TH24249Y - Sibutramine detection test kit and manufacturing process. - Google Patents

Abstract

------January 21, 2022------(OCR) The sibutramine test kit consists of a test strip in a plastic cassette, a ready-to-use extract containing ethanol in water, and a ready-to-use buffer solution containing potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), sodium chloride, disodium hydrogen phosphate (Na2HPO4), and Tween-20 in water. The manufacturing process involves the following steps: preparing sibutramine conjugate with bovine serum albumin and spraying it onto a nitrocellulose membrane; preparing the antibody conjugate by diluting the monoclonal antibody; and preparing colloidal gold for conjugation with the monoclonal antibody. The mixed colloidal gold conjugate and monoclonal antibody are dropped onto a glass fiber membrane. The prepared nitrocellulose membrane and glass fiber membrane are assembled with the support and absorbent pad, then cut to the appropriate size for placement in a plastic cassette. The test kit can be used to detect sibutramine in pharmaceutical products and dietary supplements using immunochromatography. The test kit is more sensitive than thin-layer chromatography (DL-L) test kits currently used in Thailand. The immunochromatography test kit also solves the problem of difficulty in testing, allowing the general public to perform self-testing. It is convenient to use, beneficial to society, and helps ensure public safety in the use of pharmaceutical products and dietary supplements. ------------ Page 1 of 1 page. Abstract of the Invention. This invention concerns a sibutramine test kit. The test kit can be used to detect sibutramine in pharmaceutical products and dietary supplements using immunochromatography. The test kit is more sensitive than thin-layer chromatography (DL-L) test kits currently used in Thailand. The immunochromatography test kit also solves... The problem of the difficulty in testing, which the general public can perform themselves, is addressed through a user-friendly system.

Claims (2)

