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Method for measuring soluble GPC3 protein

A determination method, protein technology, applied in immunoglobulins, chemical instruments and methods, specific peptides, etc., can solve problems such as 5-year survival rate stay

Active Publication Date: 2016-08-10
CHUGAI PHARMA CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patients with hepatocellular carcinoma who can undergo local cancer resection have a better prognosis, but their 5-year survival rate remains at 15% to 39% (Non-Patent Document 3)

Method used

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  • Method for measuring soluble GPC3 protein
  • Method for measuring soluble GPC3 protein
  • Method for measuring soluble GPC3 protein

Examples

Experimental program
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Effect test

Embodiment 1

[0177]In the mutant GPC3 core (core) formed by converting the serines of the two heparan sulfate binding sites (positions 495 and 509) of human glypican 3 (GPC3: SEQ ID NO: 70) into alanine The C-terminus of the extracellular domain (position 25-563) is endowed with a His tag to obtain the GPC3 core (sGPC3-His) protein, and the GPC3 core (sGPC3-His) protein is used for Balb / c or MRL / lpr mice A total of 6 to 9 immunizations were performed, and 3 days after the final immunization, mouse spleen cells were fused with mouse myeloma cells SP2 / 0 by a conventional method using PEG1500 or HVJ-E (Ishihara Sangyo).

[0178] Next, hybridoma cells were tested using ELISA in which sGPC3-His was directly immobilized, ELISA in which anti-His antibody was immobilized and sGPC3-His was bound, or cell ELISA (Cell ELISA) using CHO cells that constantly express GPC3. screening (see WO 2006 / 006693).

Embodiment 2

[0180] It has been found that the sGPC3-His protein can be seen in SDS-PAGE under reducing conditions with bands of about 86kDa, about 43kDa, and about 33kDa, which are the full-length type, the N-terminal fragment obtained by protease cleavage, and the C-terminal Fragment (WO 2006 / 006693). Using the mouse antibody obtained through the above screening, perform Western blotting against sGPC3-His, and select an antibody that does not recognize the C-terminal fragment. As a result, GT30, GT114, GT607 as N-terminal fragment recognition antibodies, and GT165 ( figure 1 ).

[0181] Next, in CHO cells, recombinant products with His tags attached to each of the C-terminals of GPC3 from 1 to 196 (AW2), 1 to 218 (AW3), and 1 to 357 (AW5) were expressed, and each CHO cells were pelleted for Western blotting. results, such as figure 2 , as shown in Table 1, although reactivity with AW5 or sGPC3-His was confirmed for GT30, GT114, and GT607, reactivity was not confirmed for AW2 and AW3...

Embodiment 3

[0185] In order to identify the epitope of the above-mentioned antibody in more detail, the amino acid sequence of GPC3 was divided into 30-residue overlapping peptides containing 10 residues, and the corresponding peptides were synthesized (SEQ ID NO: 1-29 in Table 2). After dissolving the synthetic peptide in dimethyl sulfoxide so as to be 1 mg / mL, it was diluted to 1 μg / mL with PBS. The diluted peptide solution was added to a 96-well plate in an amount of 70 μL per well, and immobilized at room temperature for 1 hour. Unimmobilized peptides were removed, and blocking buffer 20% Blocking-one (Nacalai Tesque, Inc.) was added in an amount of 200 μL per well for overnight blocking. After the blocking buffer was removed and washed three times with TBS-T, an antibody (diluted to 2 µg / mL with blocking buffer) was added in an amount of 70 µL / well. One hour later, the antibody was removed, TBS-T was added in an amount of 200 μL per well, and washed three times. The HRP-conjugated ...

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Abstract

The present invention relates to a method for measuring soluble GPC3 protein in a sample of interest, said method being characterized by using different two antibodies which respectively bind to different epitopes occurring in an N-terminal region of GPC3 protein.

Description

technical field [0001] The present invention relates to a method for measuring soluble GPC3 protein in a test sample, characterized by using two different antibodies that bind to different epitopes present in the N-terminal region of the GPC3 protein. Background technique [0002] It is reported that there are about 600,000 deaths per year caused by hepatocellular carcinoma, ranking fifth among the deaths caused by cancer in the world (Non-Patent Document 1). Most patients with HCC die within 1 year of diagnosis of the disease. Unfortunately, there are frequent instances of HCC being diagnosed at a later stage when curative therapies are less effective. Medical treatments, including chemotherapy, chemoembolization, cautery, and proton beam therapy, remain insufficiently effective in these patients. Most of the patients showed disease recurrence, which was accompanied by vascular invasion and multi-site intrahepatic metastases rapidly developed to the advanced stage, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCC07K16/303G01N33/57438G01N2333/4722G01N33/53
Inventor 大友俊彦天野润安达秀树铃木司水内素晶山口哲司和久井世纪
Owner CHUGAI PHARMA CO LTD
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