Latex agglutination kit for detecting streptococcus agalactiae antibodies and application of kit
A technology of Streptococcus lactis and latex agglutination, which is applied in measurement devices, immunoassays, instruments, etc., can solve the problems of decreased specificity of detection methods, difficult to guarantee antigen activity, complex protein composition, etc. specific effect
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Embodiment 1
[0069] The extraction of embodiment 1 Streptococcus agalactiae surface antigen
[0070] (1) Streptococcus agalactiae cvcc1886 was inoculated in Tryptone Soy Broth (TSB) containing 5% fetal bovine serum, and cultured with constant temperature shaking at 37°C for about 6 hours.
[0071] (2) Centrifuge at 10,000 rpm for 5 minutes to collect the bacteria, and resuspend with pre-cooled TE buffer (10mM Tris, 1mM EDTA, pH 8.0).
[0072] (3) Ultrasonic crushing, centrifugation at 5000 rpm for 15 min, and collecting the supernatant.
[0073] (4) Ultracentrifuge the supernatant at 18,000 rpm for 90 min at 4°C, collect the precipitate, resuspend with a small amount of TE buffer containing 1.5% triton-X100, and let it dissolve at room temperature for 30 min.
[0074] (5) Add an equal volume of saturated ammonium sulfate solution, and precipitate at 4°C for 2 hours.
[0075] (6) The protein precipitate was collected by centrifugation at 12000 rpm for 30 min at 4°C, resuspended in TE buff...
Embodiment 2
[0078] The Dot blotting detection of embodiment 2 surface antigen activity
[0079] Dot blotting (Zhu Caizhong et al., Clinical research on the detection of anti-Jo-1 antibody by dot blot method. Hainan Medicine, 2010, 21 (21): 3-5.) was used to detect the activity of the surface antigen. The extracted surface antigen (concentration: 600 μg / ml) was serially diluted, and 3 μl of different dilutions of the antigen were spotted on the nitrocellulose membrane, and dried in a 37°C incubator for 30 minutes. Soak in blocking solution (PBST containing 1% BSA), block in a 37°C incubator for 30 minutes, and wash twice in PBST. Add 1:20 diluted Streptococcus agalactiae positive control serum, place at 37°C for 30 minutes, and wash twice with PBST. Add goat anti-bovine IgG-HRP and place at 37°C for 30 minutes. Shake and wash 3 times, 3 minutes each time. Substrate solution (DAB-H 2 o 2 ) color. It was judged positive by the appearance of obvious brown spots. Set the same concentrat...
Embodiment 3
[0080] The preparation of embodiment 3 positive, negative control serum
[0081] Concentrate the extracted surface antigen of Streptococcus agalactiae to 2 mg / mL, and emulsify with an equal amount of Freund's adjuvant. The cows were immunized by neck intramuscular injection, 5ml each time, and then immunized again after an interval of 15 days, for a total of 3 times. The first immunization was emulsified with Freund's complete adjuvant, and the subsequent booster immunization was emulsified with Freund's incomplete adjuvant. After the serum antibody titer reached 1:1, blood was collected from the jugular vein, and the serum was separated by centrifugation. Blood was collected and the antibody titer was detected by this latex agglutination method. When the antibody titer reached 1:64, the jugular venous blood was collected with a vacuum blood collection tube, the serum was separated by centrifugation, penicillin and streptomycin were added at 1000 U / mL, and sterilized by a 0....
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