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Pepsinogen I monoclonal antibody and application thereof

A monoclonal antibody, antigen-antibody technology, applied in the direction of immunoglobulin, anti-enzyme immunoglobulin, instruments, etc., to achieve the effect of high sensitivity and high precision detection

Active Publication Date: 2021-06-22
重庆艾生斯生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide anti-PGI monoclonal antibody, solve the problem of cross-reaction with PGII, and realize high specificity binding with PGI

Method used

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  • Pepsinogen I monoclonal antibody and application thereof
  • Pepsinogen I monoclonal antibody and application thereof
  • Pepsinogen I monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Mouse immunization and antibody detection

[0034] Five 6-8 week-old SPF female BALB / c mice were selected, and Freund's complete adjuvant and PG1 protein at a concentration of 2 mg / ml were mixed and emulsified in equal volumes. The emulsified antigen was immunized with 6-8 week-old SPF grade female BALB / c mice, and each mouse was injected with 40 μg of antigen protein by sole injection or back subcutaneous injection. Two weeks after the completion of the initial immunization, the antigenic protein was mixed and emulsified with Freund's incomplete adjuvant, and 40 μg of the antigenic protein was injected per mouse again by foot injection or back subcutaneous injection. Two weeks later, blood was collected through the tail vein, the supernatant was collected by centrifugation and the serum titer was detected by ELISA. Immunize once every two weeks and test the serum titer. After the second immunization, the serum titer after a million-fold dilution has reache...

Embodiment 2

[0035] Example 2: Production and purification of monoclonal antibodies

[0036] Two groups of 6-8 week BALB / c mice were selected, and 500 μL paraffin oil was injected intraperitoneally to suppress the immune response of the mice. One week after the injection, 0.5 ml of PG1-A hybridoma cells were intraperitoneally injected into a group of mice, and the number of cells was about 1×10 6 Quantity; Another group of mice was intraperitoneally injected with 0.5ml of PG1-B hybridoma cells. Ascites collection began two weeks later. The collected ascites was subjected to ammonium sulfate precipitation and protein A affinity purification to obtain the target antibody.

Embodiment 3

[0037] Example 3: Subtype Identification of Monoclonal Antibody and Gene Sequence Cloning

[0038] The SBA Clonotyping System-HRP kit from SouthernBiothech was used to identify the subtypes of the heavy chain and light chain of the monoclonal antibody according to the instructions. The specific operation is:

[0039] 1. Dilute the capture antibody to 1 μg / mL with coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5), add 100 μL / well to the microtiter plate, and coat overnight at 4°C. The plate was washed 3 times with PBS buffer (plate washing solution) containing 0.05% Tween-20.

[0040] 2. Dilute the culture supernatant of hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the microtiter plate, and incubate at 37° C. for 30 minutes. The corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) were diluted 1:3000 with diluent.

[0041] 3. After washing the plate...

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Abstract

The invention relates to the technical field of biology, in particular to a pepsinogen I monoclonal antibody and application thereof, and discloses a group of pepsinogen I monoclonal antibodies which can be used in a matched mode. The pepsinogen I monoclonal antibodies have high specificity and stability when being combined with PGI, cannot generate cross reaction with pepsinogen II, and are suitable for various methodologies, and high-sensitivity and high-precision detection of PGI is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to pepsinogen I monoclonal antibody and its application. Background technique [0002] Pepsinogen (PG) is the precursor of pepsin, which is divided into pepsinogen I (PGI) and pepsinogen II (PGII) according to the difference in physicochemical properties and immunogenicity, and is mainly secreted by gastric mucosa. Pepsinogen has no digestive enzyme activity, and after being secreted into the gastric cavity, it is activated into pepsinogen with digestive activity under the action of gastric acid. In addition to its main function in the digestive tract, a small amount of pepsinogen will pass through the gastric mucosa and enter the blood circulation. The content of pepsinogen in serum can reflect the level of gastric juice secretion and the pathological changes of gastric mucosa. The content of pepsinogen I in serum is closely related to the function of gastric acid gland cells, and it ...

Claims

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Application Information

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IPC IPC(8): C07K16/40G01N33/577G01N33/573
CPCC07K16/40G01N33/577G01N33/573C07K2317/565C07K2317/33G01N2333/96477
Inventor 易维京蒋会会蔡方琴赵忠颢汤定斌温路新
Owner 重庆艾生斯生物工程有限公司
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