to obtain by carboxyl and bovine serum albumin or bovine gamma globulin.The toluene thiazide antigen of the present application is synthesized in preparation process, and the material and catalyst used are ordinary products, so it is more practical, suitable for industrial production and can produce economic value;And, no chloroform is used, and toxicity is lower, more security.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology; specifically, it relates to the synthesis of toluenethiazide antigen using chemical synthesis methods, a colloidal gold immunoassay rapid test kit based on antigen-antibody reaction for rapid screening of toluenethiazide substances, its preparation method, and the use of the test kit in the rapid detection of toluenethiazide drugs. Background Technology

[0002] The following background information is provided to help readers understand the present invention and should not be considered as prior art.

[0003] Xylazine (also known as thiarazine) is a veterinary drug with analgesic and muscle relaxant properties. It is suitable for various animal species, such as cattle, sheep, horses, cats, dogs, deer, mice, and elk, for sedation and to facilitate treatment, diagnosis, and surgery, as well as for pain relief or as a local anesthetic.

[0004] Reportedly, human use of xylazine began in Puerto Rico and has spread to the continental United States, occurring orally, by inhalation, or by injection. Symptoms of low-dose ingestion in humans include sedation; high doses may cause vomiting, various cardiovascular effects, miosis, central and peripheral nervous system depression, transient hypertension followed by severe hypotension, and respiratory depression. Long-term use can cause skin ulcers, slow wound healing, slurred speech, drowsiness, hypertension, and even coma, syncope, and inability to regulate body temperature. In humans, thiazides and heroin have similar pharmacological effects and may produce synergistic toxicity. Therefore, the use of toluenethiazide as an adulterant may increase the number of deaths among drug users. Xylazine alone has been proven harmful to humans, and the harm is even greater when used in combination with other abused drugs.

[0005] Currently, the conventional detection methods for toluenethiazide are mainly instrumental methods such as liquid chromatography-tandem mass spectrometry.

[0006] The high-performance liquid chromatography (HPLC) detection procedure for toluenethiazide is as follows: (Extraction) The sample is homogenized and extracted with acetonitrile-2% acetic acid; (Purification) C18 adsorbent / N-propylethylenediamine (PSA) purification treatment; (Volume Adjustment) The extract is subjected to nitrogen blowing and volume adjustment; (Separation) Separation is performed using a Waters C18 column (100 mm × 2.1 mm, 3.5 μm) with 0.1% formic acid-water solution and 0.1% formic acid-acetonitrile solution as mobile phases; (Detection and Quantification) Detection is performed in positive ion mode using multiple reaction monitoring (MRM), and quantification is performed using a matrix-matched external standard calibration curve. Due to the complexity of biological sample matrices, the sample pretreatment process is extremely critical and complex, and time-consuming (generally not less than 30 min). Furthermore, the detection procedure cannot be widely applied due to equipment cost, further limiting the detection process. Compared with widely used rapid diagnostic technologies, HPLC detection has a significant competitive disadvantage due to its cumbersome detection process.

[0007] For the high-performance liquid chromatography (HPLC) method for the detection of toluenethiazide, the core component, the chromatographic column, has a porous structure. To prevent column clogging and bubble generation, the detection process requires extremely high sample purity. The specific steps involve extraction with acetonitrile, centrifugation, concentration of the supernatant to near dryness using a rotary evaporator, dissolution of the residue with the mobile phase, and defatting with n-hexane before detection. Different extraction solvents, adsorbent types, and amounts significantly affect the flowability and recovery rate of the target analyte; otherwise, the accuracy of the detection results will be affected to varying degrees. Sample pretreatment generally takes no less than 30 minutes. In contrast, rapid diagnostic techniques generally have lower sample pretreatment requirements; most samples only require simple crushing, dissolution, and centrifugation for detection, with the entire sample sampling and processing process taking no more than 10 minutes.

[0008] In summary, the main reason for the cumbersome process of detecting benzthiazide using high-performance liquid chromatography is the strict requirements and complicated sample pretreatment procedures.

