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Antibody-linked immuno-sedimentation agent and method of isolating a target from a sample using same

a target and antibody-linked technology, applied in the field of target isolation, can solve the problems of complex and equipment-intensive cell separation, affinity agents that are not frequently used in cell separation techniques, and require multiple complex steps

Inactive Publication Date: 2013-10-10
CYTOSED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present patent is about an antibody-linked immuno-sedimentation agent (ALISA) and a method of isolating a target using it. The antibody-linked immuno-sedimentation agent comprises at least one sedimentation agent and at least one antibody linked to the sedimentation agent by a non-antigen binding region. The method involves forming a mixture of the sample, the antibody-linked immuno-sedimentation agent, and red blood cells, and incubating it under conditions sufficient to form a rouleaux, where the target is captured. The antibodies used in the patent include various ones such as anti-CD3, anti-CD19, insulin, and others. The technical effects of this patent are the improved efficiency and accuracy of target isolation using the antibody-linked immuno-sedimentation agent.

Problems solved by technology

Flow cytometry is an effective method for cellular analysis and cell separation, but is complex and equipment-intensive.
These affinity agents are not frequently used for cell separation techniques, however.
While many of these methods of cell separation are highly effective, they can be expensive, require multiple complex steps, and / or can be equipment-intensive.

Method used

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  • Antibody-linked immuno-sedimentation agent and method of isolating a target from a sample using same
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  • Antibody-linked immuno-sedimentation agent and method of isolating a target from a sample using same

Examples

Experimental program
Comparison scheme
Effect test

example 1

ALISA Preparation Using Tricholor-s-triazine

[0107]In this Example, ALISA is prepared using Tricholor-s-triazine. Anhydrous sodium carbonate (1 g) is added to 200 ml of anhydrous organic solvent (e.g., acetone, methylene chloride, dimethylsulfoxide, benzene, or 1,4-dioxane). Then, 0.55 g of Trichloro-s-triazine (TsT) is dissolved in the sodium carbonate solution. The sedimentation agent (1 mmol) (approximately 20 g of polyethylene glycol-PEG-20,000, 70 g of dextran-Dextran-70, or 180 g of hydroxyethyl starch-Hetastarch) is added to the sodium carbonate solution. The solution is then stirred overnight at room temperature. The solution is then filtered. The filtrate is then added to a sufficient amount of a non-polar organic solvent (e.g., of petroleum ether or diethyl ether) (500 ml) to produce an activated sedimentation agent precipitate that is collected by filtration. This activated sedimentation agent precipitate is further cleaned by re-dissolving in the organic solvent, filterin...

example 2

ALISA Preparation Using N-Succinimidyl Chloroformate

[0108]In this Example, ALISA is prepared using N-Succinimidyl Chloroformate. The sedimentation agent (1 mmol) (approximately 20 g of polyethylene glycol-PEG-20,000, 70 g of dextran-Dextran-70, or 180 g of hydroxyethyl starch-Hetastarch) is added to 200 ml of an anhydrous organic solvent (e.g., acetone, methylene chloride, dimethylsulfoxide, benzene, or 1,4-dioxane) at room temperature. Separately, N-succinimidyl chloroformate (6 mmol) is dissolved in 10 ml of dry acetone. Separately, 4-(dimethylamino)pyridine is dissolved in 10 ml of dry acetone. The succinimidyl chloroformate solution is added to the sedimentation agent solution with stirring. Next, the 4-(dimethylamino)pyridine solution is added to the combined succinimidyl chloroformate / sedimentation agent reaction solution. The solution is stirred for 2 hours at room temperature. The solution is filtered to remove precipitated 4-(dimethylamino)pyridine hydrochloride byproduct. ...

example 3

ALISA Preparation Using N,N′-Carbonyldiimidazole

[0109]In this Example, ALISA is prepared using N,N′-carbonyldiimidazole (CDI). The sedimentation agent (1 mmol) (approximately 20 g of polyethylene glycol-PEG-20,000, 70 g of dextran-Dextran-70, or 180 g of hydroxyethyl starch-Hetastarch) is added to 200 ml of an anhydrous organic solvent (e.g., acetone, methylene chloride, dimethylsulfoxide, benzene, or 1,4-dioxane) at room temperature. Separately, CDI (6 mmol) is dissolved in 10 ml of the organic solvent. The CDI solution is added to the sedimentation agent solution with stirring. The combined reaction solution is stirred for 2 hours at 37° C. The reaction solution is then added to a non-polar organic solvent (e.g., petroleum ether or diethyl ether) (500 ml) to precipitate the CDI activated sedimentation agent. The activated sedimentation agent is collected by filtration. The activated sedimentation agent precipitate is further cleaned by re-dissolving in the organic solvent, filteri...

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Abstract

The present disclosure is directed to antibody-linked immuno-sedimentation agent, the antibody being linked to a sedimentation agent by a non-antigen binding region of the antibody, and a method of isolating a target from a sample using the antibody-linked immuno-sedimentation agent. The methods involve forming a mixture including a sample with an antibody linked immuno-sedimentation agent and red blood cells under conditions sufficient to form red blood cell rouleaux and allow antibody-antigen binding.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of International PCT Patent Application No. PCT / US2011 / 065157, filed Dec. 15, 2011, now pending, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61 / 423,391 filed Dec. 15, 2010. These applications are incorporated herein by reference in their entireties.BACKGROUND OF THE DISCLOSURE[0002]1. Field of the Disclosure[0003]The present disclosure generally relates to the isolation of a target (e.g., cells and molecules) from a solution containing the target. More particularly, the present disclosure relates to a novel method of separating a target from a solution using an antibody-linked immuno-sedimentation agent (ALISA). Additionally, the present disclosure relates to the antibody-linked immuno-sedimentation agent itself.[0004]2. Description of the Related Art[0005]Many methods exist for the isolation of a target (e.g., cells, molecules, etc.) from a solution. Targe...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/539C07K16/00
CPCG01N33/5375G01N33/56966G01N33/56972G01N33/539G01N2333/7051C07K16/00G01N33/56988
Inventor DINGES, WARREN L.
Owner CYTOSED
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