Preparation method of liver cancer early detection kit

A technology for early detection and kits, which is applied in the field of preparation of early detection kits for liver cancer, can solve the problems of complex preparatory work for chemical cross-linking, many cross-linking by-products, and reduced enzyme activity, so as to reduce unnecessary labels and Fewer by-products and stable numerical value

Pending Publication Date: 2017-10-20
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If chemical cross-linking technology is used to label phycobiliproteins and enzymes, the activity of the enzyme may decrease after the reaction, and the preparatory work for chemical cross-linking is more complicated, and various cross-linking raw materials need to be purified separately, and there are many cross-linking by-products

Method used

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  • Preparation method of liver cancer early detection kit
  • Preparation method of liver cancer early detection kit
  • Preparation method of liver cancer early detection kit

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preparation example Construction

[0032] Please refer to figure 1 and figure 2 , the preparation method of the liver cancer early detection kit of one embodiment, comprises the following steps:

[0033] S10. Prepare the liver cancer surface antigen solution with PBST solution to form a liver cancer surface antigen solution with a concentration of 0.1-100 ng / mL, and place it at 4-6°C for use.

[0034] Wherein, the liver cancer surface antigen may be carcinoembryonic antigen (AFP) or alpha-fetoprotein (CEA).

[0035] Among them, the PBST solution contains 20mM KH 2 PO 4 , 100mM Na 2 HPO 4 12H 2 O, 1.37M NaCl, 27mM KCl, and Tween 20 with a mass fraction of 0.05% (v / v), the pH is 7.4.

[0036] S20. Prepare the monoclonal antibody solution against liver cancer surface antigen with PBST solution to a concentration of 5-10 μg / mL anti-hepatoma surface antigen monoclonal antibody solution, and store it at -4-6°C for use.

[0037] When the surface antigen of liver cancer is AFP, the monoclonal antibody against ...

Embodiment 1

[0078] The liver cancer surface antigen AFP solution was prepared with PBST solution to make a liver cancer surface antigen AFP solution with a concentration of 0.1 ng / mL, and placed at 4°C for use. Among them, the PBST solution contains 20mM KH 2 PO 4 , 100mM Na 2 HPO 4 12H 2 O, 1.37M NaCl, 27mM KCl, 0.05% (v / v) Tween 20, pH 7.4.

[0079] The anti-AFP monoclonal antibody was prepared with PBST solution to a concentration of 10 μg / mL anti-AFP monoclonal antibody solution, and stored at -4°C for use.

[0080] A 96-well plate was coated with 200 μL of an anti-AFP monoclonal antibody solution at a final concentration of 10 μg / mL at 4° C. for 20 hours, and the solution was discarded.

[0081] The 96-well plate was gently washed 3 times with PBST solution, 6 minutes each time.

[0082] A 96-well plate was blocked overnight at 4° C. using skimmed milk in PBST solution with a mass fraction of 5% (m / v).

[0083] The 96-well plate was gently washed 3 times with PBST solution, 6 ...

Embodiment 2

[0092] Liver cancer surface antigen CEA was prepared with PBST solution to make a liver cancer surface antigen CEA solution with a concentration of 100 ng / mL, and placed at 6° C. for later use. Among them, the PBST solution contains 20mM KH 2 PO 4 , 100mM Na 2 HPO 4 12H 2 O, 1.37M NaCl, 27mM KCl, 0.05% (v / v) Tween 20, pH 7.4.

[0093] The anti-CEA monoclonal antibody was prepared with PBST solution to a concentration of 5 μg / mL anti-CEA monoclonal antibody solution, and stored at -4°C for use.

[0094] A 96-well plate was coated with 250 μL of an anti-CEA monoclonal antibody solution at a final concentration of 5 μg / mL at 4° C. for 20 hours, and the solution was discarded.

[0095] The 96-well plate was gently washed 5 times with PBST solution, 5 minutes each time.

[0096] The non-binding sites of the 96-well plate were blocked overnight at 4° C. with PBST solution containing 5% (m / v) bovine serum albumin.

[0097] The 96-well plate was gently washed 5 times with PBST ...

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Abstract

The invention discloses a preparation method of a liver cancer early detection kit. The preparation method comprises the following steps: coating an ELISA (Enzyme-Linked Immunosorbent Assay) plate with a monoclonal antibody solution of an anti-liver-cancer surface antigen, and flushing with a PBST (Phosphate Buffered Solution-Tween) solution; enclosing a non-binding site of the ELISA plate with a 5 percent by mass PBST solution of skimmed milk or bovine serum albumin, and flushing with the PBST solution; in the ELISA plate, adding a liver cancer surface antigen solution into each hole for incubation, and flushing with the PBST solution; in the ELISA plate, adding a PBST solution of a monoclonal antibody of a biotinylated anti-liver-cancer surface antigen into each hole for incubation, and flushing with the PBST solution; in the ELISA plate, adding a PBST solution of recombinant phycocyanin subunit into each hole for incubation, and flushing with the PBST solution to obtain the liver cancer early detection kit. The preparation method for preparing the liver cancer early detection kit from the recombinant phycocyanin subunit is simple, is easy to operate, and is less in side products; moreover, the obtained liver cancer early detection kit has high stability, high fluorescence property and high sensitivity.

Description

technical field [0001] The invention relates to the technical field of liver cancer detection, in particular to a preparation method of a liver cancer early detection kit. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in my country, ranking second in cancer deaths. About 130,000 people die of liver cancer in my country every year, accounting for more than half of the new cases of liver cancer in the world. Most liver cancer patients are in the middle or late stage when they see a doctor, and miss the best opportunity for treatment. Early diagnosis and early treatment are the most effective ways to prevent liver tumors and reduce mortality. At present, the early diagnosis of liver cancer mainly relies on imaging examination and the determination of tumor markers. The determination of tumor markers has the advantages of high sensitivity, high specificity, and less pain, and more and more attention has been paid to them. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/577G01N33/574
CPCG01N33/533G01N33/57438G01N33/577
Inventor 焦绪栋李文军秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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