An elisa kit for detecting antibodies against Salmonella pullorum

A Salmonella and kit technology, which is applied in the field of ELISA kits for detecting Salmonella pullorum antibodies, can solve the problems of high stress in chickens, agglutination of negative serum, decreased production performance of chickens, etc., achieves good specificity and sensitivity, and improves work efficiency. Efficiency, the effect of reducing the stress of chickens

Active Publication Date: 2015-10-14
WENS FOODSTUFF GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the great stress on the chickens through blood collection by acupuncture, it is easy to cause the decline in the production performance of the chickens, and even the rupture of the liver and spleen, especially in laying hens. Agglutination can also occur, which can easily cause false positives

Method used

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  • An elisa kit for detecting antibodies against Salmonella pullorum
  • An elisa kit for detecting antibodies against Salmonella pullorum
  • An elisa kit for detecting antibodies against Salmonella pullorum

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Immunoprecipitation (pulldown) technique for screening dominant antigens of Salmonella pullorum

[0046] 1 plate agglutination test

[0047] Take 10 μL pullorum and chicken typhoid multivalent staining plate agglutination test antigen and drop it on a clean slide, then take 10 μL serum collected from the chicken farm and drop it on the slide, mix it upside down, agglutination particles appear within 2 minutes, and the liquid is clear It is judged as positive, and it is judged as negative if no aggregated particles appear, and SPF chicken is used as a negative control. The yin and yang sera of pullorum were identified and used for immunoprecipitation.

[0048] 2. Purified IgG from pullorum negative and positive serum

[0049] 2.1 Dialysis bag treatment: Take 200ml of solution I (prepared with distilled water, containing 2% (W / V) sodium bicarbonate, 1mmol EDTA, and sodium hydroxide to adjust the pH to 8.0) in a beaker, and put the beaker in a water bath to boil....

Embodiment 2

[0066] Example 2 Construction of Salmonella pullorum GroEL prokaryotic expression vector

[0067] 1 Extraction of the whole genome of Salmonella pullorum

[0068] Take 10 μL of Salmonella pullorum standard strain 533 Glycerolbacterium and inoculate 5 ml of LB liquid medium, shake overnight at 37°C.

[0069] Follow the instructions of the Bacterial Genomic DNA Rapid Extraction Kit: Take 2ml of the cultured bacteria solution, centrifuge at 10,000rpm for 30s, discard the supernatant as much as possible, and collect the bacteria. Add 200 μL of buffer RB to resuspend, centrifuge at 10,000 rpm for 30 s, and discard the supernatant. Resuspend the cells in 180 µL Buffer RB by shaking. Add 20 μL of proteinase K (20 mg / ml), mix thoroughly, then add 200 μL of binding solution CB, immediately vortex, mix well, and place at 70°C for 10 minutes. After cooling, add 100 μL of isopropanol, and immediately vortex to mix well, at this time flocculent precipitation may appear. Add the mixture...

Embodiment 3

[0101] Expression and purification of embodiment 3 recombinant protein

[0102] 1 Transformation and induced expression of recombinant prokaryotic expression vector

[0103] The constructed recombinant expression plasmid pET-28a-GroEL prokaryotic expression vector was transformed into Ecoli.BL21 competent cells, spread on LBK plates, cultured overnight at 37°C, and the recombinant transformants were screened by kanamycin resistance. Randomly pick 3 single colonies and pET-28aEcoli.BL21 empty vector to 5ml LBK liquid medium, shake and culture at 37°C for about 4h to OD600=0.6-0.8, sample 1ml respectively, then add IPTG at a final concentration of 1mmol / L, Shake at 37°C for about 6 hours, and sample 1ml each. The samples before and after induction were collected by centrifugation at 5000rpm for 10min. A certain amount of distilled water and 6×SDS loading buffer were added and boiled for 10 min, followed by SDS-PAGE electrophoresis to analyze protein expression.

[0104] SDS-P...

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Abstract

An ELISA kit for detecting a Salmonella pullorum antibody is established by screening Salmonella pullorum dominant antigen GroEL through an immunoprecipitation technology, expressing GroEL recombinant protein through utilizing a prokaryotic expression vector, and utilizing an antigen protein. The kit can reduce the response of chicken in the detection process of the Salmonella pullorum antibody, and can improve the detection specificity and the sensitivity.

Description

technical field [0001] The invention relates to antigen preparation and ELISA kit, in particular to an ELISA kit for detecting Salmonella pullorum antibody. Background technique [0002] Pullorum pullorum is a septic infectious disease caused by Salmonella pullorum pullorum. Its excrement is an important transmission medium, and it can also be transmitted vertically through eggs. It is one of the most serious diseases that endanger the chicken industry. [0003] Salmonella pullorum mostly infects young chicks within 20 days of age, causing white diarrhea, and the mortality rate is extremely high. Adult chickens can carry the bacteria without clinical symptoms. The disease can be transmitted both vertically and seriously horizontally, so the harm and economic loss caused are huge. At present, the western countries have completely eliminated pullorum, but the outbreak is still serious in our country. The conventional pullorum detection method is plate agglutination, blood is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68C12N15/70C12N15/66
CPCC12N15/70G01N33/56916G01N2333/255
Inventor 郑世军徐志超李晓齐王永强曹红
Owner WENS FOODSTUFF GRP CO LTD
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