US 12,441,764 B2
Phosphorylated antigen and antibody at SER23 site of PGAM1 protein as well as preparation methods and applications
Congyi Wang, Wuhan (CN); Chunliang Yang, Wuhan (CN); Shanjie Rong, Wuhan (CN); Fei Sun, Wuhan (CN); Jiahui Luo, Wuhan (CN); Shu Zhang, Wuhan (CN); and Qilin Yu, Wuhan (CN)
Assigned to Tongji Hospital Affiliated with Tongji Medical College of Huazhong University of Science and Technology, Wuhan (CN)
Filed by Tongji Hospital Affiliated with Tongji Medical College of Huazhong University of Science and Technology, Wuhan (CN)
Filed on Dec. 16, 2024, as Appl. No. 18/982,853.
Claims priority of application No. 202410226783.9 (CN), filed on Feb. 29, 2024.
Prior Publication US 2025/0277000 A1, Sep. 4, 2025
Int. Cl. A61K 39/395 (2006.01); C07K 7/06 (2006.01); C07K 16/40 (2006.01)
CPC C07K 7/06 (2013.01) [A61K 39/39525 (2013.01); A61K 39/3955 (2013.01); C07K 16/40 (2013.01)] 3 Claims
 
1. A method for preparing an antibody directed to a phosphorylated polypeptide, the method comprising:
immunizing an animal model to an antiserum titer of at least 1:32000 by using a phosphorylated antigen to obtain an antiserum sample; wherein the phosphorylated antigen is a phosphorylated polypeptide, and an amino acid sequence of the phosphorylated polypeptide is as shown in SEQ ID NO.3, wherein a phosphorylation site of the phosphorylated polypeptide is serine; and the phosphorylation site of the phosphorylated polypeptide is 23rd site of a phosphoglycerate mutase 1 (PGAM1) protein;
synthesizing phosphorylated polypeptide: NRFS(p)GWY as shown in SEQ ID NO. 3 and non-phosphorylated polypeptide: NRFSGWY as shown in SEQ ID NO. 1 respectively according to the amino acid sequence of the PGAM1 protein as shown in SEQ ID NO.2, and then respectively coupling the phosphorylated polypeptide and the non-phosphorylated polypeptide to a carrier protein to obtain a phosphorylated antigen and a non-phosphorylated antigen; and
purifying the antiserum sample by affinity chromatography using the phosphorylated antigen and the non-phosphorylated antigen to obtain the antibody.