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Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate

An enzyme-linked immunosorbent reagent, CA125 technology, applied in the direction of measuring devices, biological material analysis, instruments, etc., can solve the problems of lack of obvious symptoms and effective screening, low survival rate, etc., to achieve low detection of non-specific adsorption, high sensitivity, Effect of Improving Detection Sensitivity

Inactive Publication Date: 2017-02-22
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the mortality rate of ovarian cancer ranks first among gynecological tumors. Unlike breast cancer, ovarian cancer lacks obvious symptoms and effective screening in the early stage, and the survival rate is low.

Method used

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  • Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate
  • Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate
  • Preparation method of enzyme-linked immunosorbent assay kit for detecting ovarian cancer tumor marker CA125 based on trypsin fluorogenic substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0028] 1) The newly prepared 0.2mL sodium periodate NalO 4 The solution (21.39mg / mL) was added to 1mL of trypsin solution (5mg / mL), stirred at room temperature for 20min in the dark, then dialyzed overnight with 1mM pH4.4 acetate buffer, and treated by adding 20μL of 0.2M pH9. 5 carbonate buffer solution to bring the system pH to 9.0, immediately add 10mg CA125 labeled antibody Ab 2 , stirred at room temperature for 2 h in the dark, then added 0.1 mL of sodium borohydride NaBH 4 (4mg / mL) fully reacted and dialyzed overnight with 0.15MPH7.4PBS buffer to obtain the enzyme-linked detection probe Trypsin-Ab 2 ;

[0029] 2) Add 100 μL CA125-coated antibody Ab 1 (24μg / mL) was added to a 96-well ELISA plate, stood overnight at 4°C, washed the 96-well ELISA plate with washing buffer, added 300 μL BSA (10mg / mL) at 37°C for 1 hour, and then washed again with washing buffer 96-well ELISA plate, then add 100 μL CA125 standard antigen and incubate with slow shaking, wash the 96-well EL...

Embodiment example 2

[0031] 1) The newly prepared 0.2mL sodium periodate NalO 4 Solution (20mg / mL) was added to 1mL Trypsin solution (5mg / mL), stirred at room temperature for 20min in the dark, then dialyzed overnight with 1mM pH4.4 acetate buffer, and treated by adding 20μL 0.2M pH9.5 Make the pH of the system reach 9.0 with carbonate buffer, and immediately add 10mg of CA125-labeled antibody Ab 2 , stirred at room temperature for 2 h in the dark, then added 0.1 mL of sodium borohydride NaBH 4 (4mg / mL) fully reacted and dialyzed overnight with 0.15M PH7.4PBS buffer to obtain the enzyme-linked detection probe Trypsin-Ab 2 ;

[0032] 2) Add 100 μL CA125-coated antibody Ab 1 (24μg / mL) was added to a 96-well ELISA plate, stood overnight at 4°C, washed the 96-well ELISA plate with washing buffer, added 300 μL BSA (10mg / mL) at 37°C for 1 hour, and then washed again with washing buffer 96-well ELISA plate, then add 100 μL CA125 standard antigen and incubate with slow shaking, wash the 96-well ELISA ...

Embodiment example 3

[0034] 1) The newly prepared 0.2mL sodium periodate NalO 4 Solution (30mg / mL) was added to 1mL Trypsin solution (5mg / mL), stirred at room temperature for 20min in the dark, then dialyzed overnight with 1mM pH4.4 acetate buffer, by adding 20μL 0.2M pH9.5 Make the pH of the system reach 9.0 with carbonate buffer, and immediately add 10mg of CA125-labeled antibody Ab 2 , stirred at room temperature for 2 h in the dark, then added 0.1 mL of sodium borohydride NaBH 4 (4mg / mL) fully reacted and dialyzed overnight with 0.15M PH7.4PBS buffer to obtain the enzyme-linked detection probe Trypsin-Ab 2 ;

[0035] 2) Add 100 μL CA125-coated antibody Ab 1 (24μg / mL) was added to a 96-well ELISA plate, stood overnight at 4°C, washed the 96-well ELISA plate with washing buffer, added 300 μL BSA (10mg / mL) at 37°C for 1 hour, and then washed again with washing buffer 96-well ELISA plate, then add 100 μL CA125 standard antigen and incubate with slow shaking, wash the 96-well ELISA plate with w...

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Abstract

The invention relates to a preparation method of an enzyme-linked immunosorbent assay kit for detecting an ovarian cancer tumor marker CA125 based on a trypsin fluorogenic substrate. An enzyme-linked immunosorbent assay technology and a fluorescent detection technology are combined to prepare a detection probe through trypsin and a tumor marker corresponding antibody Ab2; the probe, the tumor marker and another tumor marker antibody Ab1 embedded into the enzyme-linked immunosorbent assay (ELISA) kit can form a structure similar to a sandwich through the action between antigens of the antibodies; finally fluorescence is generated through the action between enzyme and the fluorescent substrate to qualitatively and quantitatively detect the ovarian cancer related tumor marker CA125, and a novel method for detecting protein markers is established. The preparation method has the characteristics that the whole preparation process is simple and suitable for industrial production; the protein markers can be qualitatively and quantitatively detected according to the fluorescent characteristics of the fluorogenic substrate and the action of the fluorogenic substrate with the enzyme, and the specificity is good; the whole detection process is low in cost and very convenient to operate; the novel method for detecting the protein markers is established.

Description

technical field [0001] The invention relates to the technical field of disease detection, and more specifically relates to a method for preparing an enzyme-linked immunoassay kit for detecting ovarian cancer tumor marker CA125 based on a trypsin fluorescent substrate. Background technique [0002] In recent years, with the rapid changes in people's lifestyles and increasing mental pressure, the incidence of tumors in our country has been increasing year by year. Among them, the mortality rate of ovarian cancer ranks first among gynecological tumors. Unlike breast cancer, ovarian cancer lacks obvious symptoms and effective screening in the early stage, and the survival rate is low. Tumor markers of ovarian cancer can be divided into various subtypes according to their histopathological types, among which epithelial-derived tumors are the most common type, and most of the existing tumor markers are also closely related to ovarian epithelial carcinoma, such as CA125 and human e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57449G01N33/57484G01N2800/7028
Inventor 常津武玉东宫晓群
Owner TIANJIN UNIV
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