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Antibody for resisting FGF21 carboxyl terminal and application thereof

A FGF21, carboxy-terminal technology, applied in the fields of application, anti-animal/human immunoglobulin, genetic engineering, etc., to achieve the effect of clear antigen binding sites

Pending Publication Date: 2022-07-01
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the process of biomacromolecule drug development, it is challenging to establish a biological sample analysis method that can distinguish intact protein / peptide molecules from their degraded metabolites

Method used

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  • Antibody for resisting FGF21 carboxyl terminal and application thereof
  • Antibody for resisting FGF21 carboxyl terminal and application thereof
  • Antibody for resisting FGF21 carboxyl terminal and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Animal immunization

[0042] Four polypeptides were designed and synthesized, three of which contained three amino acids (YAS) at the carboxyl terminus of FGF21, and one did not contain YAS. The sequences are shown in Table 1.

[0043] Table 1 Synthetic polypeptide sequences

[0044]

[0045] The polypeptide A for immunization was coupled separately for animal immunization.

[0046] After three immunizations, blood was collected to measure serum titers. The experimental groups of immunized animals are shown in Table 2.

[0047] Table 2 Information on immunized animals

[0048]

[0049]

[0050] Using the indirect enzyme-linked immunoassay method (indirect ELISA), the animal serum with high titer for immunization polypeptide A and low titer for screening polypeptide B was screened. The specific steps include: coating the ELISA plate with polypeptide A for immunization, polypeptide B for screening (SinoA8627), protein A (SEQ ID NO: 22) and protein B...

Embodiment 2

[0059] Example 2 Fusion hybridoma cells and screening

[0060] According to the results in Table 3-5, the mouse numbered SBI180064-2#C was selected to be injected with the polypeptide SinoA8624 for booster immunization; 3 days later, the spleen was taken for fusion, and the fused cells were suspended in HAT medium, according to the ratio of 6 × 10 per well. 4 Each cell was seeded in a 96-well cell culture plate, that is, a total of 30 96-well cell culture plates were seeded. On the 6th day and the 8th day after fusion, the medium in the culture plate was discarded, fresh HAT medium was added, and the hybridoma cell culture supernatant was taken on the 10th day after fusion for primary clone screening (the results are shown in Table 6). ).

[0061] Table 6 ELISA test results of primary clone screening for the first time

[0062]

[0063] The positive cells obtained by screening were counted, and then seeded into a 96-well cell culture plate at 0.75 cells / well, and 0.5 cell...

Embodiment 3

[0068] Example 3 Monoclonal Antibody and Miniculture and Purification

[0069] Take the six hybridoma cells listed in Table 8, transfer 1 mL of hybridoma cells into 100 mL culture flasks, and periodically add a certain amount of culture medium for cell expansion, and culture for 10 days. The supernatant was obtained by centrifugal filtration of the culture medium, and purified by Protein A affinity chromatography. The specific steps include:

[0070] After collecting the culture feed liquid and centrifuging at 6000rpm for 20min, filter it with a filter, and take the supernatant;

[0071] a. Pretreatment of chromatography column: The Protein A affinity chromatography column is rinsed with ultrapure water, and then equilibrated with equilibration buffer;

[0072] b. Sample loading and equilibration: The feed liquid is loaded onto the Protein A affinity chromatography column. After the sample is loaded, rinse with equilibration buffer until the baseline is stable;

[0073] c. E...

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Abstract

The invention relates to an anti-FGF21 carboxyl terminal antibody and application thereof, the antigen binding site of the antibody is clear, and the antibody can specifically bind to the complete carboxyl terminal of FGF21; when the FGF21 lacks three amino acids (YAS) at the carboxyl terminal, the antibody is not combined with the FGF21 or the combining capacity is extremely low; the antibody can specifically bind to FGF21 or a fusion protein comprising FGF21 only if the carboxyl terminal is intact (comprising YAS). On the basis, the invention provides a biological sample analysis method capable of distinguishing complete molecules at the carboxyl terminal of the FGF21 from metabolites with three or more amino acids lost at the carboxyl terminal.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody against the carboxyl terminus of FGF21 and its application. Background technique [0002] Fibroblast growth factor 21 (FGF21) consists of 181 amino acids, including a beta clover-like core domain and random amino- and carboxy-terminal structures. Administration of recombinant FGF21 reduced plasma glucose and insulin levels, decreased liver and circulating triglyceride and cholesterol levels, and improved insulin sensitivity, energy expenditure, hepatic steatosis, and obesity in a series of animal models of insulin resistance (Xie T, Leung P S. Fibroblast growth factor21: a regulator of metabolic disease and health span[J]. American Journal of Physiology-Endocrinology and Metabolism, 2017, 313(3):E292-E302.). FGF21 has emerged as the most promising therapeutic agent for the treatment of human type 2 diabetes and related metabolic syndrome. [0003] It has been reported t...

Claims

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Application Information

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IPC IPC(8): C07K16/22C12N15/13G01N33/68G01N33/577
CPCC07K16/22G01N33/6872G01N33/577C07K2317/565C07K2317/56G01N2333/50
Inventor 刘亮林树珊李静王茜凌伊
Owner SUNSHINE LAKE PHARM CO LTD
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