[0009] For example, the method for determining residual sedative veterinary drugs in mutton (CN201410197253.2) has high requirements for test samples, complex sample processing, a cumbersome test process, and high test costs. It is not suitable for rapid detection of human body fluids or tissue samples containing a lot of impurities.

[0010] Therefore, there is a need to develop a sensitive, accurate, and efficient portable detection tool. Summary of the Invention

[0011] To address the problems existing in the current method of detecting toluenethiazide using high performance liquid chromatography, the purpose of this invention is to provide a toluenethiazide antigen that can be used to prepare toluenethiazide test strips, enabling targeted and rapid detection of toluenethiazide.

[0012] A toluthiazide antigen is obtained by deriving a carboxyl group from a toluthiazide hapten via succinic anhydride, and then conjugating the carboxyl group to bovine serum albumin or bovine gamma globulin.

[0013] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0014] A toluenethiazide artificial antigen, characterized by the following general structural formula:

[0015] .

[0016] Among them, the macromolecular proteins are bovine serum albumin (BSA) and bovine gamma globulin (BGG).

[0017] Right now:

[0018] or .

[0019] The structural formula of the toluenethiazide hapten is shown below:

[0020]

[0021] The reaction formula is as follows:

[0022] Reaction Formula 1:

[0023]

[0024] Reaction formula 2:

[0025]

[0026] The present invention also provides a method for preparing the aforementioned toluenethiazide antigen artificial antigen, comprising the following steps:

[0027] (1) At room temperature, toluenethiazide was dissolved in 5 ml of pyridine and succinic anhydride was added under vigorous stirring. The reaction was carried out overnight at room temperature. Post-treatment: pyridine was evaporated at 45 °C using a rotary evaporator. Then, 5 ml of dichloromethane and 3 ml of water were added to extract the succinic anhydride from the organic phase. The organic phase was then extracted once with 3 ml of saturated sodium chloride and dried with anhydrous sodium sulfate. The organic phases were combined and concentrated to obtain the crude product. After column purification, the product N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was obtained. The molar ratio of toluenethiazide to succinic anhydride was 1:1.2~1:2.

[0028] (2) N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was dissolved in 2 ml of N,N-dimethylformamide, and then N-hydroxysuccinimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide were added. The mixture was reacted on a turntable at 15 rpm for 24 hours. The molar ratio of H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid:N-hydroxysuccinimide:N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide is 1:1.2:1.2~1:2:2; thus, activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid is obtained.

[0029] (3) Prepare a 10 mg / ml BSA-PBS solution or BGG-PBS solution, and then slowly add the activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid solution to the BSA-PBS solution or BGG-PBS solution under ice-water bath conditions, and then place it on a turntable with a rotation speed of 15 rpm for 24 hours; wherein, the molar ratio of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid to BSA or BGG is 100:1~230:1;

[0030] (4) After coupling, dialyze with 1×PBS, changing the PBS every 2 hours. After changing the PBS 7-9 times, collect the sample;

[0031] (5) The concentration of the product toluene-thiazide antigen was determined by the Lowry method to be 8.5 mg / ml.

[0032] Another object of the present invention is to provide the application of toluenethiazide antigen in the preparation of immunoassay kits.

[0033] Another objective of this invention is to provide a toluenethiazide colloidal gold test strip prepared using the aforementioned toluenethiazide antigen, which can qualitatively and rapidly detect toluenethiazide in patients with high sensitivity and a detection limit of 1000 ng / mL. The toluenethiazide antigen can serve as a marker for the T-line of the test strip, and the toluenethiazide immunogen can also be used to generate toluenethiazide antibodies, thereby providing a source for the preparation of the colloidal gold complex in the test strip.

[0034] This invention also provides a colloidal gold rapid test strip for toluenethiazide, characterized in that it includes a sheet support plate, on which a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad are sequentially disposed. The gold-labeled pad is coated with a toluenethiazide monoclonal antibody-colloidal gold complex, and the nitrocellulose membrane has a detection line coated with toluenethiazide antigen.

