High-throughput quantitative detection method of rsv protein content and detection kit thereof

A technology for quantitative detection and protein content, which is applied in the field of quantitative detection of virus titers, can solve the problems of inappropriate compound screening and low sensitivity, and achieve the effects of reducing human factors and consumable use, high sensitivity, and improving throughput

Active Publication Date: 2015-09-30
WUXI APPTEC SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of these CPE methods is low, and the throughput is not suitable for the screening of a large number of compounds.

Method used

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  • High-throughput quantitative detection method of rsv protein content and detection kit thereof
  • High-throughput quantitative detection method of rsv protein content and detection kit thereof
  • High-throughput quantitative detection method of rsv protein content and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Non-clinical sample RSV (respiratory syncytial virus) quantitative, high-throughput ELISA detection

[0039] 1. Cell culture stage

[0040] (1) Hep2 cells (ATCC-CC123) were inoculated into 384-well cell culture plates (Greiner-781080, USA) with 3000 cells in a volume of 30 μl, and placed in a cell culture incubator (37 ° C, 5% CO 2 ) for 16 hours;

[0041] (2) Dilute non-clinical samples (i.e. compound 1, sourced from customers), according to the final concentration of 50μM, 10μM, 2μM, 0.4μM, respectively take 300nL, 60nL, 12.5nL, 2.5nL from the mother solution (10mM), add to the inoculation 384-well culture plate with cells;

[0042] (3) Dilute RSV (respiratory syncytial virus) (ATCC-VR26), the amount added is: 100 times TCID 50 Virus diluent, the volume is 30 μl;

[0043] (4) After culturing the above 384-well culture plate for 96 hours in a cell culture incubator, the cell culture supernatant was taken for ELISA detection.

[0044] 2. ELISA screening c...

Embodiment 2

[0063] Example 2 Non-clinical sample RSV (respiratory syncytial virus) quantitative, high-throughput ELISA detection

[0064] 1. Cell culture stage

[0065] (1) Hep2 cells (ATCC-CC123) were inoculated into 384-well cell culture plates (Greiner-781080, USA) with 3000 cells in a volume of 30 μl, and placed in a cell culture incubator (37 ° C, 5% CO 2 ) for 16 hours;

[0066] (2) Dilute the non-clinical sample (i.e. compound 1, the compound is from the customer), according to the final concentration of 50μM, 10μM, 2μM, 0.4μM, respectively take 300nL, 60nL, 12.5nL, 2.5nL from the mother solution (10mM), add to Inoculate a 384-well culture plate with cells;

[0067] (3) Dilute RSV (respiratory syncytial virus) (ATCC-VR26), the amount added is: 150 times TCID 50 Virus diluent, the volume is 30 μl;

[0068] (4) After culturing the above 384-well culture plate for 96 hours in a cell culture incubator, the cell culture supernatant was taken for ELISA detection.

[0069] 2. ELISA s...

Embodiment 3

[0088] Example 3 Non-clinical sample RSV (respiratory syncytial virus) quantitative, high-throughput ELISA detection

[0089] 1. Cell culture stage

[0090] (1) Hep2 cells (ATCC-CC123) were inoculated into 384-well cell culture plates (Greiner-781080, USA) with 3000 cells in a volume of 30 μl, and placed in a cell culture incubator (37 ° C, 5% CO 2 ) for 16 hours;

[0091] (2) Dilute the non-clinical sample (i.e. compound 1, the compound is from the customer), according to the final concentration of 50μM, 10μM, 2μM, 0.4μM, respectively take 300nL, 60nL, 12.5nL, 2.5nL from the mother solution (10mM), add to Inoculate a 384-well culture plate with cells;

[0092] (3) Dilute RSV (respiratory syncytial virus) (ATCC-VR26), the amount added is: 150 times TCID 50 Virus diluent, the volume is 30 μl;

[0093] (4) After culturing the above 384-well culture plate for 96 hours in a cell culture incubator, the cell culture supernatant was taken for ELISA detection.

[0094] 2. ELISA s...

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Abstract

The invention discloses a quantitative detection method of high-flux RSV (Respiratory Syncytial Virus) protein content and a detection kit thereof. The quantitative detection method comprises the following steps: A. with regard to a non-clinical sample, a detection step comprises an H2p2 cell cultivation stage and an ELISA (Enzyme-Linked Immuno Sorbent Assay) high-flux detection stage of the RSV protein content; and B. with regard to a clinical or preclinical sample, a detection step comprises the ELISA high-flux detection stage of the RSV protein content; and the quantitative detection kit comprises an ELISA detection reagent of the RSV protein content. According to the quantitative detection method of the high-flux RSV protein content and the detection kit thereof, an existing method for sieving antivirus medicines is changed, the sieving flux is improved, the sieving period is shortened and more than 60% of time can be saved. Meanwhile, the quantitative detection method of the high-flux RSV protein content and the detection kit thereof are also used for high-flux quantitative detection of RSV in a large amount of clinical or non-clinical samples.

Description

technical field [0001] The invention relates to a quantitative detection method of virus titer, in particular to a quantitative detection method and high-throughput detection of RSV (Respiratory Syncytial Virus, Respiratory Syncytial Virus) protein content for drug screening and biological sample testing Reagent test kit. Background technique [0002] Respiratory syncytial virus (RSV) is a leading cause of bronchitis and pneumonia in infancy and early childhood. The virus usually causes reinfection. Reinfection generally occurs in adults. In the elderly and immunocompromised patients, severe disease of the lower respiratory tract may result. The condition usually lasts two to three weeks. In infants and children at high risk for the condition, RSV (respiratory syncytial virus) infection can be life-threatening. Viral bronchitis is a serious infectious disease that is estimated to cause 1 million deaths per year, with the majority of cases caused by RSV (respiratory syncy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
Inventor 彭祥翔李少华陆恒
Owner WUXI APPTEC SUZHOU
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