Dog toxoplasma gondii antibody indirect ELISA detection kit

A detection kit and Toxoplasma gondii technology, applied in biological testing, measuring devices, material inspection products, etc., can solve problems such as difficult standardization, large differences in antigen preparation batches, uncertain components, etc., and achieve objective and good judgment of results Effects of immunogenicity and immunoprotection, strong specificity and sensitivity

Inactive Publication Date: 2015-10-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the antigens used in the ELISA detection of Toxoplasma gondii are parasite soluble antigens, whose composition is uncertain, and the difference between antigen preparation batches is large, so it is not easy to standardize

Method used

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  • Dog toxoplasma gondii antibody indirect ELISA detection kit
  • Dog toxoplasma gondii antibody indirect ELISA detection kit
  • Dog toxoplasma gondii antibody indirect ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of expression vector ROP5-PET32a

[0055] 1. Primer design and synthesis

[0056] According to the gene sequences published in GenBank, primers were designed to amplify the ROP5 gene sequence of Toxoplasma gondii. According to the needs of cloning, a BamHI restriction site was introduced at the 5' end of the primer ROP5F, and an Xho I restriction site was introduced at the 5' end of ROP5R. The primers were synthesized by Yingweijieji Company, and their nucleotide sequences consisted of the following:

[0057] RH strain ROP5F: 5'-TATA GGATCC ATGGCGACGAAGCTCG-3’

[0058] RH strain ROP5R: 5'-TATA CTCGAG CTCAAGCGACTGAGGGCG-3’

[0059] 2. Amplification of ROP5 target gene

[0060] The tachyzoites of Toxoplasma gondii stored in the liquid nitrogen tank in our laboratory were recovered in BALB / c mice, and the ascites of the mice was collected to extract the complete DNA of Toxoplasma gondii. Using the extracted DNA of Toxoplasma gondii as a temp...

Embodiment 2

[0074] Example 2 Induced expression and purification of ROP5 target protein

[0075] 1. Induction and expression of the target gene ROP5 in E. coli

[0076] The recombinant expression plasmid ROP5-PET32a was transformed into the expression bacteria BL21 (DE3), plated, and cultured for 12-14 hours. Pick a single white colony from the cultured plate, inoculate it in high-density bacterial growth medium (MDG), and cultivate it at 37°C and 200rpm for 6-8h. When the bacteria appear slightly cloudy, inoculate the bacterial solution at a ratio of 1:1000. with 100μg / mL Amp + In the automatic induction expression medium (ZYM-5052), shake culture at 28°C and 200rpm, take samples at different times of induction, set the induced empty vector bacteria as a control, and perform SDS-PAGE electrophoresis to detect the expression products. The electrophoresis results are shown in the appendix image 3 .

[0077] 2. Detection of the existence form of the expression product

[0078] The bact...

Embodiment 3

[0089] Example 3 Establishment of an indirect ELISA method for detecting Toxoplasma gondii antibodies

[0090] The purified expression protein ROP5 was used as the coating antigen to establish an indirect ELISA method, and the checkerboard method was used to explore the optimal antigen coating concentration, the optimal serum dilution, and the optimal reaction conditions of the ELISA method. The final conditions are as follows:

[0091] (1) Coating: Dilute the purified protein with Tris-HCl buffer (50mM pH7.6, TBS) at a ratio of 1:320 (that is, the protein coating concentration is 2.06 μg / mL), add it to a 96-well microtiter plate, 100μL / well, incubated at 37°C for 1.5h. Spin dry, wash 3 times with washing solution PBST, 2 min / time.

[0092] (2) Blocking: The blocking solution was 5% nonfat dry milk-PBST, 300 μL / well, incubated at 37°C for 1 h, the blocking solution was dried, and washed three times with PBST.

[0093] (3) Adding serum: the serum was diluted with 1% BSA-PBST...

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Abstract

The invention discloses a dog toxoplasma gondii antibody indirect ELISA detection kit, and belongs to the technical field of biological inspection and quarantine. According to the dog toxoplasma gondii antibody indirect ELISA detection kit, toxoplasma gondii ROP5 recombinant protein is taken as a detection antigen. According to the dog toxoplasma gondii antibody indirect ELISA detection kit, toxoplasma gondii recombinant protein ROP5 is obtained via gene cloning and prokaryotic expression; and a detection method comprises following steps: establishment of an indirect ELISA detection method, specificity testing, sensitivity testing, and comparison with other dog toxoplasma gondii detection kits. Large-scale standardized production of an envelope antigen of the dog toxoplasma gondii antibody indirect ELISA detection kit can be realized; detection specificity is high; sensitivity is high; repeatability is high; result determination is objective; operation process is simple; and large-scale detection can be realized. The dog toxoplasma gondii antibody indirect ELISA detection kit is suitable for dog toxoplasmosis epidemiological investigation, and detection and clinical diagnosis of dog toxoplasma gondii infection; and a rapid, simple, and specific detection method is provided for diagnosis and prevention, and epidemiological investigation of dog toxoplasmosis.

Description

technical field [0001] The invention belongs to the technical field of biological inspection and quarantine, and in particular relates to an indirect ELISA detection kit for Canis Toxoplasma antibodies. Background technique [0002] Toxoplasmosis is a global zoonotic parasitic disease caused by Toxoplasma gondii invading the nucleated cells of animals. Toxoplasma gondii is an opportunistic pathogenic parasite that does not produce obvious symptoms in normal humans. It is suitable for people with deficiencies in the immune system or in immunosuppressed patients (such as AIDS patients, malignant tumor patients, and people who have just received organ transplants). The harm is relatively large, often causing serious organ damage, and it is one of the common causes of death. The development of Toxoplasma gondii requires two hosts, and the cat is the only terminal host; the intermediate host is widely distributed, including almost all warm-blooded animals, some ectotherms and so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/45
Inventor 袁子国张秀香夏丽君李秀珍吕琳冯伟利吕淑媚文青元
Owner SOUTH CHINA AGRI UNIV
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