[0035] This invention also provides a method for preparing a toluenethiazide colloidal gold test strip, comprising the following steps:

[0036] (1) T line and C line; Goat anti-mouse IgG and toluenethiazide immunogen were diluted with phosphate buffer to 1.0 mg / ml and sprayed onto nitrocellulose membrane at a spray volume of 1.0 μl / cm to serve as the control line C line and the detection line T line;

[0037] (2) Toluenethiazide monoclonal antibody: The toluenethiazide immunogen was diluted with phosphate buffer to 1 mg / mL and mixed with the immunoadjuvant at a volume ratio of 1:1. Then, 0.5 mg was injected subcutaneously and into the hind thigh muscle of rabbits. The amount used at each site was 1 / 5 of the immunogen. The immunization was repeated every 2 weeks. The serum was separated and the antibody titer in the serum was detected by indirect ELISA. When the antibody titer reached <1:128, the serum was collected and purified to produce polyclonal antibody. Then, the spleen B cells of immunized mice were fused with tumor cells and screened by monoclonal cell technology. Ascites fluid was collected for the separation and purification of monoclonal antibody to obtain toluenethiazide monoclonal antibody.

[0038] (3) Toluenethiazide monoclonal antibody-colloidal gold complex; The pH of the colloidal gold solution was adjusted to 8.2 with 0.1 mol / L K2CO3, and the optimal amount of toluenethiazide monoclonal antibody solution from step (2) was added (10 wt% of the optimal amount of toluenethiazide monoclonal antibody to stabilize the colloidal gold was added). After stirring, 1% PEG2000 buffer was added, and the mixture was centrifuged at 100000g for 30 min. The precipitate was removed and resuspended with PEG2000 buffer. The process was repeated 3 times to obtain the colloidal gold labeled protein. The colloidal gold labeled protein was purified by gel filtration and eluted with phosphate buffer containing bovine serum albumin and sodium azide. The toluenethiazide monoclonal antibody-colloidal gold complex was collected.

[0039] (4) Gold-labeled pad; The toluene-thiazide monoclonal antibody-colloidal gold complex from step (3) was diluted in phosphate buffer containing bovine serum albumin to 1 / 6 of the initial OD value of the complex, and 50 mg / ml trehalose and 200 mg / ml sucrose were added. After mixing evenly, the mixture was sprayed evenly onto the conjugate pad at a spray rate of 1.5 μl / cm to obtain the gold-labeled pad.

[0040] (5) Assembly: After assembling the sheet support plate with coated nitrocellulose membrane, absorbent pad, gold label pad and sample pad into a large card, cut it into test strips to obtain toluenethiazide colloidal gold test strips.

[0041] Beneficial effects

[0042] The present invention has the following beneficial effects:

[0043] This invention provides a rapid method for detecting toluenethiazide residues in human urine samples. Utilizing colloidal gold immunochromatography, it allows for rapid, accurate, convenient, and efficient collection and testing of human saliva samples, and analysis of toluenethiazide residue levels in the body within a short time. Compared to conventional instrumental detection methods, this method is less expensive, simpler to operate, suitable for large-scale sample screening in practical testing scenarios, and provides accurate and reliable results.

[0044] The materials and catalysts used in the synthesis and preparation process of the toluenethiazide antigen of the present invention are all common products, thus making it more practical, suitable for industrial production and capable of generating economic value; furthermore, since chloroform is not used, it has lower toxicity and is safer.

[0045] The toluenethiazide antigen of this invention has good potency, high accuracy, and good sensitivity. When applied to test strips, it provides accurate and convenient readings. Attached Figure Description

[0046] Figure 1 This is the general structural formula for the toluenethiazide hapten.

[0047] Figure 2 This is the liquid phase spectrum of the toluenethiazide hapten.

[0048] Figure 3 This is the general structural formula for the toluenethiazide antigen.

[0049] Figure 4 This is a schematic diagram of the colloidal gold detection card.

[0050] Figure 5 This is a diagram of a color chart.

[0051] Figure 6 The graph shows the detection results of toluenethiazide antigen for small molecules.

[0052] Figure 7 The graph shows the accelerated stability results of the antigen.

