Anti-progesterone monoclonal antibody and hybridoma cell generating monoclonal antibody and application thereof

A technology of monoclonal antibody and hybridoma cells, which is applied in the direction of anti-animal/human immunoglobulin, microorganisms, biological tests, etc., can solve the problems of cumbersome steps, and achieve the effect of high specificity and affinity

Inactive Publication Date: 2013-03-27
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For example, Chinese patent CN101066998 discloses a method for preparing medroxyprogesterone acetate-specific antibodies. The method is to prepare a hapten first, then couple the obtained hapten to a protein to prepare a complete antigen, and then immunize animals to obtain Specific antibodies, but this method has the disadvantage of cumbersome steps

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  • Anti-progesterone monoclonal antibody and hybridoma cell generating monoclonal antibody and application thereof
  • Anti-progesterone monoclonal antibody and hybridoma cell generating monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Antigen immunization and cell fusion screening

[0047] 1.1 Materials

[0048]1.1.1 Reagents P4-11α-hemisuccinate-BSA and P-3-CMO-BSA were purchased from Fitzgerald Company; 6-week-old, female, BALB / c mice were purchased from the Experimental Animal Center of Peking Union Medical College. SP2 / 0 myeloma cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0049] 1.1.2 HisTrap HP and HiTrap Protein G columns were purchased from GE; Freund's adjuvant, 50% polyethylene glycol (PEG) 1450 solution, HAT, HT, and horseradish peroxidase (HRP)-labeled goat anti-mouse IgG , Tetramethylbenzidine (TMB), and progesterone (Progesterone) with a purity >90% are all produced by Sigma; DMEM medium is produced by Gibco; newborn bovine serum was purchased from Hangzhou Sijiqing Bioengineering Co., Ltd.

[0050] 1.2 Method

[0051] 1.2.1 Animal immunity

[0052] P4-11α-hemisuccinate-BSA was used as the immune antigen, and 6-week-old fe...

Embodiment 2

[0060] Antibody purification and identification

[0061] 2.1 Antibody purification

[0062] BALB / c mice sensitized by injection of paraffin oil (0.5ml / mouse) 1 week in advance were inoculated intraperitoneally with 10 hybridoma cells 6 After 7-10 days, the ascites was collected and centrifuged to obtain the supernatant. The ascites supernatant was diluted 1:10 in PBS, and loaded on a HiTrap Protein G affinity chromatography column equilibrated with PBS at 0.5ml / min. Wash with PBS, then elute with glycine buffer (pH2.9), and identify the purity by SDS-PAGE electrophoresis.

[0063] 2.2 Identification of antibodies

[0064] Competitive ELISA detection scheme: Dilute the antigen to 200ng / ml with carbonate buffer solution with a concentration of 0.01mol / L and pH9.6, coat the microtiter plate, overnight at 4°C; wash the plate 3 times with PBS / T20, 3min each time; block with PBS / T20 containing 5% calf serum, incubate at 37°C for 1h, wash the plate; add 50ul of human serum with 0...

Embodiment 3

[0066] Screening of hybridoma cell lines

[0067] 3.1 Antibody screening

[0068] Whether the obtained monoclonal antibody can be applied to the development and production of the kit needs to prepare ascites from 23 hybridoma cell lines that stably secrete anti-progesterone monoclonal antibody, and purify the antibody. After the purified antibody was obtained, the antibody titer was detected by ELISA, and the titer of the 23 strains of antibodies was between 1:6W-1:20W;

[0069] 3.2 Screening of hybridoma cell lines

[0070] 23 strains of antibodies were detected by competitive ELISA method, among which 5 strains had a reaction gradient, but only one strain (PD1) was able to reach the minimum detection limit of not more than 0.5ng / ml (results in Table 1), the concentration of antibodies secreted by the PD1 strain and The relation of OD value is shown in the appendix figure 1 (wherein, the ordinate is the antibody concentration ng / ml, and the abscissa is the OD value), from ...

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Abstract

The invention relates to an anti-progesterone monoclonal antibody with high specificity and affinity, a hybridoma cell generating the monoclonal antibody and application thereof, and particularly application in detecting the progestogen level of a human body. According to the invention, a progesterone complete antigen immune animal is adopted, cell fusion is performed after an ideal titer is achieved, and then the cell single strain with higher specificity is screened and preserved; and the strain single strain can continuously secrete a monoclonal antibody to facilitate the detection of progesterone, thereby laying a foundation for the development of a kit for detecting progesterone.

Description

technical field [0001] The invention relates to an anti-progesterone monoclonal antibody, a hybridoma cell producing the monoclonal antibody and its application, especially the application in detecting human progesterone level. Background technique [0002] Progesterone is a natural progesterone secreted by the corpus luteum of the ovary. It has a significant morphological effect on the endometrium stimulated by estrogen in the body. It is necessary to maintain pregnancy. It is clinically used for threatened abortion and habitual pregnancy. Reactive diagnosis of amenorrhea or amenorrhea causes such as miscarriage, etc. Progesterone is an important natural hormone secreted by women. This hormone plays a very important role in women. It can maintain the secretion of progesterone and help women to have a smooth pregnancy. It can also cooperate with estrogen. For other aspects of pregnancy Play a role. [0003] The clinical application of serum / plasma progesterone detection ma...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/26G01N33/74G01N33/577
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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