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Hapten compound and antibody

A compound and hapten technology, applied in the field of hapten compounds and antibodies, can solve the problems of inability to obtain antibodies, easy ionization, unclear specific conditions, etc., and achieve excellent specificity and simplicity of operation.

Inactive Publication Date: 2009-03-25
JAPAN SCI & TECH CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] However, the in vivo behavior of BSH accompanied by BNCT, especially the details of the microscopic distribution of cells on the cell surface layer, is still unclear, and there is a strong need for the development of methods that can easily and quickly determine the qualitative and quantitative behavior of BSH in vivo
As a detection and quantification method of BSH, immunological measurement method is expected to be used, but low-molecular-weight inorganic compounds such as BSH have small molecular weight and volume or are easily ionized, so they have low antigenicity, and it is not yet available to detect BSH with high sensitivity. Antibody

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] [Example 1 (synthesis of BSH hapten)]

[0141] (a) Synthesis of BSH-ethyl hexanoate (3)

[0142]BSH (101 mg, 0.46 mmol) was dissolved in acetonitrile (10 mL), then stirred at room temperature while slowly dripping ethyl bromohexanoate (0.2 mL, 1.12 mmol), and stirred at room temperature for 2 days. Then, acetonitrile was removed by concentration under reduced pressure, and the concentrated residue was purified by silica gel column chromatography (chloroform:methanol=9:1) to obtain a yellow oil (101 mg, yield 67.4%).

[0143] ·TLC: Rf=0.58 (chloroform:methanol=3:1)

[0144] · 1 H-NMR (DMSO) δ (ppm): 0.60-2.10 (m), 1.08 (t, 3H, J=7.08Hz), 1.34-1.42 (m, 2H), 1.52-1.59 (m, 2H), 1, 63-1.70(m, 2H), 2.19(t, 2H, J=7.32Hz), 2.77(m, 2H), 3.95(q, 2H, J=7.08Hz).

[0145] (b) Synthesis of BSH-hexanoic acid (4)

[0146] The compound obtained in (a) (113.7 mg, 0.31 mmol) was dissolved in methanol (2.0 mL), 2N sodium hydroxide (0.63 mL) was added dropwise, and stirred at room temp...

Embodiment 2

[0149] [Example 2 (adjustment of immunogen)]

[0150] (c) Synthesis of BSH-hexanoic acid-BSA complex (immunogen using BSA)

[0151] Add 760μL of bovine serum albumin (11mg, 164nmol) and borate buffer solution (sodium tetraborate 50mmol, sodium chloride 15.4mmol, sodium azide 0.3mmol / pure water 100mL) at pH 9.4 to the centrifuge tube, After stirring overnight at 4°C, DMF (40 μL) (solution I) was added.

[0152] In another centrifuge tube, add the compound (6.0mg, 18 μmol) N-hydroxysuccinimide (1.5mg, 13.3 μmol) obtained in said (b), 1-ethyl-3-(3-dimethyl Aminopropyl)-carbodiimide hydrochloride (2.5 mg, 13.3 μmol), DMF (200 μL), stirred overnight at room temperature (II solution).

[0153] Liquid II (10 μL / 5 minutes) was dripped into Liquid I at room temperature, stirred at room temperature for 2.5 hours, and then stirred overnight at 4°C. It was dialyzed against 10% isopropanol-phosphate buffer solution for about 60 hours (buffer solution was changed 6 times) to obtain BSH-h...

Embodiment 3

[0156] [Example 3 (production of polyclonal antibody)]

[0157] The BSH hapten-KLH conjugate adjusted in Example 2 was diluted to 1 mg / mL with phosphate buffer (pH 7.4). 600 μL of the antigen solution was mixed with an equal amount of RIBI adjuvant (MPL+TDM Adjuvant System, SIGMA M6536), and mixed thoroughly by vortexing for 2-3 minutes. Five Balb / c mice (8 weeks old, male) were immunized (50 µg / dose) by intraperitoneal injection. It was performed every two weeks, and after the third immunization, blood was collected one week after each immunization (orbital blood collection).

[0158] The antigen (BSH hapten-BSA conjugate) adjusted in Example 2 was diluted to 5.0 μg / mL with PBS (pH 7.4), 100 μL was dispensed into a microwell plate for ELISA, and allowed to stand at 37°C for 1 After 1 hour, the antigen was immobilized on the surface of the plate.

[0159] Then, wash with PBS-Tween solution (phosphate buffer pH 7.4, 0.05% Tween20), in order to prevent non-specific adsorption...

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Abstract

A compound with the structure of the formula: (1) Further, there is provided a BSH antibody obtained by using the above compound as a hapten and using as an antigen a composite composed of the hapten and a polymeric compound. Thus, there can be provided a hapten compound for production of an antibody capable of recognizing BSH with high sensitivity and high selection; a BSH antibody; and making use of the antibody and excelling in high sensitivity and quantity determination, a kit for BSH measurement and an immunological measuring method.

Description

technical field [0001] The present invention relates to a hapten compound of mercaptoundecahydrododecaborate (Japanese: mercaptoundeka hidrododecaboret, English: mercaptooundecaphydrododecaborate) (BSH), an antibody to BSH, and an immunological assay method using it, etc. , especially useful in the detection and quantification of neutron capture therapy agents used in boron neutron capture therapy (BNCT). Background technique [0002] In recent years, boron neutron capture therapy (BNCT) has attracted attention as a new cancer treatment method using radioisotopes. Boron neutron capture therapy is the boron 10 isotope ( 10 The boron compound of B) is taken into cancer cells, irradiated with low-energy neutron beams (such as thermal neutrons), and utilizes the nuclear reaction that occurs in cells to locally destroy cancer cells. In this method of treatment, the inclusion 10 The boron compound of B is important for enhancing the therapeutic effect by selectively accumulatin...

Claims

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Application Information

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IPC IPC(8): C07F5/02C07K16/00C12N5/10C12P21/08G01N33/53G01N33/577
CPCG01N33/84G01N33/94G01N33/5308C07K16/44C07F5/027A61P35/00C07F5/02C07K16/00C12N5/10C12P21/00
Inventor 切畑光统浅野智之上原幸树
Owner JAPAN SCI & TECH CORP
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