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Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies

A detection kit and technology for Toxoplasma gondii are applied in the fields of genetic engineering technology and diagnostic reagents, which can solve the problems of high false positive rate and low specificity, and achieve the effects of good specificity, high sensitivity and good repeatability.

Inactive Publication Date: 2014-06-04
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides an indirect ELISA kit for detecting antibodies to Toxoplasma gondii in dogs and cats using recombinant protein GRA7 as an antigen. Qualitative detection of insect antibodies

Method used

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  • Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies
  • Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies
  • Indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Operation steps of canine Toxoplasma gondii antibody indirect ELSIA kit

[0020] (1) After diluting the sample diluent and canine serum at 1:50, add it to the wells of the GRA7 pre-coated microtiter plate, 50 μL per well, incubator at 37°C for 1 hour;

[0021] (2) Wash with washing solution 5 times, 5 minutes each time;

[0022] (3) Add HRP-labeled rabbit anti-dog IgG (working concentration), 50 μL per well, incubator at 37°C, 1h;

[0023] (4) Wash with washing solution 5 times, 5 minutes each time;

[0024] (5) Add TMB substrate chromogenic solution, 50 μL per well, incubator at 37°C in the dark for 20 minutes;

[0025] (6) Add stop solution, 50 μL per well;

[0026] (7) Use a microplate reader to measure the optical absorption (OD) value at 450nm;

[0027] (8) Judgment of results: Measure the OD value of each well after zeroing the blank control well, and if it is greater than 2.1 times the OD value of the negative control, it is positive.

[0028] ...

Embodiment 2

[0029] Example 2 : Feline Toxoplasma gondii antibody indirect ELSIA kit operation steps

[0030] (1) After diluting the cat serum with the sample diluent at 1:64, add it to the wells of the GRA7 pre-coated microtiter plate, 50 μL per well, incubator at 37°C for 1 hour;

[0031] (2) Wash with washing solution 5 times, 5 minutes each time;

[0032] (3) Add HRP-labeled rabbit anti-cat IgG (working concentration), 50 μL per well, incubator at 37°C for 1 hour;

[0033] (4) Wash with washing solution 5 times, 5 minutes each time;

[0034] (5) Add TMB substrate chromogenic solution, 50 μL per well, incubator at 37°C in the dark for 20 minutes;

[0035] (6) Add stop solution, 50 μL per well;

[0036] (7) Use a microplate reader to measure the optical absorption (OD) value at 450nm;

[0037] (8) Judgment of results: Measure the OD value of each well after zeroing the blank control well, and if it is greater than 2.1 times the OD value of the negative control, it is positive.

[...

Embodiment 3

[0039] Example 3: Evaluation of detection results of toxoplasma gondii antibody indirect ELSIA kit

[0040] 259 positive and negative sera of the canine Toxoplasma gondii antibody determined by the MAT method and the IFAT method were detected with the kit. The test results are shown in Table 1. Statistical analysis showed that there was no significant difference between the ELISA test results and the actual results (P>0.05; Kappa=0.8631, 95% CI: 0.7803, 0.9459), and the result consistency rate was 96.1%. Canine Toxoplasma gondii antibody indirect ELSIA kit has a sensitivity of 88.6% and a specificity of 97.7%.

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Abstract

The invention discloses an indirect enzyme-linked immuno sorbent assay (ELISA) kit for detecting canine and feline Toxoplasma gondii antibodies, relates to an immunological detection technology for human and animal infectious diseases and is applicable to qualitative detection of canine and feline Toxoplasma gondii antibodies. The kit comprises a Toxoplasma gondii recombinant antigen GRA7 pre-coated ELISA plate, wherein the sample diluent is 0.01 mol / L of PBS (with the pH of 7.2) containing 0.5 percent of BSA and 0.05 percent of NaN3; the enzyme bonder is a horse radish peroxidase (HRP)-rabbit anti-dog / cat IgG bonder; the substrate developing solution is a TMB developing solution; the stopping solution is a 2mol / L of sulfuric acid solution, positive control and negative control. The kit is high in sensitivity, high in specificity and high in repeatability and can serve as a method for detecting canine and feline Toxoplasma gondii antibodies.

Description

technical field [0001] The invention discloses an indirect ELISA kit for detecting toxoplasma gondii antibody in dogs and cats, and belongs to the field of genetic engineering technology and diagnostic reagents. Background technique [0002] Toxoplasmosis is an important zoonotic parasitic disease caused by Toxoplasma gondii, which leads to abortion, teratogenicity, and stillbirth in humans and animals, and seriously endangers public health safety and animal husbandry production. Cat is the terminal host of Toxoplasma gondii, and dog is the intermediate host of Toxoplasma gondii, which plays an important role in the transmission and infection of Toxoplasma gondii. At present, there is no effective vaccine against Toxoplasma gondii, and the monitoring of Toxoplasma gondii infection in dogs and cats is of great significance for the prevention and control of the disease. [0003] The diagnosis of Toxoplasma gondii infection mainly includes methods of etiology, molecular biolog...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/56905G01N2333/45
Inventor 刘全王泽东蔡玉峰魏峰
Owner JILIN AGRICULTURAL UNIV
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