Probe complex

A technology of complexes and probes, which can be used in biological testing, material inspection products, measuring devices, etc., and can solve problems such as hindering sensitivity

Active Publication Date: 2007-07-04
ADVANCED LIFE SCI INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In immunohistochemistry and immunoassays, the strong hydrophobicity of the probe complex will lead to non-specific binding of the complex to the tissue or plate due to the hydrophobic bond, resulting in increased background that prevents adequate sensitivity from being obtained

Method used

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  • Probe complex

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Production of Biotin-anti-HCV Core Antigen Monoclonal Antibody Complex Using Ficoll 400 as Carrier

[0049] 44 mg of Ficoll 400 (Amersham Bioscienses) was weighed and dissolved in 0.8 mL of 0.1 M phosphate buffer (pH 7.0), 0.4 mL of sodium periodate solution was added and mixed. After incubation at room temperature for 2 hours, excess sodium periodate was removed by gel filtration (SephadexG25, Amersham Bioscienses), bovine serum albumin (BSA) solution was added and reacted at room temperature for 3 hours, and BSA was added to Ficoll. To stabilize the reaction product, dimethylamine borate (DMAB; Seikagaku Corporation) was added and mixed, reacted at room temperature for 1 hour, and then Tris solution was added to block the unreacted aldehyde groups on Fenco. After overnight reaction at room temperature, the reaction product was purified by gel filtration (Sephacryl S300, 1.6×30), and the absorbance at 280 nm was measured to calculate the concentration of the carrier-BS...

Embodiment 2

[0051] Preparation of biotinylated anti-HCV core antigen monoclonal antibody by conventional method

[0052] Biotinylated antibodies were prepared as described in the attached document Sulfo-NHS-LC-Biotin (Pierce #21335). Mix sulfo-NHS-LC-biotin and anti-HCV core antigen monoclonal antibody (a mixture of equal amounts of C11-14 IgG and C11-9 IgG) in PBS and incubate at room temperature for 1 h, then pass through gel filtration ( Sephadex G25, Amersham Biosciences) to remove excess sulfo-NHS-LC-biotin. The absorbance at 280 nm of the biotinylated anti-HCV core antigen monoclonal antibody was measured to calculate the antibody concentration.

Embodiment 3

[0054] Comparison of the biotin-anti-HCV core antigen monoclonal antibody complex prepared in Example 1 and the biotinylated anti-HCV core antigen monoclonal antibody prepared by conventional methods in Example 2.

[0055] Adjust the anti-HCV core antigen monoclonal antibody to 4 μg / mL with 0.1M acetate / 0.1M phosphate buffer (pH 4.8), add 250 μl to each well of a 96-well microplate, and place each well at 4 Incubate overnight at °C. After washing with PBS, 350 μl of 0.5% casein was added to each well, and each well was incubated at room temperature for 3 hours. Add recombinant HCV core antigen (c11) with concentrations of 0fmol / L, 148fmol / L, 444fmol / L, 1333fmol / L, 4000fmol / L, 12000fmol / L and 36000fmol / L as samples, and incubate each well with stirring at room temperature 1 hour. After washing 6 times with 10 mM phosphate buffer pH 7.3 (washing solution) containing 0.05% Tween 20, the biotin-anti-HCV core antigen monoclonal prepared in Example 1 with a concentration of 1 μg / m...

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Abstract

To provide a soluble highly sensitive probe complex with high functional capability ensuring usability in immunoassays of high sensitivity. A highly sensitive probe complex can be produced by linking hydrophilic intermediums to a carrier and linking detection markers such as biotin, haptens or low-molecular-weight peptides and antibodies to the intermediums. By virtue of the linking of hydrophilic intermediums to a carrier, a multiplicity of hapten molecules can be linked and the probe complex as a whole becomes hydrophilic, so that there can be obtained a probe complex with high functional capability which would not induce nonspecific reactions.

Description

[Field of invention] [0001] The present invention relates to a technique for producing a probe complex in which a probe and a detection label such as a hapten or a low molecular weight peptide are linked to a carrier through an intermediary. The probe complex thus prepared can be widely used in immunomeasurement using immune response, including enzyme immunoassay and immunohistochemistry. [Background technique] [0002] Immunohistochemistry and immunoassay have been used as a method for detecting self-antigens or foreign antigens in organisms using an immune reaction between an antigen and an antibody. The high specificity and sensitivity of these methods make it possible to detect small amounts of substances in organisms without isolating them. [0003] Immunohistochemistry is a method of detecting the localization of antigens in cells and tissues through the specific reaction between antigens and antibodies. The widely used ABC method of immunohistochemistry is performed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/53G01N33/547
CPCG01N33/542G01N33/54393G01N33/53G01N33/543G01N33/547
Inventor 青柳克巳饭田久美子松原直子石田雄彦
Owner ADVANCED LIFE SCI INST
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