TH24888Y - Test kit for the detection of IgM and IgG antibodies against the SARS-CoV-2 virus and its manufacturing process. - Google Patents

Abstract

The SARS-CoV-2 antibody detection kit, consisting of pre-mixed buffer solutions and a test cassette containing test strips, comprises five types of paper. The nitrocellulose membrane paper holds mouse antibodies specific to human IgM and human IgG antibodies at positions M and G, respectively, and mouse antibodies specific to the flag polypeptide (FLAG) at position C. These are printed on the cassette. When assembled with the other paper components, a complete test strip is formed within the cassette. This allows for rapid preliminary detection of SARS-CoV-2 antibodies without the need for specialized laboratory equipment, contributing to more effective disease control and prevention.

Claims (2)

------11/02/2565------(OCR) 1. The test kit for detecting IGM and IGG antibodies against SARS-CoV-2 virus consists of: 1.1 A plastic cassette for holding the test strip, measuring 20 mm x 90 mm (width x length). The bottom plastic piece has a central plate for placing the test strip. The top plastic piece has a round opening at the end for dropping buffer solution, marked with the embossed letter "B". Next is a rectangular opening for inserting the test sample, marked with the embossed letter "S". A rectangular opening along the length of the membrane displays the test results, marked with the letters "M", "G", and "C". Finally, there are 20 small square openings containing five types of test paper: - Type 1 paper is a nitrocellulose membrane measuring 4 mm x 25 mm, covering the center of a polystyrene sheet. It has a pore size of 8 micrometers and serves as the result display area. The test cassette will support mouse antibodies that are specific to human IgM antibodies and mouse antibodies that are specific to human IgG antibodies at positions M and G, respectively, and mouse antibodies that are specific to the flag polypeptide (FLAG) at position C, written on the test cassette. - The second type of paper is a glass fiber paper, 4 mm x 8 mm, which acts as a conjugate layer for the recombinant nucleocapsid protein (NP) of the SARS-CoV-2 virus, which is connected to the flag polypeptide and overlaps the first type of paper by 2-3 mm. - The third type of paper is a fine microglass fiber paper, 4 mm x 10 mm, which absorbs the sample and supports the solution, overlapping the second type of paper by 2-4 mm. - The fourth type of paper is cotton fiber paper. It has dimensions of 4 mm x 22 mm, with a water absorption capacity of 31 mg/cm². It is the part that absorbs the sample and holds the solution, overlapping the 32-4 mm paper type, attached to the bottom edge of the test sheet. - Paper type 5 is made of absorbent material, with dimensions of 4 mm x 22 mm, with a water absorption capacity of 128 mg/cm². It is placed on the top end of the test sheet and overlaps the paper type 1 by 2-3 mm, serving to absorb the entire sample and solution. 1.2 Ready-made Tuppher solution consists of 0.3% this base, 0.5% casein, and 0.1% sodium azide, packaged in a plastic bottle. 2. Manufacturing process of the SARS-CoV-2 antibody detection test kit, IgChem and IgGME types, according to claim 1, which has the following steps: 2.1 Antibody preparation. 2.2 Spraying the capturing antibodies: - Prepare a mouse antibody that specifically binds to human IgM antibody at a concentration of 0.5 mg/ml in phosphate-buffered solution at position M. - Prepare a mouse antibody that specifically binds to human IgG antibody at a concentration of 0.8 mg/ml in phosphate-buffered solution at position G. - Prepare a mouse antibody that specifically binds to polypeptide flag at a concentration of 0.2 mg/ml in phosphate-buffered solution at position C. The mouse antibodies that specifically bind to the human IgM antibody at position M, the mouse antibody that specifically binds to the human IgG antibody at position G, and the mouse antibody that specifically binds to the polypeptide flag at position C are printed on the plastic casing. The sprayed nitrocellulose membranes were then dried for 2 hours at 37°C and stored at room temperature in sealed aluminum foil bags with a desiccant. 