US 12,247,085 B2
Hybridoma cell line of secreting cyproheptadine monoclonal antibodies and preparation method thereof
Chuanlai Xu, Wuxi (CN); Hua Kuang, Wuxi (CN); Wei Jiang, Wuxi (CN); Liguang Xu, Wuxi (CN); Wei Ma, Wuxi (CN); Liqiang Liu, Wuxi (CN); Xiaoling Wu, Wuxi (CN); Shanshan Song, Wuxi (CN); and Yongming Hu, Wuxi (CN)
Assigned to JIANGNAN UNIVERSITY, Jiangsu (CN)
Filed by JIANGNAN UNIVERSITY, Wuxi (CN)
Filed on Dec. 27, 2022, as Appl. No. 18/146,783.
Application 18/146,783 is a division of application No. 16/221,873, filed on Dec. 17, 2018, granted, now 11,603,414.
Claims priority of application No. 201810219839.2 (CN), filed on Mar. 16, 2018.
Prior Publication US 2023/0133974 A1, May 4, 2023
Int. Cl. C07K 16/44 (2006.01); C07K 16/06 (2006.01); C07K 16/12 (2006.01); C12N 5/09 (2010.01); G01N 33/12 (2006.01)
CPC C07K 16/44 (2013.01) [C07K 16/065 (2013.01); C07K 16/1289 (2013.01); C12N 5/0693 (2013.01); G01N 33/12 (2013.01)] 1 Claim
 
1. A method for preparing a hybrid tumor cell line that secretes cyproheptadine monoclonal antibodies with Deposit Number CGMCC No. 14697, comprising:
synthesis of cyproheptadine hapten, synthesis of cyproheptadine complete antigen, animal immunization, cell fusion, cell screening and cell line establishment, the hybrid tumor cell line was obtained, and named monoclonal cell line with the Deposit Number CGMCC No. 14697;
wherein the cyproheptadine hapten is as follows:

OG Complex Work Unit Chemistry
the synthesis of cyproheptadine hapten comprises:
1 the compound 1 is dissolved in methanol solution, and ethyl chloroformate is added for stirring to obtain mixture 1, wherein the molecular formula of compound 1 is as follows:

OG Complex Work Unit Chemistry
2 the mixture 1 in 1 mentioned above is cooled, and hydrochloride (HCl) is added in to obtain mixture 2, and then extracted with ethyl acetate, the extracted organic layer is concentrated, and the compound 2 is obtained;
3 an aqueous solution of potassium hydroxide is added drop by drop to the ethanol mixture of the compound 2 and stirred to get mixture 3, wherein the molecular formula of compound 2 is as follows:

OG Complex Work Unit Chemistry
4 the mixture 3 in 3 is concentrated and extracted with ethyl acetate, and the extracted organic layer is dried, and the compound 3 is obtained, wherein the molecular formula of the compound 3 is as follows:

OG Complex Work Unit Chemistry
5 the compound 3 is dissolved in tetrahydrofuran (THF) solution containing 3-chloropropionic acid, add triethylamine and stir under N2 to get mixture 4;
6 the mixture 4 in 5 is concentrated and extracted with ethyl acetate, and the extracted organic layer is dried, and the crude product of cyproheptadine hapten is obtained, after purification by silica gel chromatography (DCM/MeOH=20:1), cyproheptadine hapten was obtained.