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Method for preparing CysC-paired monoclonal antibody

A monoclonal antibody and cystatin technology, applied in the field of protein engineering, can solve the problems of difficult to obtain paired monoclonal antibodies, heavy workload, narrow screening range, etc., and achieve the effect of increasing the probability, reducing the workload, and expanding the screening range

Active Publication Date: 2012-01-18
BEIJING LEADMAN BIOCHEM
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  • Abstract
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AI Technical Summary

Problems solved by technology

This method has a large workload and a relatively narrow screening range. Since CysC is an antigen with a small molecular weight, it is difficult to obtain paired monoclonal antibodies by this method.

Method used

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  • Method for preparing CysC-paired monoclonal antibody
  • Method for preparing CysC-paired monoclonal antibody
  • Method for preparing CysC-paired monoclonal antibody

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Experimental program
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Embodiment Construction

[0023] 1. Experimental materials

[0024] 1) Expression vector: pET28a(+) was purchased from (Novagen); Figure 4 As shown, also refer to the product manual provided by Novagen.

[0025] 2) Expression host bacteria: Escherichia coli BL21(DE3), purchased from Beijing Tiangen Biotechnology Co., Ltd.;

[0026] 3) Yeast powder and peptone: both are products of Oxoid Company;

[0027] 4) The CysC gene was synthesized by Shanghai Xuguan Biotechnology Co., Ltd.; the CysC gene sequence with NcoI and XhoI restriction sites is shown in SEQ ID NO: 1 ( Figure 5 ), italics are the added NcoI and XhoI restriction sites.

[0028] 5) SP 2 / 0 myeloma cells were purchased from the Cell Center of Shanghai Institute of Biochemistry and preserved by the R&D Department of our company;

[0029] 6) Balb / c mice were purchased from the Experimental Animal Center of Chinese Academy of Medical Sciences.

[0030] 7) Fetal bovine serum: purchased from Hangzhou Sijiqing Company;

[0031] 8) DMEM mediu...

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Abstract

The invention relates to the field of protein engineering, in particular to a method for preparing CysC-paired monoclonal antibodies, which comprises the following steps of: screening hybridoma cells having specific binding with CysC after fusing mouse splenic cells and myeloma cells capable of generating CysC antibodies, preparing cells for generating the monoclonal antibodies, finishing first-time cell fusion, further purifying out the monoclonal antibodies, and selecting one monoclonal antibody to be marked with horse radish peroxidase; then implementing the second-time fusion of the mouse splenic cells and the myeloma cells; finishing CysC-antigen coating on an elisa plate, adding a hybridoma-cell cultural supernatant obtained through the second-time cell fusion to elisa-plate holes, arranging a blank control hole, subsequently adding the monoclonal antibody marked with the horse radish peroxidase to the elisa-plate holes, incubating at the temperature of 37 DEG C for 30 minutes, adding a substrate to each hole for color rendering, and measuring a light absorption value under 450nm after stopping reaction through sulfuric acid; and selecting the uninhibited holes as the paired monoclonal antibodies.

Description

technical field [0001] The invention relates to the field of protein engineering, in particular to a method for preparing CysC paired monoclonal antibodies. Background technique [0002] Cystatin C (Cystatin C, CysC) is Cystatin C, with a molecular weight of 13kD. It is a low-molecular-weight protein that can be produced by all nuclear cells in the body with a constant yield. CysC in circulation only passes through the glomerulus filtered and removed. Therefore, CysC is an ideal endogenous marker to reflect changes in glomerular filtration rate (GRF) (Zhang Yaozu. The significance of serum CysC as an indicator of glomerular filtration rate in chronic renal failure. Modern Laboratory Medicine Journal, 2005, 20 (6): 72-73.), the determination of its serum concentration is a new renal function evaluation index that is superior to the traditional measurement of blood urea, uric acid, creatinine and endogenous creatinine clearance. In 2002, the FDA announced 26 brand-new detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/38
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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