Indirect ELISA kit for cat toxoplasma gondii serum antibody and preparation method thereof

A serum antibody and Toxoplasma gondii technology, applied in the direction of biological testing, material inspection products, measuring devices, etc., can solve the problems of complicated operation process, laborious, low sensitivity, etc., and achieve easy to use, reliable immunological diagnosis method, strong specificity sexual effect

Inactive Publication Date: 2017-01-11
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological methods are time-consuming, labor-intensive, and low-sensitivity
Molecular biology techniques have high sensitivity and specificity, but require expensive instruments and equipment, and the operation process is complicated

Method used

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  • Indirect ELISA kit for cat toxoplasma gondii serum antibody and preparation method thereof
  • Indirect ELISA kit for cat toxoplasma gondii serum antibody and preparation method thereof
  • Indirect ELISA kit for cat toxoplasma gondii serum antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Preparation of Coating Antigen SAG3 Protein

[0035] Using genetic engineering technology to induce expression of the target gene in Escherichia coli, after the correct expression is identified, colonies are picked, expanded and cultivated for bacteria preservation. After inducing the recombinant protein identified correctly, the cells were collected by centrifugation, and the expression results were observed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (see figure 1 );

[0036] 1) Prepare 300mL of LB medium and autoclave it;

[0037] 2) Transfer 5 mL of the bacterial liquid shaken overnight to the culture medium in (1), add 500 μl of kanamycin and place it in a shaker at 37°C for 1.5-2 hours to observe at any time;

[0038] 3) Add 300 μl IPTG, continue to place in a shaker for induction for 4-5 hours and observe at any time;

[0039] 4) Collect the bacterial liquid, and centrifuge at 8000rpm×10min at 4°C (the same below);

[0040] 5) The pellet was resuspen...

Embodiment 2

[0046] Establishment of indirect ELISA method

[0047]1. Antigen coating concentration and serum dilution

[0048] The coating amount of antigen is 0.375μg / well and the serum dilution factor is 1:800;

[0049] 2. Working concentration of enzyme-labeled secondary antibody

[0050] The optimal working concentration of the labeled secondary antibody is 1:5000;

[0051] 3. Selection of the best sealing agent

[0052] The sealing agent is 5% skimmed milk powder;

[0053] 4. The best incubation time for the enzyme-labeled secondary antibody

[0054] Secondary antibody incubation time is 45min;

[0055] 5. Determination of performance

[0056] (1) Sensitivity test

[0057] The sensitivity is 1:25600 (see Table 1);

[0058] Table 1

[0059] Sensitivity Test Results

[0060]

[0061] (2) Specificity test

[0062] The established indirect ELISA method was used to detect the standard positive serum of feline calicivirus and the standard positive serum of feline transmissibl...

Embodiment 3

[0070] Assembling the components of an indirect ELISA kit for detecting serum antibodies to Toxoplasma gondii in cats

[0071] The test kit includes the following components:

[0072] 1. Toxoplasma gondii surface antigen SAG3 protein (stored at -20°C);

[0073] 2. Coating solution (0.05M carbonate buffer) PH (9.6): weigh NaCO 3 1.59g; NaHCO 3 2.93g; 80mL of distilled water, after it dissolves, adjust the pH value to 9.6, and dilute to 100mL;

[0074] 3. Blocking solution: Weigh 5g of skimmed milk powder and add PBST to dissolve it, then dilute to 100mL;

[0075] 4. Washing solution and sample diluent (PBST): Weigh 8g of NaCl; 0.2g of KCl; NaHPO 4 .12H 2 O 2.9g; KH 2 PO 4 0.2g; Tween-200.5Ml;

[0076] 5. TMB substrate chromogenic solution: Substrate A solution (weigh TMB 200mg, add absolute ethanol 100mL, adjust pH value to 5.0, add double distilled water to make up to 1000mL), and substrate B solution (weigh lemon Acid 9.33g, Na 2 HPO 4 14.60g, add 6.4mLH 2 o 2 , ...

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Abstract

The invention relates to an indirect ELISA kit for cat toxoplasma gondii serum antibody and a preparation method thereof. The toxoplasma gondii in serum is detected by adopting toxoplasma gondii surface protein SAG3 as a coating antigen; an indirect ELISA method is established by adopting HRP marked goat anti-cat IgG as a detection antibody, which has relatively strong specificity, sensitivity and repeatability; according to the invention, except for the fact that the washing liquid is powder, the main reagents can be directly applied after being dissolved in water; for the kit, the laboratory detection washing liquid is used as a basic solution, and the rest all exists in a form of liquid working solution, thus the kit is convenient to use; and the invention provides a convenient and reliable immunological diagnosis method for large sample detection and epidemiological survey and provides a basis for the diagnosis of cat toxoplasmosis.

Description

technical field [0001] The invention relates to a detection kit for detecting feline toxoplasma gondii serum antibody and a preparation method thereof, belonging to the technical field of veterinary biology. Applied to the detection of cat toxoplasmosis. Background technique [0002] Feline toxoplasmosis is an important zoonotic parasitic disease. Cats are the final host of the disease and can infect almost all warm-blooded animals, which can lead to abortion of pregnant women (animals), stillbirth, deformity, stunted growth and development of fetuses (young animals), and death. Humans are insidiously infected. The infection rate in the population is high, with about one in six people worldwide infected with toxoplasmosis. Toxoplasma gondii infection will seriously affect the immunocompromised population, because it will pose a serious threat to human health, so it is an important disease in human medicine. [0003] Studies have found that Toxoplasma gondii has been foun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/531
CPCG01N33/53G01N33/531G01N2333/45
Inventor 宫鹏涛刘伟建杨正涛张西臣寇金华李建华杨举李赫李棕松
Owner JILIN UNIV
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