Rs virus detecting kit using anti-rs virus monoclonal antibody, immuno-chromatographic test device, and new anti-rs virus monoclonal antibody
A technology of monoclonal antibody and immunochromatography, which is applied in the direction of immunoglobulin, analytical materials, measuring devices, etc., and can solve problems such as insufficient sensitivity
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Embodiment 1
[0117] Hybridoma preparation
[0118] (Mouse Immunization)
[0119] 100 μl of Freund's complete adjuvant (FCA) was added to 100 μl of RSV antigen-containing phosphate buffer solution (PBS) commercially available from Capricon, and mixed and emulsified to prepare 200 μl of FCA RSV antigen solution. Then, 200 µl of FIA RSV antigen solution was prepared using Freund's incomplete adjuvant (FIA) in the same manner as above without using FCA.
[0120] BALB / C female mice aged 7-8 weeks were intraperitoneally injected with 200 μl of FCA RSV antigen solution for the first immunization. After the first immunization, booster immunization was carried out with 200 μl of FIA RSV antigen solution every 2 to 3 weeks. 200 µl of RSV antigen (manufactured by Capricon) was intravenously injected 10 days and 3 days before splenocyte isolation. Three days after the last injection, splenocytes were isolated, and splenocytes were fused with P3 X63-Ag8·653 mouse myeloma cells by PEG method to prepa...
Embodiment 2
[0128] Confirmation of Reactivity of Anti-RS Virus-F Protein Monoclonal Antibody
[0129] (confirmation of reactivity of 1565 antibody, 412 antibody and 255 antibody)
[0130] RS virus-F antigen used Long strain (10[6.7]TCID(50) / ml), A2 strain (10[5.5]TCID(50) / ml), 9320 strain (10[5.5]TCID(50) / ml ml) and RSV of the B1 wild-type (BWV) strain (10[4.5]TCID(50) / ml). These RSV4 strains are commercially available from the American Standard Biological Collection (ATCC). The above-mentioned RSV was diluted to 1 / 10 with a phosphate buffer solution containing bovine serum albumin (BSA) (hereinafter referred to as the first buffer solution).
[0131]Equal amounts of each diluted antigen solution and sample extract (100mM citric acid-0.4M NaCl, 10mM dithiothreitol, 0.1(v / v)% polyoxyethylene octyl phenyl ether) Mix and extract for 5 minutes to prepare various antigen extracts.
[0132] A sepharose solution containing commercially available anti-mouse antibody IgG bound to sepharose bea...
Embodiment 3
[0149] Confirmation of Antigen Recognition Site of Anti-RS Virus-F Protein Monoclonal Antibody
[0150] (Inhibition test against B016 antibody)
[0151] Strain A2 (10[5.5]TCID(50) / ml) was diluted 1 / 10 with the first buffer. The diluted antigen solution and the sample extract were mixed in equal amounts, and extracted for 5 minutes to prepare the A2 strain antigen extract. Then dilute the A2 strain antigen extract to 1 / 20 with the first buffer solution to prepare the antigen sample.
[0152] 100 μL of the 133-1H antibody (DHEMICON) solution diluted to a concentration of 10 μg / mL with 0.1 M sodium phosphate buffer was added to each well of the immune module (NUNC). The wells were washed three times with the second buffer. After washing, add 300 μL of the first buffer solution to each well to block (hereinafter this immune module is referred to as the 133-1H antibody sensitization plate).
[0153] Remove the first buffer in the 133-1H antibody-sensitized plate. After removal...
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