[0053] Figure label:

[0054] Sample pad 201, gold label pad 202, nitrocellulose membrane 203, absorbent pad 204, sheet support plate 205, test strip 200, test line (T line) 206, control line (C line) 207 Detailed Implementation

[0055] Example 1: Preparation of Toluenethiazide Antigen

[0056] (1) At room temperature, 100 mg of toluenethiazide was dissolved in 5 ml of pyridine. Under vigorous stirring, 55 mg of succinic anhydride was added and the reaction was carried out overnight at room temperature. Post-treatment: Pyridine was evaporated at 45 °C using a rotary evaporator. Then, 5 ml of dichloromethane and 3 ml of water were added to extract the succinic anhydride from the organic phase. The organic phase was then extracted once with 3 ml of saturated sodium chloride and dried with anhydrous sodium sulfate. The organic phases were combined and concentrated to obtain 150 mg of crude product. After column purification, 120 mg of product N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was obtained.

[0057] (2) 120 mg of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylphenyl)-succinic acid was dissolved in 2 ml of N,N-dimethylformamide, and then 53.4 mg of N-hydroxysuccinimide and 88.5 mg of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide were added. The mixture was reacted on a turntable at 15 rpm for 24 hours. Activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylphenyl)-succinic acid was obtained.

[0058] (3) Prepare 24 ml of 10 mg / ml BSA-PBS solution, and then slowly add the activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid solution to the BSA-PBS solution under ice-water bath conditions, and then place it on a turntable with a rotation speed of 15 rpm for 24 hours; the molar ratio of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid to BSA is 100:1~230:1.

[0059] (4) After the coupling is completed, dialyze with 1×PBS, changing the PBS every 2 hours. After changing the PBS 7-9 times, collect the sample.

[0060] (5) The product concentration was determined by the Lowry method: 8.5 mg / ml, total 26 ml.

[0061] The liquid chromatogram of the toluenethiazide hapten is shown below. Figure 2 (Ultraviolet detector, wavelength 254nm).

[0062] Example 2: Preparation of Toluenethiazide Antigen

[0063] (1) At room temperature, 100 mg of toluenethiazide was dissolved in 5 ml of pyridine. Under vigorous stirring, 68 mg of succinic anhydride was added and the reaction was carried out overnight at room temperature. Post-treatment: Pyridine was evaporated at 45 °C using a rotary evaporator. Then, 5 ml of dichloromethane and 3 ml of water were added to extract the succinic anhydride from the organic phase. The organic phase was then extracted once with 3 ml of saturated sodium chloride and dried with anhydrous sodium sulfate. The organic phases were combined and concentrated to obtain 150 mg of crude product. After column purification, 60 mg of product N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was obtained.

[0064] (2) Dissolve 60 mg of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylphenyl)-succinic acid in 1 ml of N,N-dimethylformamide, then add 26 mg of N-hydroxysuccinimide and 44 mg of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, and react on a turntable at 15 rpm for 24 hours. Activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylphenyl)-succinic acid is obtained.

[0065] (3) Prepare 12 ml of 10 mg / ml BSA-PBS solution, and then slowly add the activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid solution to BGG-PBS solution under ice-water bath conditions, and then place it on a turntable with a rotation speed of 15 rpm for 24 hours; the molar ratio of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid to BSA is 100:1~230:1.

[0066] (4) After the coupling is completed, dialyze with 1×PBS, changing the PBS every 2 hours. After changing the PBS 7-9 times, collect the sample.

[0067] (5) The product concentration was determined by the Lowry method: 8.5 mg / ml, total 15 ml.

[0068] Example 3: Preparation of Toluenethiazide Antigen

[0069] (1) At room temperature, 100 mg of toluenethiazide was dissolved in 5 ml of pyridine. Under vigorous stirring, 90 mg of succinic anhydride was added and the reaction was carried out overnight at room temperature (the molar ratio of thiazide to succinic anhydride was 1:2). Post-treatment: Pyridine was evaporated at 45 °C using a rotary evaporator. Then, 5 ml of dichloromethane and 3 ml of water were added to extract the succinic anhydride from the organic phase. The organic phase was then extracted once with 3 ml of saturated sodium chloride and dried with anhydrous sodium sulfate. The organic phases were combined and concentrated to obtain 150 mg of crude product. After column purification, 60 mg of product N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was obtained.

