Pepsinogen I and pepsinogen II combined detection kit and preparation method and detection method thereof

A pepsinogen and combined detection technology, applied in the field of in vitro diagnostic reagents, can solve the problems of large error in detection results, time-consuming and laborious, complicated operation steps and the like

Pending Publication Date: 2018-08-17
上海铭源数康生物芯片有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, imported reagents are relatively expensive, which causes a certain economic burden for patients; while the main detection method of traditional domestic chemiluminescence method is a single target detection for a single person, and it takes a long time to obtain PGⅠ/PGⅡ test results, wh

Method used

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  • Pepsinogen I and pepsinogen II combined detection kit and preparation method and detection method thereof
  • Pepsinogen I and pepsinogen II combined detection kit and preparation method and detection method thereof
  • Pepsinogen I and pepsinogen II combined detection kit and preparation method and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The preparation steps of the enzyme-linked plate coated with anti-PGⅠ and anti-PGⅡ monoclonal antibodies are as follows:

[0070] 1) Spotting: adjust PGⅠ monoclonal antibody and PGⅡ monoclonal antibody to 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL respectively with spotting buffer. mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL, 1.0mg / mL, preferably 0.4mg / mL; through the GeSim Nano-PlotterTM chip sampling system, absorb PGⅠantibody and PGⅡantibody solutions and add them to the enzyme-linked plate In the same well, PGⅠ and PGⅡ are three replicate points each, and the sample volume of each point is 2nl, 4nl, 6nl, 8nl, 10nl, preferably 8nl, arranged in order according to the spot shape;

[0071] 2) Fixation: Cover the wells of the enzyme-linked plate with a cover film, place at 37°C for 3 hours, overnight at -20°C, and then place at -80°C for 2 hours for antibody fixation;

[0072] 3) Blocking: add blocking solution according to the amount of 200ul per well, block overn...

Embodiment 2

[0074] The preparation steps of the enzyme conjugate labeled with another anti-PGⅠ and another anti-PGⅡ monoclonal antibody are as follows (the PGⅠ antibody and the PGⅡ antibody are labeled separately):

[0075] 1) Weigh 0.4 mg of horseradish peroxidase (HRP) and dissolve in 80 μL of 0.2 M, pH 5.6 acetate buffer to an enzyme concentration of 5 mg / mL;

[0076] 2) Add 22.4 μL of 0.1M sodium periodate, at this time the solution changes from original brown to dark green, and react in the dark at 4°C for 25 minutes;

[0077] 3) Add 16 μL of 2.5% ethylene glycol solution, vortex and mix well, and stop the reaction at 4°C in the dark for 1 hour;

[0078] 4) Take the corresponding antibody, add the antibody solution according to the mass ratio of HRP enzyme and antibody at 1:1, 2:1, 3:1, preferably 2:1, buffer with 0.2M carbonate pH9.5 Adjust the pH value of the solution to 9.0-9.5, and then dialyze through 0.01M carbonate buffer at 4°C in the dark overnight;

[0079] 5) Add 24 μL o...

Embodiment 3

[0086] The preparation steps of PGⅠ and PGⅡ calibrator dilutions are as follows:

[0087] 1) Weigh 8 g of disodium hydrogen phosphate, 0.27 g of sodium dihydrogen phosphate, 8 g of sodium chloride, and 0.2 g of potassium chloride in a 1L container, add an appropriate amount of purified water and stir to dissolve completely;

[0088] 2) Use a pipette to pipette 1mL ProClin 300 into a beaker of 10mL purified water, dissolve it completely and pour it into the above 1L container, add purified water to 900mL and stir thoroughly;

[0089] 3) Adjust the pH meter to measure the pH value of the solution, and adjust the pH value to be controlled at 7.2-7.4;

[0090] 4) Weigh 30g of bovine serum albumin and 1g of protein protectant GH, add them to the above 1L container, stir evenly, and dissolve them fully;

[0091] 5) Dilute to 1L, filter with a 0.2μm filter, label and store under a sterile environment at 2-8°C after filtration.

[0092] Table 2 Calibrator diluent formula

[0093] ...

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Abstract

The invention discloses a pepsinogen I and pepsinogen II combined detection kit and a preparation method and detection method thereof. On the basis of enzyme immunoassay, a high sensitivity chemiluminescence detection technology is adopted, PG I and PG II can be simultaneously measured in one circulation test; compared with a method that measures PG I and PG II individually in sequence, the PG I/PG II detection time is shortened in clinic; furthermore, universal reagents are used during whole detection process, the detection cost is largely reduced; a Hamliton automatic detection and analysisinstrument is used, automation (from sample loading to result analysis) is realized; the interference of manual operation is avoided maximally; the time and error of operation such as sample loading,and the like are both reduced; the repeatability is strong, the detection becomes faster, and the results are more stable and reliable.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnostic reagents, in particular to a combined detection method for pepsinogen I and pepsinogen II and a kit thereof, in particular to a preparation and measurement method of the kit. Background technique [0002] Gastric cancer is one of the most common malignant tumors, and the death rate of gastric cancer ranks second in the mortality rate of common malignant tumors worldwide. China is a high-incidence area of ​​gastric cancer, and its morbidity and mortality rank first among all kinds of malignant tumors. Early detection, early diagnosis, and early treatment are one of the key measures to reduce the mortality rate of gastric cancer. At present, the diagnosis of gastric cancer and other gastric diseases still relies on gastroscopy and histopathology. However, gastroscopy is painful and often not accepted by patients, which easily leads to delays in diagnosis and treatment. In recent years,...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/577G01N33/558
CPCG01N33/535G01N33/558G01N33/577
Inventor 李亨芬季海鹏施晓燕唐博柳飞舟
Owner 上海铭源数康生物芯片有限公司
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