------21/01/2565------(OCR) 1. Sibutramine Test Kit consists of: 1.1 A plastic cassette for holding the test strip. This consists of a bottom plastic piece for placing the test strip and an top plastic piece with various openings. The outermost opening is an oval-shaped well for dropping the sample extracted with a ready-made reagent containing 50% (volume/volume) ethanol in water, contained in a glass bottle. Next is a rectangular opening, a window displaying the test results, labeled "C" and "T". Inside are four test paper sections. Section 1 is a nitrocellulose membrane measuring 4 mm x 25 mm, covering the center of a 140 µm porous polystyrene sheet. This section acts as the display area, receiving the sibutramine conjugate with bovine serum albumin at position "T" and the goat Iggg antibody specific to mouse at position "C". These positions are indicated on the test cassette. Part 2 is a glass fiber paper measuring 4 mm x 8 mm, overlapping 2-3 mm of the nitrocellulose membrane. This layer serves as the sheet coated with the antibody conjugate. Part 3 is a glass fiber paper measuring 4 mm x 20 mm, overlapping 2-4 mm of Part 2. The bottom edge of the test sheet is attached to act as the absorbent and solution-holding area. Part 4 is a nitrocellulose membrane paper measuring 4 mm x 20 mm (width x length). It is placed over the top end of the test plate, overlapping the nitrocellulose membrane by 2-3 mm. It serves to absorb all the sample and solution. 1.2 Ready-made buffer solution consists of 0.1 g (g) potassium chloride (KCl), 0.12 g (g) potassium dihydrogen phosphate (KH2PO4), 4 g (g) sodium chloride (NaCl), 1.81 g (g) disodium hydrogen phosphate (Na2HPO4), and 250 µl (m³) Tween-20. The volume is adjusted with water to a total volume of 500 ml (m³). The ratios of the components can be adjusted according to the volume of the ready-made buffer solution to be prepared. 2. Sibutramine test kit manufacturing process. According to claim 1, the following procedure is followed: 2.1 Prepare sibutramine conjugate with bovine serum albumin at a concentration of 0.05 mg/ml and mouse-specific goat IGG antibody at a concentration of 0.5 mg/ml. Spray onto a 0.9 µL/cm² nitrocellulose membrane, dry for 24 hours at 37°C, and store at room temperature in a sealed aluminum foil bag containing a desiccant. 2.2 The antibody conjugate can be prepared by diluting the monoclonal antibody at 0.2 mg/ml. The monoclonal antibody was diluted with HEPES solution. A colloidal gold solution was then prepared for conjugating the monoclonal antibody by adding 80 μL of 0.1 M potassium carbonate (K2CO3) to 10 ml of colloidal gold solution. 300 μL of the diluted monoclonal antibody was then added and left to stand at room temperature for 50 minutes. 500 μL of 10% w/v bovine serum albumin was added, and the mixture was centrifuged for 45 minutes at 8,000 rpm at 4°C. The supernatant was then discarded, and a buffer suspension was added. (Resuspension buffer) containing 0.02 molar (M) tris hydrochloride (Tris-HCl), 0.1% w/w Tween-20, 0.1% w/w PEG, 1% w/w PVP, 5% w/w sucrose, 4% w/w trehalose, 2% w/w sorbitol, 1% w/w mannitol, 0.04% w/w sodium azide (NaN3), and 0.2% w/w BSA. (BSA) Then, mix 300 µl (µg) colloidal gold conjugated with the monoclonal antibody with 900 µl (µg) 5% w/w Tween-20 in resuspension buffer. Next, drop the mixed colloidal gold conjugated with the monoclonal antibody onto a glass fiber membrane. The glass fiber membrane was then dried for 24 hours at 37°C and stored at room temperature in an aluminum foil bag with a desiccant, sealed tightly. 2.3 Assembly of the test kit: Assemble the paper components prepared above as follows: Part 4: Nitrocellulose membrane paper, placed over the top end of the polystyrene sheet with the nitrocellulose membrane coated with sibutramine. The conjugate antibody with bovine serum albumin and mouse-specific goat Iggg antibody is then overlaid on a 2-3 mm portion of the nitrocellulose membrane. A second portion of glass fiber paper coated with the antibody conjugate is then placed over the bottom end, overlapping the nitrocellulose membrane by 2-3 mm. A third portion of glass fiber paper is attached to the bottom edge of the polystyrene sheet, overlapping the second portion. This results in a test strip measuring 60 mm x 300 mm. Cut the strip into pieces measuring 4 mm x 60 mm. Place the cut test strips into the test cassette. Store in a sealed aluminum foil pouch containing a desiccant. ------------ Page 1 of 2 Claim 1. Sibutramine Test Kit and Manufacturing Process 1.1 The plastic casing for the test strip consists of a bottom plastic piece for placing the test strip and an top plastic piece with various openings. The outermost opening is an oval-shaped well for dropping the sample extracted with a ready-made extraction solution consisting of 50 percent (volume) of ethanol in water, contained in a glass bottle. Next is a rectangular opening, a window displaying the test results, labeled "C" and "T". Inside, there are four test paper sections. Section 1 is a nitrocellulose membrane measuring 4 mm x 25 mm, covering the center of a 140 µm pore-bearing polystyrene sheet. This section acts as the display area, receiving the sibutramine conjugate with bovine serum albumin at position "T" and the goat Iggg antibody specific to mouse at position "C". These positions are indicated on the test casing. Part 2 is a glass fiber paper measuring 4 mm x 8 mm, overlapping 2-3 mm of the nitrocellulose membrane. This layer acts as the sheet coated with the antibody conjugate. Part 3 is a glass fiber paper measuring 4 mm x 20 mm, overlapping 2-4 mm with Part 2. The bottom edge of the test sheet is attached as the absorbent and solution-holding part. Part 4 is a nitrocellulose membrane measuring 4 mm x 20 mm, covering the top end of the test sheet and overlapping the... A 2-3 mm nitrocellulose membrane acts as an absorbent for all samples and solutions. 1.2 A ready-made buffer solution consists of 0.1 g (g) potassium chloride (KCl), 0.12 g (g) potassium dihydrogen phosphate (KH2PO4), 4 g (g) sodium chloride (NaCl), 1.81 g (g) disodium hydrogen phosphate (Na2HPO4), and 250 µl (µg) Tween-20. Adjust the volume with water until the total volume is 500 ml (ml). The ratios of the components can be adjusted according to the desired volume of the ready-made buffer solution. 2. The manufacturing process for the sibutramine test kit and the manufacturing procedure as per claim 1 are as follows: 2.1 Prepare sibutramine conjugate with bovine serum albumin at a concentration of 0.05 mg/ml and mouse-specific goat IGG antibody at a concentration of 0.5 mg/ml. Spray onto a 0.9 µL/cm² nitrocellulose membrane, dry for 24 hours at 37°C, and store at room temperature in a sealed aluminum foil bag with a desiccant. 2.2 The antibody conjugate can be prepared by diluting the monoclonal antibody at 0.2 mg/ml with HEPES solution, then preparing... To conjugate the monoclonal antibody with colloidal gold, add 80 µl of 0.1 M potassium carbonate (K2CO3) to 10 mL of colloidal gold solution. Then add 300 µl of diluted monoclonal antibody and let it stand at room temperature for 50 minutes. Add 500 µl of 10% w/v bovine serum albumin and centrifuge for 45 minutes at 8,000 rpm at 4 °C. After that, discard the supernatant and add a suspension buffer. (Resuspension buffer) Page 2 of 2, containing a mixture of 0.02 molar (M) tris hydrochloride (Tris-HCl), 0.1% w/w Tween-20, 0.1% w/w PEG, 1% w/w PVP, 5% w/w sucrose, 4% w/w trehalose, 2% w/w sorbitol, 1% w/w mannitol, 0.04% w/w sodium azide (NaN3), and 0.2% w/w BSA. Then mix 300 µl. ((Symbol)l) Colloidal gold conjugated with monoclonal antibody and 900 µl ((Symbol)l) 5% w/w Tween-20 in resuspension buffer. The mixed colloidal gold conjugated with monoclonal antibody is then dropped onto a glass fiber membrane. The glass fiber membrane is then dried for 24 hours at 37°C and stored at room temperature in an aluminum foil bag with a desiccant and sealed tightly. 2.3 Assembly of the Test Kit Components: The paper components prepared above are layered as follows: Part 4: Nitrocellulose membrane paper is placed over the top end of the polystyrene sheet containing the nitrocellulose membrane coated with sibutramine conjugated with bovine serum albumin and mouse-specific goat Igg antibody, overlapping each other. Overlap the nitrocellulose membrane by 2-3 millimeters. Then, take the second layer of glass fiber paper coated with the antibody conjugate and cover the bottom end, overlapping it by 2-3 millimeters. Next, attach the third layer of glass fiber paper to the bottom edge of the polystyrene sheet, overlapping the second layer of glass fiber paper. This will create a test sheet measuring 60 mm x 300 mm. Cut the test sheet to a size of 4 mm x 60 mm. Place the cut test sheets into the test cassette. Store them in an aluminum foil pouch containing a desiccant and seal it tightly.