[0070] (2) Dissolve 60 mg of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylphenyl)-succinic acid in 1 ml of N,N-dimethylformamide, then add 26 mg of N-hydroxysuccinimide and 44 mg of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, and react on a turntable at 15 rpm for 24 hours. Activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylphenyl)-succinic acid is obtained.

[0071] (3) Prepare 12 ml of 10 mg / ml BGG-PBS solution, and then slowly add the activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid solution to the BGG-PBS solution under ice-water bath conditions, and then place it on a turntable with a rotation speed of 15 rpm for 24 hours.

[0072] (4) After the coupling is completed, dialyze with 1×PBS, changing the PBS every 2 hours. After changing the PBS 7-9 times, collect the sample.

[0073] (5) The product concentration was determined by the Lowry method: 8.5 mg / ml, total 15 ml.

[0074] Experimental Example 4: Performance Determination of Toluenethiazide Artificial Antigen

[0075] Generally, healthy female Balb / c mice aged 6-8 weeks were selected and immunized according to the following animal immunization steps. The toluenethiazide-BSA conjugate synthesized by Hangzhou Anxu Biotechnology Co., Ltd. in Example 1 was used for the animal immunization experiment. The immunization method is the immunization procedure disclosed in the prior art, and will not be specifically described in this invention. A total of 4 mice were immunized, and the mice were numbered A0, A1, A2, and A3 in sequence. Their ocular serum titers were measured (the specific detection method is also the procedure disclosed in the prior art).

[0076] Antibody serum titer data are shown in Table 1:

[0077] Table 1

[0078]

[0079] Conclusion: After three immunizations, the ocular blood titers of the four mice were all >312500. Using 100 ng / ml toluenethiazide for competitive detection, the serum of the four mice achieved an inhibitory effect of more than 50% at a dilution of 1:62500.

[0080] Example 5: Application of toluenethiazide antigen in colloidal gold test strips

[0081] (1) T line and C line; Goat anti-mouse IgG and toluenethiazide immunogen were diluted with phosphate buffer to 1.0 mg / ml and sprayed onto nitrocellulose membrane at a spray volume of 1.0 μl / cm to serve as control line C line 207 and detection line T line 206.

[0082] (2) Toluenethiazide monoclonal antibody: The toluenethiazide immunogen was diluted with phosphate buffer to 1 mg / mL and mixed with the immunoadjuvant at a volume ratio of 1:1. Then, 0.5 mg was injected subcutaneously and into the hind thigh muscle of rabbits. The amount used at each site was 1 / 5 of the immunogen. The immunization was repeated every 2 weeks. The serum was separated and the antibody titer in the serum was detected by indirect ELISA. When the antibody titer reached <1:128, the serum was collected and purified to produce polyclonal antibody. Then, the spleen B cells of immunized mice were fused with tumor cells and screened by monoclonal cell technology. Ascites fluid was collected for the separation and purification of monoclonal antibody to obtain toluenethiazide monoclonal antibody.

[0083] (3) Toluenethiazide monoclonal antibody-colloidal gold complex; The pH of the colloidal gold solution was adjusted to 8.2 with 0.1 mol / L K2CO3, and the optimal amount of toluenethiazide monoclonal antibody solution from step (2) was added (10 wt% of the optimal amount of toluenethiazide monoclonal antibody to stabilize the colloidal gold was added). After stirring, 1% PEG2000 buffer was added, and the mixture was centrifuged at 100000g for 30 min. The precipitate was removed and resuspended with PEG2000 buffer. The process was repeated 3 times to obtain the colloidal gold labeled protein. The colloidal gold labeled protein was purified by gel filtration and eluted with phosphate buffer containing bovine serum albumin and sodium azide. The toluenethiazide monoclonal antibody-colloidal gold complex was collected.

[0084] (4) Gold-labeled pad; The toluene-thiazide monoclonal antibody-colloidal gold complex from step (3) was diluted in phosphate buffer containing bovine serum albumin to 1 / 6 of the initial OD value of the complex, and 50 mg / ml trehalose and 200 mg / ml sucrose were added. After mixing evenly, the mixture was sprayed evenly onto the conjugate pad at a spray rate of 1.5 μl / cm to obtain the gold-labeled pad.

