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Indirect ELISA method for detecting haemophilus parasuis antibody by recombining Neu protein and kit of method

A technology for detection of Haemophilus suis and protein, which is applied in the field of indirect ELISA method and its kit, can solve the problems of lack of cross-protection, risk of spreading poison, high detection cost, etc., to avoid a large number of false negatives and false positives, and avoid spreading poison Danger, the effect of reducing the cost of detection

Inactive Publication Date: 2017-03-15
永州职业技术学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the clinical signs of Haemophilus parasuis and other causes of respiratory disease in pigs are very similar and confusing
It has been confirmed that Haemophilus parasuis is a common bacterium in the upper respiratory tract of pigs, but its isolation rate is low when the bacteria are isolated and identified clinically, mainly because the isolation and culture of this bacteria is easily affected by factors such as antibiotics or chemotherapeutic agents. High nutritional requirements, short storage time
Therefore, traditional diagnostic techniques can no longer meet the needs of the development of modern swine diseases. Apply modern molecular biology techniques and immunological techniques to find a candidate antigen for the detection or diagnosis of Haemophilus parasuis, and establish a rapid and simple diagnosis. method is very important
[0004] At this stage, among the many detection methods for Haemophilus parasuis, ELISA detection method is the most widely used and most commercially mature detection method; however, only the Synbiocits company in the United States currently produces ELISA for detecting this disease in the market. Kits, commercial diagnostic kits for the detection of this disease have not yet been produced in China. This kit uses whole HPS bacteria as the coating antigen, which has the potential risk of spreading the virus, and the detection cost is high. The detection fee for each serum sample is about 60 RMB. This makes many breeding companies discouraged; in addition, due to the large number of serotypes of Haemophilus parasuis, there is a lack of cross-protection between the sera, and there are large differences in the prevalence of bacterial strains in various places. If the traditional ELISA diagnostic method is used to coat the whole bacteria, the The method is only highly diagnostic for bacteria of the same serotype, and the accuracy is very strong, but for strains of different serotypes, the diagnostic performance is significantly reduced, and even a large number of false negatives and false positives occur

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The concrete steps of the indirect ELISA method of its recombinant Neu protein detection Haemophilus parasuis antibody are as follows:

[0045] S1: Dilute the prepared antigen to 100ng / 100μl with 0.05 mol / L pH 9.6 carbonate buffer, coat the microplate plate, 100uL per well, lightly shake the microplate plate to spread the antigen evenly on the bottom of the well, 37 ℃ for 2 h, transfer to 4 ℃ for overnight adsorption;

[0046] S2: The next day, wash with PBST washing solution 3 to 5 times, each time for 5 minutes, and pat dry;

[0047] S3: Then block with 0.15% BSA, 200uL per well, incubate at 37°C for 1h, then wash the plate with washing solution for 3 to 5 times;

[0048] S4: Add the serum to be tested diluted with PBS, 100uL per well, set up negative and positive control wells at the same time, act at 37°C for 1 hour, wash the plate as above;

[0049] S5: Add diluted SPA-HRP, act at 23 °C for 1 hour, wash the plate as above;

[0050] S6: Add freshly prepared OPD-H...

Embodiment 2

[0054] The concrete steps of the indirect ELISA method of its recombinant Neu protein detection Haemophilus parasuis antibody are as follows:

[0055] S1: Dilute the prepared antigen to 25ng / 100μl with 0.05 mol / L pH 9.6 carbonate buffer, coat the microtiter plate, 100uL per well, lightly shake the microtiter plate to spread the antigen evenly on the bottom of the well, 37 ℃ for 2 h, transfer to 4 ℃ for overnight adsorption;

[0056] S2: The next day, wash with PBST washing solution 3 to 5 times, each time for 5 minutes, and pat dry;

[0057] S3: Then block with 0.15% BSA, 200uL per well, incubate at 37°C for 1h, then wash the plate with washing solution for 3 to 5 times;

[0058] S4: Add sample after sealing, add 100uL of serum to be tested diluted with PBS to each well, set up negative and positive control wells at the same time, act at 37 °C for 1 hour, wash the plate as above;

[0059] S5: Add diluted SPA-HRP, act at 23 °C for 1 hour, wash the plate as above;

[0060] S6...

Embodiment 3

[0064] The concrete steps of the indirect ELISA method of its recombinant Neu protein detection Haemophilus parasuis antibody are as follows:

[0065] S1: Dilute the prepared antigen to 50ng / 100μl with 0.05 mol / L pH 9.6 carbonate buffer, coat the microplate plate, 100uL per well, lightly shake the microplate plate to spread the antigen evenly on the bottom of the well, 37 ℃ for 2 h, transfer to 4 ℃ for overnight adsorption;

[0066] S2: The next day, wash with PBST washing solution 3 to 5 times, each time for 5 minutes, and pat dry;

[0067] S3: Then block with 0.15% BSA, 200uL per well, incubate at 37°C for 1h, then wash the plate with washing solution for 3 to 5 times;

[0068] S4: Add sample after sealing, add 100uL of serum to be tested diluted with PBS to each well, set up negative and positive control wells at the same time, act at 37 °C for 1 hour, wash the plate as above;

[0069] S5: Add diluted SPA-HRP, act at 23 °C for 1 hour, wash the plate as above;

[0070] S6...

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Abstract

The invention relates to the field of antibody detection, in particular to an indirect ELISA method for detecting a haemophilus parasuis antibody by recombining Neu protein and a kit of the method. The indirect ELISA method for detecting the haemophilus parasuis antibody by recombining the Neu protein comprises the steps that recombinant expressed and purified haemophilus parasuis Neuraminidase (Neu) protein serves as a coating antigen, accurate detection can be conducted on various serotype strains by optimizing the ELISA reaction condition, and the phenomenon that a large number of false negative results and false positive results appear can be better avoided; meanwhile, the phenomenon that the toxic risk exists in ELISA detection of a haemophilus parasuis disease can be effectively avoided, and the detection cost is greatly lowered.

Description

technical field [0001] The invention relates to the field of antibody detection, in particular to an indirect ELISA method and kit for detecting Haemophilus parasuis antibody with recombinant Neu protein. Background technique [0002] Haemophilus parasuis (HP) can cause polyarthritis, serositis, and meningitis in pigs, often in the nursery stage of piglets, and the mortality rate can reach 50%. With the rapid expansion of the scale of the pig industry and the change of the pig raising model, the epidemic trend of this disease has become increasingly serious in recent years, which has brought huge economic losses to the pig industry in my country, and has become the most serious bacterial disease in the pig industry. one of the diseases. Therefore, rapid and accurate diagnosis of the disease in order to take appropriate measures is the key to successful prevention and control of the disease. [0003] At present, the diagnosis of this disease in primary laboratories is limite...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C12N9/24
CPCG01N33/56911C12N9/2402C12Y302/01018G01N2333/924
Inventor 刘俊琦
Owner 永州职业技术学院
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