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In vitro test tool and method for evaluating biological activity of RANKL target spot compound

A technology of biological activity and detection method, applied in the field of in vitro detection of RANKL target compounds, can solve the problems of difficulty in ensuring the stability and reliability of detection results, poor method accuracy and precision, and long experimental period, and achieve serum Highly Tolerable, Accurate, and Specific Effects

Pending Publication Date: 2018-11-23
JIANGSU T MAB BIOPHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional method has a long experimental cycle, cumbersome operation, poor method accuracy and precision, and it is difficult to guarantee the stability and reliability of the test results.

Method used

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  • In vitro test tool and method for evaluating biological activity of RANKL target spot compound
  • In vitro test tool and method for evaluating biological activity of RANKL target spot compound
  • In vitro test tool and method for evaluating biological activity of RANKL target spot compound

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Resistance Drug Concentration Screening in Example 1 Tool Cell Construction

[0036] HEK293 cells (purchased from ATCC) were plated on 24-well plates, and when the cells grew to a confluence of 90%, the cells were digested, and 50% of the cells were mixed with different concentrations of Hygromycin B (20 μg / ml, 25 μg / ml, 40 μg / ml ml, 75 μg / ml) and G418 (200 μg / ml, 400 μg / ml, 600 μg / ml, 800 μg / ml) were mixed and spread on a 24-well plate. Culture for 1 to 2 weeks, so that the lowest concentration of Hygromycin B and G418 used to make all the cells die is the resistance screening concentration. During this period, when the confluence of the cells in the plate reached about 90%, they were passaged at a ratio of 1:4. The results showed that the concentrations of resistance screening drugs G418 and Hygromycin B were 800 μg / ml and 25 μg / ml, respectively.

Embodiment 2

[0037] The construction of embodiment 2 tool cell construction

[0038] Transfer the blank HEK293 cells to a T75 flask and culture them until the confluence reaches over 90%, collect the cells, add plasmid A (self-constructed) and plasmid B (purchased from Promega) at a certain ratio at the same time, electric shock at 250V for 30ms, and culture after 24 hours The base was replaced with fresh DMEM medium (containing 10% FBS). The cell density reached 90% after 72 hours of transfection, inoculated in a new T25 flask at a ratio of 1:1, and replaced with a medium containing Hygromycin B and G418 for screening, and screened for a period of time to obtain a stable transfected cell line Afterwards, monoclonal cells were picked and expanded using the limiting dilution method. Add RANKL stimulation to the candidate monoclonal cell line, detect the response value, and select the monoclonal cell line that meets the requirements, that is, the clone strain with high background value and ...

Embodiment 3

[0041]Embodiment 3 specificity evaluation experiment

[0042] Using the monoclonal antibody drug against the RANKL target (TK006, refer to CN 103232539B for the specific preparation method), the monoclonal antibody drug against the VEGF target (TK001, refer to CN101935349 B for the specific preparation method), and the drug excipient solution as materials, the above detection The method steps are tested, and the results are as follows figure 1 , when this method is used for anti-VEGF monoclonal antibody and drug excipients buffered saline solution, it is impossible to perform four-parameter fitting on the results, but the detection results of anti-RANKL monoclonal antibody in the figure can draw a complete dose-effect curve, and at the same time meet the Combined coefficient R 2 >0.98, CV% of multiple holes <20%. The experimental results show that the method of the present invention has good specificity.

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Abstract

The invention provides an in vitro test tool and method for evaluating biological activity of RANKL target spot compound. The in vitro test tool and method transfers two plasmids with target genes into HEK293 cell (human embryonic kidney cell), then using the tool cell to evaluate biological activity of monoclonal drug in vitro through drug screening to obtain the tool cell for detection, which compared with traditional test method, the method has advantages of short experimental period, convenient operation, high accuracy and precision, so as to guarantee the stability, reliability and the like of the test results. The test tool cell provided by the invention can be used for biological activity evaluation of therapeutic monoclonal antibodies taking RANKL (receptor activator of NF-kappaB ligand) as target spot, antidrug neutralizing antibodies test in animal trials and clinical trials and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a tool and method for evaluating RANKL target compounds in vitro. Background technique [0002] Bone metastasis of malignant tumors or metastatic bone disease (Metastatic Bone Diseases, MBD) is a common disease of advanced tumors. Bone metastases of malignant tumors often lead to severe bone lesions, which can greatly reduce the quality of life of tumor patients. In severe cases, the condition will deteriorate rapidly or even die, which greatly affects the prolongation of the patient's survival period. Therefore, while controlling the primary disease, active prevention and treatment of SREs (skeletal metastases and bone-related events) also have important clinical significance in the treatment of malignant tumor bone metastases. [0003] Preclinical studies have found that the occurrence of bone metastasis is a complex system involving multiple protein interactio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/577
CPCG01N21/76G01N33/577
Inventor 丁满生方鹏严守升游猛沈红顾迎迎朱海雯岳会亭刘大涛谢宁
Owner JIANGSU T MAB BIOPHARMA
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