[0085] (5) Assembly: After assembling the sheet support plate 205 with the coated nitrocellulose membrane 203, the absorbent pad 204, the gold label pad 202, and the sample pad 201 into a large card, cut it into test strips to obtain the toluenethiazide colloidal gold test strip 200. See Figure 4 .

[0086] Example 6: Validation of the sample detection performance of the toluenethiazide colloidal gold test strip.

[0087] Different concentrations of toluenethiazide solutions were prepared and tested using the test paper prepared in Example 5. The color intensity of the T line in the judgment segment was then compared using a colorimetric card. Each concentration was measured three times, and the average value was taken. The results are shown in Table 2 below.

[0088] Table 2. Sample detection results of the toluenethiazide colloidal gold assay kit

[0089]

[0090] The results show that the kit of the present invention has a detection sensitivity up to 1000 ng / ml and high accuracy in the concentration range of 1500-3000 ng / ml. This indicates that the actual detection sensitivity performance of the kit meets expectations.

[0091] The results of this colloidal gold test strip are interpreted using a colorimetric card method. G1-G10 represent the color development intensity of the T line, where G1 indicates no color development of the T line, representing a strong positive result; G10 indicates a dark T line, representing a strong negative result. As can be seen from the table, the test strip prepared in this invention can detect concentrations as low as 1000 ng / mL, exhibiting high sensitivity, especially in the concentration range of 1000–1500 ng / mL. Furthermore, the color intensity of the T line is negatively correlated with the concentration of toluenethiazide; the higher the concentration, the lighter the color.

[0092] Note: Standard color chart: The color range of the standard color chart is between 0 and 10, i.e., G1-G10. Colors increase in depth from none to all. See [link / reference]. Figure 5 As shown.

[0093] Table 3. Detection sensitivity results of three antigens against toluenethiazide small molecules (test graphs shown) Figure 6 )

[0094]

[0095] Table 4: Accelerated stability of antigens (after baking the assembled test cards at 55°C for 72 hours) results (see Table 4) Figure 7 )

[0096]

[0097] As shown in Tables 2, 3, and 4, the product meets the QC standard, has good thermal stability, and passes the antigen test.

[0098] Example 7: Application of Toluenethiazide Colloidal Gold Test Strip in Urine Detection

[0099] The toluenethiazide colloidal gold test strip prepared in Example 5 above was used to repeatedly test 89 clinical urine samples.

[0100] (Including 30 positive samples, with concentration distributions of 1000-1250 ng / mL (4 samples), 1250-1500 ng / mL (10 samples), and 1500-3000 ng / mL (16 samples), the detection results are shown in Table 5 below.

[0101] Table 5. Detection results of samples using the toluenethiazide colloidal gold assay kit.

[0102]

[0103] The results show that the kit of the present invention achieved an accuracy of over 90% in testing 89 urine samples. This indicates that the actual detection accuracy performance of the kit meets expectations.

Claims

1. A toluenethiazide antigen, characterized in that, Its general structural formula is: Among them, the macromolecular proteins are bovine serum albumin (BSA) and bovine gamma globulin (BGG).

2. A method for preparing the toluenethiazide antigen as described in claim 1, characterized in that, Among them, the macromolecular proteins are bovine serum albumin (BSA) and bovine gamma globulin (BGG).

3. The preparation method according to claim 2, characterized in that, The specific preparation steps are as follows: (1) At room temperature, toluenethiazide was dissolved in 5 ml of pyridine and succinic anhydride was added under vigorous stirring. The reaction was carried out overnight at room temperature. Post-treatment: pyridine was evaporated at 45 °C using a rotary evaporator. Then, 5 ml of dichloromethane and 3 ml of water were added to extract the succinic anhydride from the organic phase. The organic phase was then extracted once with 3 ml of saturated sodium chloride and dried with anhydrous sodium sulfate. The organic phases were combined and concentrated to obtain the crude product. After column purification, the product N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was obtained. The molar ratio of toluenethiazide to succinic anhydride was 1:1.2~1:

2. (2) N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid was dissolved in 2 ml of N,N-dimethylformamide, and then N-hydroxysuccinimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide were added. The mixture was reacted on a turntable at 15 rpm for 24 hours. The molar ratio of H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid:N-hydroxysuccinimide:N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide is 1:1.2:1.2~1:2:2; thus, activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid is obtained. (3) Prepare a 10 mg / ml BSA-PBS solution or BGG-PBS solution, and then slowly add the activated N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid solution to the BSA-PBS solution or BGG-PBS solution under ice-water bath conditions, and then place it on a turntable with a rotation speed of 15 rpm for 24 hours; wherein, the molar ratio of N-(5,6-dihydro-4H-[1,3]thiazin-2-yl)-N-(2,6-dimethylbenzene)-succinic acid to BSA or BGG is 100:1~230:1; (4) After coupling, dialyze with 1×PBS, changing the PBS every 2 hours. After changing the PBS 7-9 times, collect the sample; (5) The concentration of the product toluene-thiazide antigen was determined by the Lowry method to be 8.5 mg / ml.

4. The use of the toluenethiazide antigen as described in claim 1 in the preparation of an immunoassay kit.

5. A colloidal gold rapid test strip for toluenethiazide as described in claim 1, characterized in that, The device includes a sheet support plate, on which a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad are sequentially arranged. The gold label pad is coated with a toluene-thiazide monoclonal antibody-colloidal gold complex, and the nitrocellulose membrane has a detection line coated with toluene-thiazide antigen.

6. A method for preparing the toluenethiazide colloidal gold test strip as described in claim 5, comprising the following steps: (1) T line and C line; Goat anti-mouse IgG and toluenethiazide immunogen were diluted with phosphate buffer to 1.0 mg / ml and sprayed onto nitrocellulose membrane at a spray volume of 1.0 μl / cm to serve as the control line C line and the detection line T line; (2) Toluenethiazide monoclonal antibody: The toluenethiazide immunogen was diluted with phosphate buffer to 1 mg / mL and mixed with the immunoadjuvant at a volume ratio of 1:

1. Then, 0.5 mg was injected subcutaneously and into the hind thigh muscle of rabbits. The amount used at each site was 1 / 5 of the immunogen. The immunization was repeated every 2 weeks. The serum was separated and the antibody titer in the serum was detected by indirect ELISA. When the antibody titer reached <1:128, the serum was collected and purified to produce polyclonal antibody. Then, the spleen B cells of immunized mice were fused with tumor cells and screened by monoclonal cell technology. Ascites fluid was collected for the separation and purification of monoclonal antibody to obtain toluenethiazide monoclonal antibody. (3) Toluenethiazide monoclonal antibody-colloidal gold complex; The pH of the colloidal gold solution was adjusted to 8.2 with 0.1 mol / L K2CO3, and the optimal amount of toluenethiazide monoclonal antibody solution from step (2) was added (10 wt% of the optimal amount of toluenethiazide monoclonal antibody to stabilize the colloidal gold was added). After stirring, 1% PEG2000 buffer was added, and the mixture was centrifuged at 100000g for 30 min. The precipitate was removed and resuspended with PEG2000 buffer. The process was repeated 3 times to obtain the colloidal gold labeled protein. The colloidal gold labeled protein was purified by gel filtration and eluted with phosphate buffer containing bovine serum albumin and sodium azide. The toluenethiazide monoclonal antibody-colloidal gold complex was collected. (4) Gold-labeled pad; The toluene-thiazide monoclonal antibody-colloidal gold complex from step (3) was diluted in phosphate buffer containing bovine serum albumin to 1 / 6 of the initial OD value of the complex, and 50 mg / ml trehalose and 200 mg / ml sucrose were added. After mixing evenly, the mixture was sprayed evenly onto the conjugate pad at a spray rate of 1.5 μl / cm to obtain the gold-labeled pad. (5) Assembly: After assembling the sheet support plate with coated nitrocellulose membrane, absorbent pad, gold label pad and sample pad into a large card, cut it into test strips to obtain toluenethiazide colloidal gold test strips.

Images