2.3 Preparation of Conjugated Nucleocapsid Proteins and Glass Fiber Paper - Prepare 10 µg of nucleocapsid protein in 100 µl of borate buffer solution. - Prepare colloidal gold with an absorbance (OD) of 1.0 ± 0.1 at a wavelength of 525 nm, adjusting the pH to 9.00 ± 0.1. - Conjugation: Add 100 µl of nucleocapsid protein to 1,000 µl of colloidal gold and mix thoroughly for 30 minutes. Stop the reaction by adding 10% BSA. To achieve a final concentration of 1%, centrifugate at 12000g for 30 minutes at 4°C. Collect the precipitate and dissolve it in a phosphate buffer solution. The absorbance at a wavelength of 540 nm was 19 (positive or negative 1-). Prepare fiberglass paper with dimensions of 100 mm x 300 mm by immersing it in a borate buffer solution. Then, dry it at 37°C for 3 hours. 2.4 Spray the protein conjugate onto the prepared fiberglass paper using a sprayer at a rate of 10 µL/cm², spraying each strand to be 300 mm long. Dry it at 37°C for 2 hours. Then, use a blade to cut along the conjugate lines in each row to create 8 mm x 300 mm sheets. Store at room temperature in aluminum foil bags. 2.5 Assembling the Test Kit - Assemble all the paper components prepared above as follows: A polystyrene sheet has a nitrocellulose membrane coated with various antibodies attached to the center. One end of the nitrocellulose membrane is overlaid with an absorbent material, and the other end is overlaid with glass fiber paper coated with nucleocapsid proteins, followed by fine glass fiber paper, and the bottom edge is secured with cotton fiber paper. Each type of paper overlaps by 2-3 millimeters. One polystyrene sheet measures 80 millimeters wide by 300 millimeters long. - Cut the test sheet widthwise into small pieces using a cutter to a width of 4 millimeters. This will result in test sheets sized to fit the test kit: 4 millimeters wide by 80 millimeters long. Then, place the cut test sheets into the test cassettes in the designated positions. The lower plastic cassette is for placing the test sheets, and the upper plastic piece has various openings: a circular well at the end for dropping buffer solution, a rectangular opening for inserting the test sample, and rectangular openings along the length of the membrane. This is a window displaying the test results, with the letters "MG" and "C" printed on it. It is stored in a sealed aluminum foil pouch containing a desiccant. 1. Components of the SARS-CoV-2 IgM and IgG Antibody Test Kit include: 1.1 A plastic cassette for holding the test strip, approximately 20 mm x 90 mm (width x length). It features a central base for placing the test strip, a round opening at the end for dropping buffer solution (marked with the embossed letter "B"), a rectangular opening for inserting the test sample (marked with the embossed letter "S"), and a rectangular opening along the length of the membrane displaying the test results (marked with the letters "M," "G," and "C"). Finally, there are 20 small square openings containing five types of test paper: - Type 1 paper is a nitrocellulose membrane measuring 4 mm x 25 mm, covering the center of a polystyrene sheet. It has an 8-micrometer pore size and serves as the display layer. It holds mouse antibodies that specifically bind to human IgM antibodies and other antibodies. The mice that specifically bound to human IgG antibodies at positions M" and G", respectively, and the mice that bound to the flag polypeptide (FLAG) at position C" are indicated on the test cassette. - The second type of paper is a glass fiber paper, 4 mm x 8 mm in size, which acts as an absorbent layer for the recombinant nucleocapsid protein (NP) of the SARS-CoV-2 virus, which is attached to the flag polypeptide. It overlaps the first type of paper by approximately 2-3 mm. - The third type of paper is a fine glass fiber paper (microglass fiber), 4 mm x 10 mm in size, which absorbs the sample and holds the solution. It overlaps the second type of paper by approximately 2-4 mm. - The fourth type of paper is cotton fiber paper. It measures 4 mm x 22 mm (width x length) and has a water absorption capacity of 31 mg/cm². It acts as the sample absorbent and solution support layer, overlapping the third type of paper by approximately 2-4 mm, and secured to the bottom edge of the test sheet. - The fifth type of paper is made from absorbent material, measuring 4 mm x 22 mm and having a water absorption capacity of 128 mg/cm². It is placed on the top end of the test sheet and overlaps the first type of paper by approximately 2-3 mm, absorbing all of the sample and solution. 