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Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof

A technology of pepsinogen and kits, applied in chemiluminescence/bioluminescence, analysis by making materials react chemically, measurement devices, etc., can solve the problems of long test time, environmental pollution, ECLIA and dCLIA, and meet the requirements of Clinical requirements, good measurement repeatability, and the effect of shortening operation time

Pending Publication Date: 2021-07-13
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ELISA is time-consuming and has poor reproducibility, and cannot be used for high-throughput detection. RIA detection methods are radioactive and are likely to cause environmental pollution. ECLIA and dCLIA are high in instrument and reagent costs, making them unsuitable for use in areas with limited resources. massive health check
[0005] However, the existing PGI detection kits require a long test time, and most of them are semi-automatic tests, which are easy to cause sample contamination, cannot be continuously added to the test, and the storage time is greatly affected by environmental conditions, and the stability and sensitivity of long-term storage are poor.

Method used

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  • Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof
  • Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof
  • Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] A pepsinogen I assay kit, said kit comprising (1) a solution labeled with a magnetic microsphere conjugate of a PGI monoclonal antibody; (2) a solution labeled with an enzyme-labeled conjugate of a PGI antibody; (3 ) substrate solution; (4) PGI calibrator.

[0050] The magnetic microsphere conjugate marked with PGI monoclonal antibody is a conjugate of magnetic particle and PGI antibody; the diameter of the magnetic particle is 1000-2000 nanometers; In the solution of the substance, the concentration of the magnetic microsphere conjugate labeled with the PGI monoclonal antibody was 4 μg / mL.

[0051] In the solution of the enzyme-labeled conjugate labeled with the PGI antibody, the concentration of the enzyme-labeled conjugate labeled with the PGI antibody is 0.5 μg / mL.

[0052] The substrate solution is AMPDD substrate solution; the manufacturer of the substrate solution is Wanfu Biotechnology Co., Ltd.

[0053] The solution of the magnetic microsphere conjugate label...

Embodiment 2

[0065] A preparation method based on PGI kit, comprising the following:

[0066] Preparation of a solution of magnetic microsphere conjugates labeled with PGI monoclonal antibody: Wash 1 mL of Tosyl magnetic particles 3 times with 0.1 mol / L borate buffer solution with a pH of 9.5 for activation; the activated magnetic particles Suspend in 550 μL of 3 mol / L ammonium sulfate buffer with a pH of 9.5; then add 200 μL of 1 mg / mL PGI monoclonal antibody solution to the ammonium sulfate binding buffer; place the test tube in a 37°C incubator and incubate for 18 hours with rotation After the reaction, put the test tube on the magnetic stand to separate the supernatant, then add 100 μL of 1wt% BSA solution to the supernatant, and continue to rotate and incubate at 37°C for 6 hours; after the reaction, use 0.15mol / L NaCl, 0.05% Tween20, 0.025mol / L Tris-HCl buffer solution with a pH of 7.2 for 5 washes; finally, wash with pH 7.5 containing 0.15mol / L NaCl, 0.5% BSA, 0.08% Tween-20, 0.05 ...

Embodiment 3

[0070] The performance evaluation of embodiment 3 kit of the present invention

[0071] 1. Standard curve

[0072] Dilute the PGI antigen with the standard diluent to prepare different concentrations of calibrator solutions S0-S6, the concentrations are 0, 3, 6, 12, 25, 50, and 100 ng / mL, and store them at -20°C for later use. Then use the kit provided in the embodiment of the present invention to detect the calibrator, and read the luminescence intensity value corresponding to each calibrator. With the concentration as the abscissa and the luminous intensity as the ordinate, a standard curve is obtained by fitting, and the results are as follows figure 2 .

[0073] 2. Minimum detection limit

[0074] Use the zero-concentration calibrator dilution as a sample for detection, repeat the measurement 20 times, and obtain the optical signal value of the 20 measurement results, calculate the average value (M) and standard deviation (SD), and obtain M+2SD, according to zero Conce...

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Abstract

The invention discloses a pepsinogen I enzymatic chemiluminescence detection kit and a preparation method and application thereof. The kit comprises a solution of a magnetic microsphere conjugate marked with a PGI monoclonal antibody, a solution of an enzyme-labeled conjugate marked with a PGI antibody and a substrate solution. According to the kit, a pepsinogen antibody is connected to the surface of a magnetic bead microsphere to serve as a solid-phase reagent, pepsinogen in a sample is captured, and an enzyme-labeled anti-pepsinogen monoclonal antibody reagent is added, so that an enzyme-labeled antibody sandwich immune complex is formed. The kit has the advantages that a high-sensitivity chemiluminescence technology and a high-stability magnetic particle technology are combined together, and the prepared kit is high in sensitivity and good in stability and can meet clinical requirements; meanwhile, the whole reaction detection process can be matched with a full-automatic detection analysis instrument, continuous sample adding is achieved, the operation time is shortened, and the kit can be used for large-scale community health examination and has a certain application prospect.

Description

technical field [0001] The invention belongs to the technical field of diagnostic kits, and in particular relates to a pepsinogen I enzymatic chemiluminescent detection kit and a preparation method and application thereof. Background technique [0002] In recent years, the incidence of gastric cancer has been on the rise, which is mainly attributed to changes in people's lifestyles and diets. 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. Meanwhile, gastric cancer cannot be diagnosed until it reaches an advanced stage in most cases. [0003] Pepsinogen (PG) is the inactive precursor of pepsin (proteolytic enzyme) in gastric juice. It is a protein polypeptide chain composed of 375 amino acids, with an average relative molecular mass of 42000. There are 7 groups of pepsin in human g...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/543
CPCG01N21/76G01N33/54326
Inventor 何小维柳方方王羽
Owner SOUTH CHINA UNIV OF TECH