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Method for determining leptin

A technology of leptin and cells, applied in the field of leptin active system, can solve problems such as libraries that are not suitable for screening different substances

Inactive Publication Date: 2002-11-06
YEDA RES & DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, as mentioned above, the biological activity of leptin can only be determined using animal experiments, which are cumbersome and therefore unsuitable for screening libraries containing millions of different substances
Although several in vitro assays have been described, they are not leptin-specific

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Mouse Swiss3T3 F442A cells were grown and differentiated on 96-well plates. Leptin (positive control) or a putative leptin-like substance was added to the cultures to a final concentration of 1 μg / ml and culture was continued for 24 hours. Aliquots of leptin- or leptin-like induction medium were then transferred to ELISA plates pre-coated with a mouse Ang-2-specific capture monoclonal antibody. Microtiter plates (Dynatech or Maxisorb, by Nunc) were coated overnight at 4°C with anti-mouse Ang-2 monoclonal antibody (serum-free hybridoma supernatant or ascites immunoglobulin). Plates were washed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked with the same solution for at least 2 hours at 37°C. Then the medium induced by leptin or leptin-like substances was taken out, transferred to an ELISA plate (100 μl / well), and kept at 37° C. for 4 hours. The plate was washed three times with PBS containing Tween 20 (0.05%), then rabbit anti-mouse Ang-2 serum (1:1000...

Embodiment 2

[0028] Mouse Swiss3T3 F442A cells were grown and differentiated on 96-well plates. Leptin (positive control) or a putative leptin-like substance was added to the cultures to a final concentration of 1 μg / ml and culture was continued for 24 hours. Aliquots of leptin- or leptin-like induction medium were then transferred to ELISA plates pre-coated with a mouse Ang-2-specific capture monoclonal antibody. Microtiter plates (Dynatech or Maxisorb, by Nunc) were coated overnight at 4°C with anti-mouse Ang-2 monoclonal antibody (serum-free hybridoma supernatant or ascites immunoglobulin). Plates were washed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked with the same solution for at least 2 hours at 37°C. Then the medium induced by leptin or leptin-like substances was taken out, transferred to an ELISA plate (100 μl / well), and kept at 37° C. for 4 hours. The plate was washed three times with PBS containing Tween 20 (0.05%), then rabbit anti-mouse Ang-2 serum (1:1000...

Embodiment 3

[0032] Mouse Swiss3T3 F442A preadipocytes were stably transfected with a reporter vector consisting of the entire promoter region of mouse Ang-2 followed by the green fluorescent protein (GFP) gene. Cells were grown and differentiated on 96-well plates. Samples of leptin (positive control) or a putative leptin-like substance were added to the culture and the culture continued. The appearance of green fluorescence indicates that the cell culture has been exposed to leptin or a leptin-like substance. Example 4 Determination of Leptin and Leptin-like Substances Using Reporter Carriers

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Abstract

The present invention provides a method for the determination of leptin in a sample, based on the detection of leptin-induced angiopoietin in certain cells or cell lines, such as Swiss 3T3 F442A mouse preadipocytes or human bone marrow stromal cell line hMS2-12 -2 Detection of expression. The detection of leptin is as follows: cells are transfected with a vector containing a reporter gene (such as green fluorescent protein (GFP)) under the control of angiopoietin-2 promoter.

Description

field of invention [0001] The present invention relates to a leptin activity system, more specifically, the present invention relates to in vitro detection of bioactive substances of leptin or leptinoids, and the detection method is suitable for high-efficiency screening of molecular libraries. Background of the invention [0002] Obesity, defined as excess body fat relative to a lean body mass, is associated with important psychiatric and medical disorders, including hypertension, hyperlipidemia, and type II or non-insulin-dependent diabetes mellitus (NIDDM). There are 6-10 million NIDDM patients in the United States, 18% of whom are 65 years old (Kamel, HK, eta1, Clin Geriatr. Med., 1999, 15, 265). Among obese patients with NIDDM, about 45% of men and 70% of women. By losing weight, their diabetes symptoms were substantially improved or eliminated (Harris 1991, DiabetesCare, 14, 639). [0003] Leptin is the product of obesity genes (Zhang, Y., et al.1994, Nature 372, 425...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12N15/09C12QC12Q1/02C12Q1/68G01N33/15G01N33/50G01N33/577G01N33/68G01N33/74
CPCC12Q1/6883G01N33/6893G01N33/74C12Q2600/136C12Q2600/158
Inventor M·鲁宾斯坦B·科恩
Owner YEDA RES & DEV CO LTD
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