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Monoclonal antibody for resisting GPC3

A monoclonal antibody, cgmccno.3232 technology, applied in the field of biomedicine, can solve the problems of limited specificity and sensitivity, few types of hepatocyte-specific antibodies, inconvenient pathological diagnosis and differential diagnosis of liver cancer, etc. Effect

Inactive Publication Date: 2010-01-27
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few types of liver cell-specific antibodies to choose from in immunohistochemical experiments
At present, the diagnostic markers of hepatocellular carcinoma widely used in clinical pathology are mainly Hep Par-1, CD34, pCEA, AFP, etc., but their specificity and sensitivity have certain limitations, which brings great difficulties to the pathological diagnosis and differential diagnosis of liver cancer. Great inconvenience [Wee A.. Appl Immunohistochem Mol Morphol. 2006; 14: 266-272]

Method used

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  • Monoclonal antibody for resisting GPC3
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  • Monoclonal antibody for resisting GPC3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Expression of Human GPC3 Protein Fragment

[0052] 1.1 Amplification of GPC3-N-terminal gene fragment

[0053] A 981bp GPC3-N-terminal gene fragment was cloned from human placenta cDNA by PCR method as an amplification template.

[0054] Design the following PCR primers:

[0055] Primer 1:

[0056] 5’-CGGC GAA TTC AA GCC ACC TGT CAC CAA GTC-3’

[0057] Primer 2:

[0058] 5’-CGC CTC GAG TCA AAA TCT ATA TTG GCG TTG-3’

[0059] PCR reaction system:

[0060] KOD enzyme, 1 μl; KOD buffer night, 5 μl; 25mmol / L MgCl 2 , 3 μl; 10 μmol / L P1, 1 μl; 10 μmol / L P2, 1 μl; dNTP, 5 μl; template, 0.5 μl; ddH 2 O, 34.5 μl; a total of 50 μl.

[0061] Pre-denaturation at 94°C for 4 minutes; then denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, chain extension at 68°C for 60 sec, 30 cycles.

[0062] The resulting PCR product was purified with a gel extraction kit (Qiagen Gel Extraction Kit). With reference to the method described in "Molecular Cloning" J. Sambrook,...

Embodiment 2

[0071] Preparation and purification of Z1C15 monoclonal antibody

[0072] 2.1 Immunization of mice with GPC3 fusion protein

[0073] Mix the GPC3 protein purified in Example 1 with complete Fred's adjuvant (CFA) and immunize 5-6 week-old female Bab / c mice (Experimental Animal Center of Second Military Medical University), intradermal multipoint injection, 100 μg / Only. After 4 weeks, the first booster immunization was injected intraperitoneally, 100 μg per mouse. After 6 weeks, the second booster immunization was injected intraperitoneally, 100 μg per mouse. At the 8th week of immunization, the tail blood of the immunized mice was taken to measure the titer, and the GPC3 antigen was applied to coat with 3 μg / ml, and 10% FCS was blocked. color, using a microplate reader (Bio-RAD 550) at OD 492 down reading.

[0074] 2.2 Establishment and screening of hybridoma cell lines secreting anti-GPC3 protein antibody

[0075] Mouse myeloma SP2 / 0 cells were prepared at the same ...

Embodiment 3

[0083] Identification of Z1C15 mAb

[0084] 3.1 In vitro culture of Z1C15 cells

[0085] Use the Z1C15 hybridoma cell CGMCC No.3232 prepared in Example 2 to carry out in vitro cell culture (DMEM, 10% FBS medium, 37 ° C, 5% CO 2 cultured in an incubator) to obtain a large amount of hybridoma cell supernatant.

[0086] 3.2 Type identification of monoclonal antibodies

[0087] Mouse Monoclonal Antibody Ig Class and Subclass Detection Kit (HyCult biotechnology bv MouseMonoclonal Antibody Isotyping Kit, P / N: HL2010) was used to identify Ig class and subclass. figure 2 . Studies have shown that the monoclonal antibody heavy chain is IgG2b type, and the light chain is κ chain.

[0088] 3.3 Affinity determination of monoclonal antibody to GPC3 protein

[0089] To identify whether the monoclonal antibody can recognize the exogenous GPC3 protein. With reference to the method described in "Molecular Cloning" J. Sambrook, D.W. Russell (U.S.), translated by Huang Peitang, etc., ...

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Abstract

The invention discloses a monoclonal antibody of GPC3, which is generated by the secretion of a human GPC3 resistant monoclonal antibody hybrid tumor cell strain with the preservation number of CGMCC No.3232. The monoclonal antibody can be used for an immunohistochemical test of hepatocellular carcinoma.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a monoclonal antibody against glypican protein 3 (GPC3) and a kit for immunohistochemical detection of hepatocellular carcinoma. Background technique [0002] Primary liver cancer (HCC) is one of the most common malignant tumors, and its incidence is on the rise worldwide. In recent years, there are 500,000 to 600,000 new cases of liver cancer in the world every year, and about 50% of them occur in my country. Due to its insidious onset, atypical clinical symptoms, and difficulty in early diagnosis, most liver cancers are found in the middle and late stages, not only missing the best period of treatment, but also due to the lack of specific treatment methods and high recurrence, the prognosis is poor . Currently, liver cancer ranks second in the mortality rate of malignant tumors in my country. [0003] At present, image diagnosis, pathologic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12P21/08G01N33/577
Inventor 王红阳周赟谈治雄
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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