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Antihuman SCGN monoclone antibody, preparation, application and hybrid tumor cell strain

A monoclonal antibody and recombinant protein technology, applied in the direction of anti-animal/human immunoglobulin, anti-hormone immunoglobulin, biochemical equipment and methods, etc., can solve diagnostic difficulties, small cell lung cancer does not express neuroendocrine markers, etc. problem, to achieve the effect of significant application prospects

Active Publication Date: 2008-09-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to factors such as the number of biopsy tissues and the extrusion of materials, it is difficult to diagnose, and immunological markers are often used
However, as mentioned earlier, at least 10% of small cell lung cancers do not express various neuroendocrine markers, which makes diagnosis difficult and even leads to unnecessary surgical treatment

Method used

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  • Antihuman SCGN monoclone antibody, preparation, application and hybrid tumor cell strain
  • Antihuman SCGN monoclone antibody, preparation, application and hybrid tumor cell strain
  • Antihuman SCGN monoclone antibody, preparation, application and hybrid tumor cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Expression and purification of GST-SCGN fusion protein

[0032] 1. Induced expression of GST-SCGN fusion protein

[0033] A. Pick the pGEX-4T-1 / SCGN plasmid (constructed by Shanghai Yuhua Life Science and Technology Development Co., Ltd.) containing the nucleotide sequence shown in SEQ ID No.3 to the LB culture medium (containing Amp50 μg / ml) overnight at 37°C.

[0034] B. Dilute the overnight bacteria at a ratio of 1:100, and culture with shaking at 37°C until the OD600 of the bacterial solution reaches 0.4-1.0.

[0035] C. Add IPTG inducer to a final concentration of 1 mM, and induce for 4 hours at 30°C.

[0036] D. Centrifuge at 4500rpm for 5 minutes, collect the cells and suspend them in pre-cooled PBS (Phosphate Buffer Solution) phosphate buffer solution, add phenylmethylsulfonamide fluoride (PMSF) to a final concentration of 2 mmol / L.

[0037] E. Sonicate the bacterial cells in an ice bath (5 seconds each time, 5 seconds in between, 30 minutes in...

Embodiment 2

[0060] Embodiment 2: the preparation of antiserum

[0061] Use 50 μg of purified SCGN protein and an equal volume of complete Freund's adjuvant to fully mix and emulsify it, and the formation of water-in-oil is a sign of successful mixing. Female BALB / c mice aged 6-8 weeks were injected subcutaneously at multiple points on the back for primary immunization. Two weeks later, BALB / c mice were boosted with an equal volume of incomplete Freund's adjuvant and 50 mg of SCGN protein emulsified, and boosted every two weeks thereafter. After boosting immunization for 3 times, blood was collected from the eyes of the mice and the serum was separated, and the titer was determined by indirect ELISA method. When it reached 10 -4 Then ready to blend. Immunization was boosted once three days before fusion, and immunized mice were obtained.

Embodiment 3

[0062] Embodiment 3: Preparation of monoclonal antibody

[0063] 1. Feeder cell plating

[0064] A 16-week-old BALB / c immunized mouse was killed by pulling its neck, and then disinfected by soaking in 75% alcohol. The peritoneal cavity was opened, feeder cells were taken, plated, cultured in a 37°C incubator, and reserved for the next day.

[0065] 2. Fusion of immunized splenocytes with mouse myeloma cells

[0066] The spleen of the immunized BALB / c mouse was taken under aseptic conditions, the capsule was peeled off to free the cells, and the filtered spleen cells were collected by passing through a 100-mesh steel mesh. Mix SP2 / 0 cells in a good growth state and in the logarithmic growth phase with splenocytes at a ratio of 1:10, fuse with 50% PEG as the mediator, resuspend the cells with HAT medium after centrifugation, and use the containing Spread the HAT culture medium of the cells on a 96-well plate, 2 drops per well. Placed at 37 degrees, 5% CO 2 cultured in an in...

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Abstract

The present invention provides an anti-human secretagogin (SCGN) monoclonal antibody, a preparation method and applications thereof as well as a hybridoma cell line producing the monoclonal antibody. The monoclonal antibody has specificity in respect to the 204th to 1034th nucleotide-encoded polypeptides of the nucleotide sequence shown as SEQ ID No.1 and is secreted by cell strains with an accession number as CCTCC No: C200729. On the basis of the present invention, a sensitive, reliable peripheral blood test method, which can test neuroendocrine tumors, particularly the peripheral blood of small cell lung cancer, can be established, providing a theoretic foundation for assessing the anti-human secretagogin monoclonal antibody in terms of diagnosis, prognosis and clinic treatment effect test.

Description

(1) Technical field [0001] The invention relates to a monoclonal antibody against human secretagogin (SCGN for short), its preparation and application, and a hybridoma cell strain producing the monoclonal antibody. (2) Background technology [0002] Malignant tumors are one of the most common diseases that seriously endanger the health of the people in our country. About 1.3 million people die of malignant tumors every year, which is the second leading cause of death in our country. Due to the limitations of the understanding of tumor occurrence and development, there are still few ideal tumor markers in clinical practice. Secretagogue is a down-regulated protein in colorectal cancer that we screened for the first time by two-dimensional gel electrophoresis, and was verified by Western blotting and immunohistochemical feedback. Secretagogue is a new gene screened from the cDNA library of human pancreatic β cells by Wagner et al. in 2000 by using immune screening technology....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26C12N5/18G01N33/577
Inventor 来茂德李风英徐恩萍马裕
Owner ZHEJIANG UNIV
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