1.2 Ready-made buffer solution consists of 0.3% thiamine base, 0.5% casein, and 0.1% sodium azide, packaged in a plastic bottle. 2. The manufacturing process of the SARS-CoV-2 IgChem and IgG antibody test kit involves the following steps: 2.1 Preparation of capture antibodies: - Prepare mouse antibodies that specifically bind to human IgM antibodies at a concentration of 0.5 mg/ml in phosphate buffer solution at position M. - Prepare mouse antibodies that specifically bind to human IgG antibodies at a concentration of 0.8 mg/ml in phosphate buffer solution at position G. - Prepare mouse antibodies that specifically bind to polypeptide flag at a concentration of 0.2 mg/ml in phosphate buffer solution at position C. 2.2 Spraying of capture antibodies: Peel off the adhesive paper from the center of an 80 mm wide x 300 mm long polystyrene sheet. Then, cover it with a 25 mm wide x 300 mm long nitrocellulose membrane in a straight line along the length of the nitrocellulose membrane. Spray the solution at a rate of 1 microliter per centimeter. Mouse antibodies that specifically bind to human IgM antibodies at the M position, mouse antibodies that specifically bind to human IgG antibodies at the G position, and mouse antibodies that specifically bind... With the polypeptide flag at position "C" as printed on the plastic cartridge, the sprayed nitrocellulose membrane was then dried for 2 hours at 37°C. It was then stored at room temperature in a sealed aluminum foil bag with a desiccant. 2.3 Preparation of Conjugated Protein Nucleocapsids and Glass Fiber Paper - Prepare 10 µg of nucleocapsid protein in 100 µl of borate buffer solution. - Prepare colloidal gold with an absorbance (OD) of 1.0 (symbol) 0.1 at a wavelength of 525 nm, by adjusting the pH to 9.00 (symbol) 0.1. - Conjugation: Add 100 µl of nucleocapsid protein to 1,000 µl of colloidal gold. Mix thoroughly for 30 minutes. Stop the reaction by adding 10% BSA to achieve a final concentration of 1%. Centrifugation was performed at 12000g for 30 minutes at 4°C. The precipitate was then dissolved in phosphate buffer solution, yielding an absorbance of 19 (symbol) at 540 nm wavelength. 1. Preparation of glass fiber paper: 100 mm x 300 mm, immersed in borate buffer solution, then dried at 37°C for 3 hours. 2.4 Spraying of protein conjugates onto the prepared glass fiber paper using a sprayer at a rate of 10 µL/cm², spraying each strand 300 mm long. Dried at 37°C for 2 hours. Then, using a blade, cut along the conjugate lines in each row to create 8 mm x 300 mm strips. Store at room temperature in an aluminum foil bag with a desiccant and seal tightly. 2.5 Assembling the test kit: Assemble all the prepared papers as follows: The polystyrene sheet has a nitrocellulose membrane coated with various antibodies attached to the center. One end of the nitrocellulose membrane is overlaid with an absorbent paper, while the other end is overlaid with glass fiber paper coated with nucleocapsid proteins, followed by fine glass fiber paper, and the bottom edge is secured with cotton fiber paper. Each type of paper overlaps by 2-3 millimeters. A single polystyrene sheet measures 80 millimeters wide by 300 millimeters long. The test sheet is cut widthwise into small pieces using a cutter, each 4 millimeters wide, resulting in test strips sized to fit a 4-millimeter wide by 80-millimeter long test kit. The cut test strips are then placed in the designated positions within the test cassette. The lower plastic cassette holds the test strips, and the upper plastic cassette has various openings: a circular well at the end for dispensing buffer solution, a rectangular opening for inserting the test sample, and rectangular openings along the length of the membrane displaying the test results. The letters "M", "G", and "C" are printed on the openings. The cassette should be stored in a sealed aluminum foil pouch containing a desiccant.