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High-flux detection method of O-shaped foot-and-mouth disease antibodies

A detection method and technology for foot-and-mouth disease, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of low detection throughput, slow detection speed, and inability to meet the needs of fast high-throughput detection, so as to improve detection throughput, improve Intuitive and fast effect of detection speed and judgment process

Inactive Publication Date: 2018-07-27
BAIMING BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the above problems, the present invention provides a high-throughput detection method of O-type foot-and-mouth disease antibody, which can solve the problem of slow detection speed and low detection throughput of the liquid-phase blocking ELISA method, which cannot meet the requirements of rapid high-throughput under a large number of samples. Technical issues with detection requirements

Method used

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  • High-flux detection method of O-shaped foot-and-mouth disease antibodies

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Effect test

Embodiment 1

[0042] A detection method for O-type foot-and-mouth disease antibody, which comprises the following steps,

[0043] (1) Use the sample diluent to perform a 32-fold single dilution of the serum to be tested, the negative quality control serum and the positive quality control serum, and add 50 μl / well to the antigen-antibody reaction plate;

[0044] (2) Add biotinylated foot-and-mouth disease virus type O antigen working solution, 50 μl / well, set 4 antigen control wells at the same time, add 100 μl / well antigen working solution, shake, and incubate at 37°C for 90 minutes;

[0045] (3) Transfer the entire solution in the reaction plate to a rabbit polyclonal antibody-coated plate, 50 μl / well, and incubate at 37°C for 40 minutes;

[0046] (4) Wash the plate, dry it, add enzyme-labeled streptavidin working solution to the rabbit polyclonal antibody plate, 50 μl / well, and incubate at 37°C for 30 minutes;

[0047] (5) Wash the plate, dry it, mix substrate A and substrate B in equal ...

Embodiment 2

[0059] 2.1 Determination of the optimal coating concentration of rabbit polyclonal antibody and the optimal working concentration of antigen

[0060] Use checkerboard titration to determine the optimal coating concentration of rabbit polyclonal antibody and the optimal working concentration of antigen. The coating concentration of rabbit polyclonal antibody was serially diluted from 32 μg / ml to 1 μg / ml for lateral coating, and the concentration of antigen was serially diluted to 0.08 μg / ml from 40 μg / ml to add samples vertically. Finally, the signal value was considered to be sufficiently large ( RLU>50000000) and the antigen concentration is at the singular point of the change under the corresponding coating concentration, the coating concentration is selected as 2 μg / ml, and the corresponding antigen working concentration is 15 μg / ml, the titration results are shown in Table 3.

[0061] Table 3: Titration Results

[0062]

[0063] 2.2 Determination of serum dilution fact...

Embodiment 3

[0073] 3.1 Conformity rate test

[0074] In order to verify the performance of the kit, 244 field sera (referring to the serum collected from breeding farms, farms and other places of origin) were selected for determination under the above-mentioned conditions, and at the same time, the O-type foot-and-mouth disease antibody liquid phase of Lanzhou Veterinary Research Institute Blocking ELISA detection kit for detection as a comparison, the results are as follows image 3 As shown, the results show that the correlation between the two is y=0.9435+0.0831, and the correlation coefficient r is 0.9551, which is highly correlated, indicating that the present invention has good specificity and sensitivity, and the detection results have high reliability.

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Abstract

The invention provides a high-flux detection method of O-shaped foot-and-mouth disease antibodies. The technical problems that the detection speed of a liquid phase blocking ELISA method is low; the detection flux is low, and the fast high-flux detection requirements under the condition of a great number of samples cannot be met are solved. The detection method is characterized in that a chemiluminescence immunosorbent method is used for testing the relative light intensity RLU of the serum to be tested and the antigen reference relative light intensity RLU of the sample to be tested; the inhibition rate PI is calculated according to the relative light intensity RLU of the serum to be tested and the antigen reference relative light intensity RLU. The method has the characteristics that inthe sample treatment process by the chemiluminescence immunosorbent method, the sample serum to be tested, negative quality control serum and positive quality control serum are subjected to single time dilution; the inhibition rate PI statistic interval corresponding to each valence serum does not have the overlapping phenomenon at the single dilution time; the inhibition rate PI is subjected to comparison with the critical inhibition rate corresponding to each valence serum, so that the valence of the serum to be tested is determined.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a high-throughput detection method for O-type foot-and-mouth disease antibody. Background technique [0002] Foot-mouth disease (FMD) is an acute, febrile, contagious infectious disease common to cloven-hoofed animals (pigs, cattle, sheep, camels, etc.) caused by foot-and-mouth disease virus. Epizooties, OIE) is listed as the first class A infectious disease, which is extremely harmful to animal husbandry production. Elimination and control of foot-and-mouth disease is a worldwide issue of common concern to governments of all countries. [0003] Serological diagnostic technology is one of the core technologies in the prevention and control of foot-and-mouth disease, including liquid-phase blocking ELISA, solid-phase competitive ELISA, non-structural protein 3ABC antibody detection ELISA and many other technologies. Liquid-phase blocking ELISA is the most widely use...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558
CPCG01N33/558G01N33/56983
Inventor 杨艳坤王世杰卢菲白仲虎刘秀霞詹锦玲
Owner BAIMING BIOTECH CO LTD
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