Indirect ELISA (Enzyme-linked Immuno Sorbent Assay) kit for detecting type A haemophilus paragallinarum antibody as well as detection method and application thereof

A technology of Haemophilus gallinaceus and kits, applied in the direction of measuring devices, color/spectral characteristic measurement, instruments, etc., to achieve the effects of good immune reactivity, easy preparation and purification, and reliable technical means

Inactive Publication Date: 2017-09-29
YANGLING VOCATIONAL & TECHN COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the diagnosis method of chicken infectious rhinitis and the evaluation method of vaccine immune effect, such as clinical diagnosis, hemagglutination test, etc., it is not suitable for large-scale sample detection. For the monitoring of chicken infectious rhinitis epidemic situation and the effective evaluation of vaccines, it is necessary to establish a more sensitive, fast detection method

Method used

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  • Indirect ELISA (Enzyme-linked Immuno Sorbent Assay) kit for detecting type A haemophilus paragallinarum antibody as well as detection method and application thereof
  • Indirect ELISA (Enzyme-linked Immuno Sorbent Assay) kit for detecting type A haemophilus paragallinarum antibody as well as detection method and application thereof
  • Indirect ELISA (Enzyme-linked Immuno Sorbent Assay) kit for detecting type A haemophilus paragallinarum antibody as well as detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Construction of Haemophilus paragallinarum HA (HA refers to hemagglutinin) protein expression vector type A:

[0035] Genomic DNA of Haemophilus paragallinarum Hpg-8 strain (purchased from China Veterinary Drug Administration) was extracted, and the synthetic primer HAF:5'-CGC was designed GGATCC ATGAAAAAAAACTGCAATCG-3' (the underline is the BamHI restriction site), HAR:5'-CCG CTCGAG CTCGTTACCTTTAACTGAGAT-3' (the underline is the XhoI restriction site), amplified to obtain the HA gene amplification product, the length of the amplification product is 1032bp, its nucleotide sequence is detailed in SEQ.ID.No.1, the purpose of HA gene recovery from gel The fragment (amplified product) was ligated to the cloning vector pEASY-T1, and ligated overnight at 16°C. The ligated product was transformed into Trans5α clone competent cells, and spread onto a kanamycin-resistant (100mg / ml) LB plate, and placed at 37°C Cultivate overnight, pick a single clone, shake culture in kana...

Embodiment 2

[0063] Specific test of ELISA kit:

[0064] According to the established indirect ELISA operation method, use the ELISA kit to detect positive serum samples of known Haemophilus paragallinarum type A, Newcastle disease, avian influenza, Salmonella, Staphylococcus aureus, Pasteurella multocida and Escherichia coli respectively ; Measure the OD value of each hole with a microplate reader at a wavelength of 450nm to determine the specificity of the indirect ELISA detection method;

[0065] The test results showed that only the OD of the positive serum of Haemophilus paragallinarum type A 450 >0.34, other serum OD 450 All are less than 0.34, indicating that the specificity of the indirect ELISA test results is good.

Embodiment 3

[0067] Repeated tests within the same batch of ELISA kits:

[0068]Use the recombinant HA protein of Haemophilus paragallinarum type A prepared by induction and purification in the same batch, coat the microtiter plate according to the optimal coating concentration, and use the established indirect ELISA operation method to detect the different levels of Haemophilus paragallinarum type A antibodies 3 copies of the positive serum and 3 copies of the negative serum of Haemophilus paragallinarum type A, the 6 randomly selected sera were detected at different times, and the OD value was measured at a wavelength of 450nm with a microplate reader; according to the OD value of each well 450 Value, calculate the average value of each serum Standard deviation S, coefficient of variation CV; test results showed that 6 serum OD 450 The coefficient of variation of the value is between 1.5% and 3.7%, indicating that the detection results of the recombinant HA protein of Haemophilus parag...

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Abstract

The invention discloses an indirect ELISA kit for detecting Haemophilus paragallinarum type A antibody, a detection method and an application thereof, and relates to the technical field of biological detection. The ELISA kit of the present invention includes: coated microplate, washing solution, serum diluent, substrate chromogenic solution, rabbit anti-chicken enzyme-labeled secondary antibody, stop solution, type A Hpg standard positive serum and type A Hpg standard negative serum Serum; wherein, the coated microtiter plate uses the recombinant hemagglutinin protein of Haemophilus paragallinarum type A as the coated antigen. The ELISA kit of the present invention is used to detect the antibody of Haemophilus paragallinarum type A, and has the advantages of high efficiency, good sensitivity, specificity and repeatability, and the kit is easy to operate, fast and low in cost, and is suitable for clinical applications and promote.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an indirect ELISA kit for detecting the antibody of Haemophilus paragallinarum type A, a detection method and an application thereof. Background technique [0002] Haemophilus paragallinarum (Hpg) is the pathogen of infectious rhinitis in chickens. The clinical symptoms of the disease are acute upper respiratory tract infection in chickens, inflammation of the nasal cavity and sinuses, inflammation of the conjunctiva, tearing, decreased egg production, growth slow. Chicken infectious rhinitis occurs frequently in various regions of the world and is endemic. Although the fatality rate of the disease is relatively low, Haemophilus paragallinarum can exist in chicken flocks for a long time, reducing the production performance of chickens and resulting in a decrease in egg production rate , the growth of the bred chicken is slow, and the ability of the sick chicken to r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/535G01N33/543G01N21/31
CPCG01N33/56922G01N21/31G01N33/535G01N33/543G01N2333/465
Inventor 韩飞张曼杨书会
Owner YANGLING VOCATIONAL & TECHN COLLEGE
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