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A monoclonal antibody for serological diagnosis of liver cancer and its use

A monoclonal antibody, liver cancer technology, applied in the field of oncology and medicine, can solve the problems of missed diagnosis, no detection and or treatment of liver cancer, large differences in antibody affinity and specificity, etc.

Active Publication Date: 2011-12-14
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recent studies have reported that the positive rate of using AFP to diagnose HCC is only 50%, and the sensitivity is significantly lower in small liver cancers with a diameter <3cm, and the positive rate is less than 40%, which is likely to cause missed diagnosis; secondly, in some benign liver diseases, reproductive abnormalities It can also be increased in patients with fetal tumors and lung cancer, which is easy to cause misdiagnosis
However, the affinity and specificity of existing antibodies against cancer (such as HCC) markers vary greatly, and most of them are difficult to meet the needs of practical applications.
For example, taking GPC3 as an example, there are foreign reports that the content of GPC3 in peripheral blood is low, and the increase in the concentration of GPC3 in patients with liver cancer is small, and it is difficult to distinguish it from normal controls, especially those with liver cirrhosis.
[0006] Since there is still no effective method for detection and treatment of liver cancer, there is an urgent need in this field to develop highly specific anti-human liver cancer monoclonal antibodies and corresponding effective methods for detecting liver cancer

Method used

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  • A monoclonal antibody for serological diagnosis of liver cancer and its use
  • A monoclonal antibody for serological diagnosis of liver cancer and its use
  • A monoclonal antibody for serological diagnosis of liver cancer and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Preparation and purification of 7C8 monoclonal antibody

[0088] (1). Polypeptide synthesis

[0089] The antigenicity of the full-length GPC3 protein was analyzed, and based on the antigenicity and accessibility of the protein, the polypeptide antigen GPC3350-364 amino acids was finally selected as the immunogen, and the short peptide was named GPC3-Ag2. The short peptide GPC3-Ag2 was prepared by artificial synthesis, and its sequence is AHSQQRQYRSAYYPE (SEQ ID NO: 17).

[0090] Table 1

[0091] name

GPC3-Ag2

starting point

350-364

molecular weight

1884.01 Da

quality

14.3mg

purity

97.70％

[0092] After synthesis, the synthesized short peptide was coupled with KLH by using the glutaraldehyde method to obtain the coupled polypeptide as an immunogen.

[0093] (2) Preparation of hybridoma

[0094] Take 6-8 week-old BALB / c mice, mix the synthetic conjugated polypeptide with an equal volume of Freund's ...

Embodiment 2

[0104] Identification and Typing of Anti-GPC3(350-364aa) Monoclonal Antibody 7C8

[0105] Three hybridomas 3D4, 3G6, and 7C8 with higher OD values ​​in ascites fluid were selected, and the three monoclonal antibodies 3D4, 3G6, and 7C8 purified from ascites were diluted 1:10,000 with GPC3 standard protein and negative control protein. (IL-15) was detected by direct ELISA, and the results are shown in Table 3.

[0106] Table 3 Reaction (OD value) of monoclonal antibody and GPC3 standard substance protein

[0107] GPC3 standard

IL-15

3D4

0.149

0.135

3G6

2.622

1.013

7C8

2.839

0.102

[0108] The results showed that monoclonal antibody 7C8 could recognize GPC3 standard protein more specifically than 3D4 and 3G6, but had no obvious reaction with IL-15. The subtype of 7C8 was further identified as IgG1.

[0109] The hybridoma cell GPC3-7C8 producing the high-titer monoclonal antibody 7C8 was deposi...

Embodiment 3

[0111] ELISA detection

[0112] 1. HRP-labeled secondary antibody

[0113] The second antibody used in this example is HRP-labeled anti-GPC3 (GPC3 protein 25-358aa) monoclonal antibody GP9 (the preparation method refers to the method described in the example of CN 200710039562.7), and 0.6 mg of HRP was weighed and dissolved in 120 μl of water ; add NaIO 4 (12.8mg / ml) 120μl, 4 ℃ for 30 minutes; add ethylene glycol (9μl in 1ml) 120μl room temperature for 30 minutes; .6) Slowly stir and dialyze overnight to make it combine; add NaBH the next day 4 (5mg / ml) 24μl, 2 hours at 4°C; put in a dialysis bag, dialyze against 0.05M / L PBS (pH7.2) at 4°C overnight and centrifuge at 4°C, take the supernatant to detect the titer.

[0114] 2. Linear relationship of ELISA detection method

[0115] The anti-GPC3-Ag2 monoclonal antibody 7C8 prepared in Example 1 was used to coat the plate, and the HRP-labeled anti-GPC3 (GPC3 protein 25-358aa) monoclonal antibody GP9 (see step 1) was used as th...

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Abstract

The invention relates to a monoclonal antibody for serological diagnosis of liver cancer and application thereof, and particularly provides an anti-human liver cancer specific monoclonal antibody 7C8. The monoclonal antibody has the characteristics of high stability and high specificity in combination with liver cancer antigen. The invention also provides 7C8 immunoglobulin and a fragment as well as an immunological conjugate thereof and a medicinal composition containing the immunoglobulin, the fragment or the immunological conjugate. The invention also provides a kit for detecting liver cancer.

Description

technical field [0001] The present invention relates to the fields of oncology and medicine. More specifically, the present invention relates to a monoclonal antibody for serological diagnosis of liver cancer and its use. Background technique [0002] Primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) is one of the most common malignant tumors in my country, accounting for the second place in cancer deaths among residents. Liver cancer is mostly in the middle and late stage when symptoms appear, and the recurrence and metastasis rate after resection is high. Therefore, early diagnosis of liver cancer is of great significance to prolong the survival time of patients and reduce the mortality of liver cancer. [0003] At present, the diagnosis of liver cancer mainly depends on imaging examination, liver biopsy histological examination and laboratory examination. Imaging diagnosis plays an important role in the diagnosis of liver cancer, but it has certain limit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20C12N15/13G01N33/577G01N33/574C12R1/91
Inventor 屠红陈敏张菁
Owner SHANGHAI INST OF ONCOLOGY
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