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ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using monoclonal antibody

A monoclonal antibody, rapid technology, used in measurement devices, instruments, scientific instruments, etc.

Inactive Publication Date: 2013-04-17
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Currently, there is no report on ELISA for the rapid detection of c-Myc using two monoclonal antibodies

Method used

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  • ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using monoclonal antibody
  • ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using monoclonal antibody
  • ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The specific method steps are as follows:

[0045] 1. Preparation and purification of C-myc antigen

[0046] Bacteria solution OD 600nm When the value is 0.4, after being induced by IPTG for 1 hour, the cells are collected by centrifugation at 6500rmp for 10 minutes, suspended, and the cells are broken by an ultrasonic breaker (20% power, broken for 10 minutes), denatured by 2-8M urea for 2 hours, and then refolded, and used by AKATA instrument The protein was purified by nickel ion affinity chromatography, and finally c-Myc protein with a purity of more than 95% was obtained ( figure 1 ).

[0047] 2. Preparation of Monoclonal Antibody

[0048] 2.1 Animal immunity

[0049] The prokaryotic expressed and purified C-myc protein was fully emulsified with an equal volume of complete Freund's adjuvant, and the antigen was injected into the peritoneal cavity of healthy Balb / c mice, 0.4ml / mouse, the amount of antigen was 50μg / mouse; the first immunization 14 Two days later...

Embodiment 2

[0085] The specific method steps are as follows:

[0086] 1. Preparation and purification of C-myc antigen

[0087] Bacteria solution OD 600nm When the value is 0.6, after being induced by IPTG for 1 hour, the cells are collected by centrifugation at 6500rmp for 10 minutes, suspended, and the cells are broken by an ultrasonic breaker (20% power, broken for 10 minutes), denatured by 2-8M urea for 2 hours, and then refolded, and used by AKATA instrument The protein was purified by nickel ion affinity chromatography, and finally c-Myc protein with a purity of more than 95% was obtained ( figure 1 ).

[0088] 2. Preparation of Monoclonal Antibody

[0089] 2.1 Animal immunity

[0090] The prokaryotic expressed and purified C-myc protein was fully emulsified with an equal volume of complete Freund's adjuvant, and the antigen was injected into the abdominal cavity of healthy Balb / c mice, 0.6ml / mouse, and the amount of antigen was 100μg / mouse; the first immunization was 14 Two da...

Embodiment 3

[0121] The specific method steps are as follows:

[0122] 1. Preparation and purification of C-myc antigen

[0123] Bacteria solution OD 600nm When the value is 0.6, after being induced by IPTG for 1 hour, the cells are collected by centrifugation at 6500rmp for 10 minutes, suspended, and the cells are broken by an ultrasonic breaker (20% power, broken for 10 minutes), denatured by 2-8M urea for 2 hours, and then refolded, and used by AKATA instrument The protein was purified by nickel ion affinity chromatography, and finally c-Myc protein with a purity of more than 95% was obtained ( figure 1 ).

[0124] 2. Preparation of Monoclonal Antibody

[0125] 2.1 Animal immunity

[0126] The prokaryotic expressed and purified C-myc protein was fully emulsified with an equal volume of complete Freund's adjuvant, and the antigen was injected into the abdominal cavity of healthy Balb / c mice, 0.6ml / mouse, and the amount of antigen was 100μg / mouse; the first immunization was 14 Two da...

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Abstract

The invention relates to an ELISA (enzyme-linked immunosorbent assay) method for quickly testing C-Myc by using a monoclonal antibody. According to the method, prokaryotic expression C-Myc protein is purified through protein purification technology; immune mouse spleen cells are performed cell fusion with SP2 / 0 myeloma cell, and a clone capable of stably secreting an antibody against C-myc IgG1 and a Cmyc IgM antibody clone are obtained based on ELISA screening and the subtype identification of subclone and monoclonal antibody Ig. A sandwich ELISA method and the two direct ELISA methods for quickly testing c-Myc are built by using an antibody against c-Myc IgG1 and an antibody against c-Myc Ig. With the method, a C-myc protein sample is accurately detected, the detected C-myc protein concentration range is 0.01-1000 pmol / L, and the detection limit is 0.01-0.05 pmol / L; the specificity and the sensitivity of C-Myc detection are greatly improved, the operation is simple, and the repeatability is high; and the method detects the expression level of C-Myc in tumor tissues, and provides experimental data for screening of malignant tumor.

Description

technical field [0001] The invention relates to the field of developing a tumor detection kit, more specifically, relates to an ELISA method for rapidly detecting C-Myc by using a monoclonal antibody. Background technique [0002] Proto-oncogene C-myc is an adjustable gene that makes cells proliferate indefinitely, obtains immortalization function, and promotes cell division. It is highly expressed in many tumors [1] . In cancerous cells, the expression of C-myc protein is abnormally increased, which makes the cells break away from the regulation restriction of normal growth, transforms into a malignant phenotype, and becomes cancerous. It has been reported that the overexpression of C-myc gene is closely related to the occurrence and development of tumors, such as leukemia, lung cancer, liver cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, certain neuroblastosis, retinoblastoma, osteosarcoma , chondrosarcoma, chordoma, liposarcoma, rhabdomyosarcoma, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 李文哲金锦花
Owner DALIAN UNIV
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