A double-antibody sandwich kit for detecting a toxoplasma gondii circulating antigen and a preparing method thereof

A double anti-sandwich and Toxoplasma gondii technology is applied in the field of immunological detection to achieve the effects of low detection cost, convenient use and improved sensitivity

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

At present, there is no report on the establishment of double-antibody sandwich ELISA method using monoclonal antibody prepared by SAG3 to detect circulating antigen in porcine Toxoplasma gondii serum

Method used

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  • A double-antibody sandwich kit for detecting a toxoplasma gondii circulating antigen and a preparing method thereof
  • A double-antibody sandwich kit for detecting a toxoplasma gondii circulating antigen and a preparing method thereof
  • A double-antibody sandwich kit for detecting a toxoplasma gondii circulating antigen and a preparing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Monoclonal antibody labeled with HRP

[0034] Anti-A-SAG3-23 monoclonal antibody was labeled according to the instructions of the Glue activated horseradish peroxidase kit. Proceed as follows:

[0035] (1) Take McAb-23 solution 100ug, add 100ul REAGENTⅠA type activated horseradish peroxidase.

[0036] (2) After adding REAGENT II, ​​mix thoroughly and adjust the pH value to 9.5 with pH test paper, and place it at 37°C for 30 minutes.

[0037] (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.

[0038] (4) Add REAGENⅢ (about 3 times the volume of REAGENTⅡ) and ensure that the pH of the enzyme conjugate is around 7.0.

[0039] (5) Add glycerol to 50% of the total volume to stabilize the activity of the enzyme conjugate, and store at -20°C.

Embodiment 2

[0041] Determination of optimal dilution of labeled antibody

[0042] Dilute the standard positive and negative samples and coat the microtiter plate overnight at 4°C. Block with 5% skimmed milk powder diluted with PBST at 1:400, 1:800, 1:1600, 1:3200, 1 :6400 dilution ratio to dilute HRP-Anti-A-SAG3-23, then develop color, and terminate the reaction. Determination of OD 450nm value, take OD 450nm The dilution ratio of the well whose value is closest to 1 is the optimal working concentration of HRP-labeled antibody. The dilution ratio of 1:3200 is its optimal working concentration.

Embodiment 3

[0044] Establishment of double-antibody sandwich ELISA method

[0045] 1. Determination of the optimal dilution ratio of coating antibody and serum antigen

[0046] Using the checkerboard method, use Anti-A-SAG3-7 as the capture antibody at a ratio of 1:400 to 1:12800 to coat the microtiter plate vertically, 100 μL / well, overnight at 4 °C. The next day, the plate was shaken and washed 3 times with PBST, 3 minutes each time. 5% skimmed milk powder diluted with PBST was used as a blocking agent, 100 μL / well, and incubated at 37 °C for 2 h. Wash the plate as above, dilute the Toxoplasma gondii positive and negative sera with PBST at 1:10, 1:20, 1:40, and 1:80, then add 100 μL / well of the enzyme plate horizontally, and incubate at 37°C for 2 hours. Wash the plate as above, add the HRP-labeled Anti-A-SAG3-23 monoclonal antibody whose optimal dilution factor has been determined at a ratio of 1:3200 to the microtiter plate, 100 μL / well, and incubate at 37°C for 1 hour. Wash the plat...

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Abstract

A double-antibody sandwich kit for detecting a toxoplasma gondii circulating antigen and a preparing method thereof are disclosed. A double-antibody sandwich ELISA process is established by utilizing two anti-SAG3 monoclonal antibodies and used for detecting the toxoplasma gondii circulating antigen in pig serum. The kit and the method overcome disadvantages, such as low detection specificity, low sensitivity and high requirements on persons and instruments in pig serum toxoplasma gondii circulating antigen detection at present, and provide a process for detecting early infection of toxoplasmosis in pigs.

Description

technical field [0001] The invention discloses a double-antibody sandwich ELISA kit for detecting toxoplasma circulating antigen and a preparation method thereof, relates to a toxoplasma detection method, and belongs to the technical field of immunological detection. Background technique [0002] Toxoplasmosis is a serious zoonotic parasitic disease caused by Toxoplasma gondii with a worldwide distribution. Infects almost all warm-blooded animals including humans and domestic animals. One-third of the world's population is infected with the pathogen, and pigs, cattle, sheep, etc. among livestock can be infected with the disease. According to Dubey (2009), the infection rate of Toxoplasma gondii in various countries in the world is as high as 90.4%. From 2009 to 2013 in my country, the infection rate of pigs among animals reached 23.4%, and the mortality rate was as high as 60%. The disease has been reported in various places in my country. Toxoplasma gondii is also a food...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56905G01N33/577G01N2333/45
Inventor 张西臣杨正涛寇金华宫鹏涛李建华杨举李赫李棕松高珺珊
Owner JILIN UNIV
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