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Dog toxoplasma infection double antibody sandwich ELISA detection kit and preparation method

A double-antibody sandwich and Toxoplasma gondii technology, which is applied in the field of canine disease detection, can solve the problems of unsatisfactory detection of circulating antigens of Toxoplasma gondii infection in dogs, and achieve good market prospects, simple operation, and easy-to-judgment effects

Inactive Publication Date: 2016-10-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention discloses a double-antibody sandwich ELISA detection kit for toxoplasma gondii infection circulating antigen (CAg), which is used for detecting whether dogs are infected with toxoplasma gondii, and solves the problem that there is no ideal method for detecting the circulating antigen of canine toxoplasma gondii infection

Method used

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  • Dog toxoplasma infection double antibody sandwich ELISA detection kit and preparation method
  • Dog toxoplasma infection double antibody sandwich ELISA detection kit and preparation method
  • Dog toxoplasma infection double antibody sandwich ELISA detection kit and preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Monoclonal antibody labeled with HRP

[0035] Label 29# anti-toxoplasma SAG3 monoclonal antibody according to the instructions of the Glue activated horseradish peroxidase kit. Proceed as follows:

[0036] (1) Take McAb-29 solution 100ug, add 100ul REAGENTIA type activated horseradish peroxidase.

[0037] (2) After adding REAGENTII, mix well and adjust the pH value to 9.5 with pH test paper, and place at 37°C for 30 minutes.

[0038] (3) Insert a 200ul pipette tip into the sodium borohydride powder, and when the white powder can be seen at the tip of the pipette, transfer it to the enzyme standard and mix well.

[0039] (4) Add REAGENIII (about 3 times the volume of REAGENTII) and ensure that the pH of the enzyme conjugate is around 7.0.

[0040] (5) Add glycerol to 50% of the total volume to stabilize the activity of the enzyme conjugate, and store at -20°C.

Embodiment 2

[0042] Determination of optimal dilution of labeled antibody

[0043] Select the best blocking agent, dilute the capture antibody and reference positive serum of Toxoplasma gondii according to the optimal working concentration, and dilute the detection antibody to different concentrations of 1:3000, 1:6000, 1:9000. Add substrate buffer to develop color and measure OD 450 nm value, determine the optimal working concentration of the detection antibody as 1:3000.

Embodiment 3

[0045] Establishment of double-antibody sandwich ELISA method

[0046] 1. Determination of the working concentration of the capture antibody and reference positive serum of Toxoplasma gondii

[0047] Using the checkerboard square array method, take different concentrations of capture antibodies: 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 / well dilution times to coat the microtiter plate, select The best blocking agent, incubated with Toxoplasma gondii reference positive sera with different dilutions of 1:6, 1:12, 1:24, 1:48, 1:96, 1:192, performed double-antibody sandwich ELISA, and determined the capture antibody and Optimal working concentration in serum. The optimal coating concentration of capture antibody was 1:800 / 100 μL, and the optimal dilution of Toxoplasma gondii reference positive serum was 1:12.

[0048]Table 1 The optimal coating concentration of antibody and the optimal dilution concentration of Toxoplasma gondii reference positive serum determined by checkerbo...

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Abstract

The invention provides a double antibody sandwich ELISA detection kit for detecting dog toxoplasma circulating antigens and a preparation method of the kit. Two anti-toxoplasma SAG3 monoclonal antibodies including a 42# anti-toxoplasma SAG3 monoclonal antibody and a 29# anti-toxoplasma SAG3 monoclonal antibody are used, and a double antibody sandwich ELISA method for detecting dog toxoplasma CAg is built. The kit is high in detection specificity, good in sensitivity, high in accuracy and good in stability, has the advantages of being easy to operate, quick in detection, easy to judge and the like, and is suitable for clinical quick diagnosis, basic-level epidemiological survey and the like.

Description

technical field [0001] The invention relates to a method for detecting circulating antigen (CAg) infected by Toxoplasma gondii in dogs, and further provides a double-antibody sandwich ELISA detection kit for Toxoplasma gondii infection in dogs. The invention also discloses a preparation method of the kit, belonging to canine The technical field of animal disease detection. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic opportunistic pathogen. It can widely infect vertebrates including humans. Most people with normal immune function become asymptomatic hosts after being infected with Toxoplasma gondii; some infected people will have more typical symptoms of toxoplasmosis. [0003] Dogs are the intermediate host of toxoplasmosis and an important source of infection of toxoplasmosis. In recent years, due to the increasing number of pet dogs, dogs infected with Toxoplasma gondii pose a great potential threat to human...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 李建华宋慧琦张西臣杨升高珺珊寇金华宫鹏涛杨举李赫李棕松杨正涛尹继刚
Owner JILIN UNIV
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