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Anti-prostate-specific-antigen PSA monoclone antibody and its use

A monoclonal antibody and prostate-specific technology, applied in anti-animal/human immunoglobulin, instruments, measuring devices, etc., can solve the problems of high price, insufficient sensitivity of PSA detection, unsuitable for large-scale clinical application, etc., and achieve good results The effect of antibody characteristics and sensitive detection range

Inactive Publication Date: 2007-11-14
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The technical problem to be solved in the present invention is to provide a class of anti-prostate specific antigen PSA monoclonal antibody and its application, to overcome the lack of sensitivity or high price of PSA detection carried out by the existing similar monoclonal antibody, which is not suitable for large-scale clinical application defect

Method used

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  • Anti-prostate-specific-antigen PSA monoclone antibody and its use
  • Anti-prostate-specific-antigen PSA monoclone antibody and its use
  • Anti-prostate-specific-antigen PSA monoclone antibody and its use

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Experimental program
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Effect test

Embodiment 1

[0054] animal immunity

[0055] BALB / c healthy mice homologous to the used myeloma cells were selected, and the age of the mice was 8-12 weeks, male or female. Antigen was human free PSA (Biocheck, INC. CAT#4001). Antigen stock solution concentration: 1.84mg / mL, Balb / c mouse immunization dose: 100μg PSA each time. The injection method is multi-point intramuscular injection. Dilute with PBS or normal saline before use. Immunization program: 0, 3, 6 three times immunization. Three days before the fusion, take 100μg and dilute it with PBS to 0.5mL for intraperitoneal injection for memory stimulation. Three days after the last immunization, splenocytes were isolated for fusion.

[0056] Antibody titers in mouse serum after table 1 is immunized with PSA for the third time

[0057] Detection of antibody titer in mouse serum by ELISA after the third immunization with PSA

Embodiment 2

[0059] Construction of hybridoma cell lines and preparation of monoclonal antibodies

[0060] 1. Culture of myeloma cell lines

[0061] The most important point in selecting a tumor cell line is homology with the B cells to be fused. If spleen cells are to be fused, various myeloma cell lines can be used, and we use SP2 / 0 cell line. The cell line has good growth and fusion efficiency. In addition, the cell line itself does not secrete any immunoglobulin heavy chain or light chain. The highest growth scale for cells is 9×10 5 / mL, the doubling time is usually 10-15h. Fusion cells are selected in the logarithmic growth phase, with good cell shape and activity (activity greater than 95%). Myeloma cell lines should be adapted to culture medium containing 8-azaguanine before fusion, and the cell concentration should be adjusted to 2×10 with fresh medium the day before cell fusion. 5 / mL, the next day is generally the logarithmic growth phase cells.

[0062] 2. Preparation of f...

Embodiment 3

[0089] Applications of Monoclonal Antibodies

[0090] 1. Antigen coating amount saturation curve

[0091] Use the cell supernatant (dilution one time) as the detection sample, the amount of coated antigen varies from 0.125 to 4g / mL, the primary antibody is the cell supernatant of each clone, and the dilution is one time, and the secondary antibody is detected with goat anti-mouse-HRP . It can be seen from the slope of the curve that when the antigen is coated at 2 μg / mL, the antibody in the supernatant begins to be excessive. In the range from 0.125 μg / mL to 1 μg / mL, the slope of the curve is relatively high, indicating that the antigen coating is more sensitive in this range, so we choose the subsequent antigen coating concentration to be 0.5 μg / mL.

[0092] The curve of PSA coating concentration exploration is shown in Fig. 3 .

[0093] 2. Antibody competition ELISA experiment to determine whether the five cell lines are determined by an antigenic determinant. One times ...

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Abstract

This invention relates to three kinds of monoclonal antibody of prostate specific antigen(PSA), and corresponding hybridoma cell strain. The preservation number respectively are: CCTCC-C200620, CCTCC-C200621 and CCTCC-C200622. The detection range of this monoclonal antibody can reach 0 to 2048ng / mL, possess feature of sensitivity, idiosyncratic and low cost. The invention has important application prospects at cytobiology, biomacromolecule detection and medicine clinical diagnosis etc.

Description

Background technique [0001] Prostate specific antigen (PSA) is an antigen related to prostate cancer. It is a glycoprotein substance with a MW of about 34KD, a pH of 6.8-7.5, an isoelectric point of 6.9, and a half-life of 2.2±0.8 days. PSA exists in the prostate endoplasmic reticulum and prostate epithelial cells and secretions. Both normal prostate and diseased prostate tissue contain PSA. It is recognized as a better tumor marker for diagnosing prostate cancer, and its specificity for diagnosing prostate cancer is 82%-97%. Prostate cancer accounts for 10% to 20% of all types of cancer in men, and is the most common cancer in men. The disease progresses slowly and is the main cancer that threatens the lives of men over 50 years old, accounting for the second male mortality rate in Western countries. Epidemiological surveys show that with the improvement of the living standard of Chinese residents, the aggravation of environmental pollution and the change of diet structure,...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/53G01N33/574
Inventor 孙兵张秀琴田林朱静燕季永镛
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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