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

Active Publication Date: 2014-01-22
武汉市畜牧兽医科学研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The total bacterial protein composition is complex, and the pertinence is not strong, which will easily lead to a decrease in the specificity of the established detection method; the cost of purifying and expressing the antigen from E. coli is high, and the antigen activity is difficult to guarantee

Method used

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  • Latex agglutination kit for detecting streptococcus agalactiae antibodies and application of kit
  • Latex agglutination kit for detecting streptococcus agalactiae antibodies and application of kit
  • Latex agglutination kit for detecting streptococcus agalactiae antibodies and application of kit

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a latex agglutination kit for detecting streptococcus agalactiae antibodies of dairy cows as well as a preparation method and an application of the kit. The kit comprises latex agglutination particles, positive control serum, negative control serum, a sample diluent, a glass slide and a graduated pipette. The kit is used for detecting whether the dairy cows suffer from mastitis, is simple, convenient and fast in method, has strong specificity and high sensitivity and is suitable for being used for detection by grass-roots veterinarians; and instruments and equipments are not needed.

Description

technical field [0001] The invention relates to the technical field of animal disease detection, in particular to a kit for detecting antibodies to Streptococcus agalactiae in dairy cows and the application of the kit in the detection of Streptococcus agalactiae antibodies. technical background [0002] For a long time, dairy cow mastitis has been the number one disease affecting the development of dairy farming industry and the food safety of dairy products. Among the existing dairy herds in my country, the incidence rate of clinical mastitis reaches 9.7%-55.6%, and the positive rate of recessive mastitis reaches 61.03%-79.62% (Feng Wanyu et al., Application of biological agents in the prevention and treatment of dairy cow mastitis, Journal of Animal Husbandry and Veterinary Medicine, 2010, 4:31-33). Streptococcus agalactiae accounts for a large proportion of the pathogenic bacteria causing mastitis in cows. According to the survey results of domestic scholars, dairy cow m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/566G01N33/544
CPCG01N33/531G01N33/544G01N33/566G01N33/56944G01N2333/315G01N2469/20G01N2800/7095
Inventor 卢顺高其双向敏黄海军陈志华占才耀彭霞周莉钱运国童伟文陶弼菲夏瑜王莲芳华娟
Owner 武汉市畜牧兽医科学研究所
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