Methods and compositions for improving detection and/or capture of a target entity

Patent metadata

Field	Value
Publication number	US-10551379-B2
Application number	US-201414766575-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 15, 2013
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

Methods, compositions, kits and systems for detecting and/or capturing a target entity in a sample are disclosed. In particular, the methods, compositions and kits described herein can be used for pretreatment of target-binding agents with a blocking agent to reduce non-target binding in a complex matrix (e.g., blood). Methods and compositions for detecting and/or capturing a microbe in a test sample, including bodily fluids such as blood and tissues of a subject, food, water, and environmental surfaces are also disclosed.

First claim

What is claimed is:

1. A method of capturing at least one target entity in a sample, the method comprising:
contacting a sample with a composition comprising a target-binding agent attached to a solid substrate, the target-binding agent having a blocking agent bound thereto, wherein the blocking agent comprises a hexose, and wherein the target-binding agent comprises a mannan-binding lectin (MBL) consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 8; and
allowing the target entity, wherein the target entity comprises a microbe or a microbial fragment, to displace the blocking agent and to bind to the target-binding agent, thereby capturing the target entity comprising a microbe or the microbial fragment.

2. The method of claim 1 , wherein the sample comprises blood and the blocking agent has an effective binding affinity for the target-binding agent that is lower than an effective binding affinity of the microbe or the microbial fragment for the target-binding agent, and that is higher than the effective binding affinity of a blood cell or a fragment thereof for the target-binding agent.

3. The method of claim 1 , wherein the blocking agent is a monomer, the monomer having no free binding site after binding to the target-binding agent.

4. The method of claim 1 , wherein the blocking agent is a multimer, the multimer having at least one free-binding site after binding to the target-binding agent.

5. The method of claim 1 , wherein the blocking agent comprises glucose.

6. The method of claim 1 , wherein the blocking agent inhibits binding of the target-binding agent to a blood cell or a fragment thereof present in the sample prior to the binding of the microbe or the microbial fragment to the target-binding agent.

7. The method of claim 1 , wherein the effective binding affinity of the blocking agent for the target-binding agent is characterized by a dissociation constant, wherein the dissociation constant ranges from about 1 mM to about 50 mM.

8. The method of claim 7 , wherein the blocking agent is pre-bound to the target-binding agent prior to the contacting.

9. The method of claim 1 , further comprising separating the target-binding agent from the sample after the microbe or the microbial fragment is captured on the target-binding agent.

10. The method of claim 1 , further comprising detecting the microbe or the microbial fragment that is bound to the target-binding agent.

11. The method of claim 10 , wherein the microbe or the microbial fragment is detected by a method comprising contacting the bound microbe or microbial fragment with a detection agent, wherein the detection agent does not bind to the blocking agent.

12. The method of claim 1 , wherein target-binding agent comprises a fusion protein that includes an Fc portion of an immunoglobulin linked to the MBL (FcMBL), the Fc portion being attached to the solid support, and further wherein the blocking agent comprises a glucose bound to a carbohydrate recognition domain of the MBL to inhibit binding of a blood cell or a fragment to the target-binding agent.

13. The method of claim 1 , wherein the solid substrate is selected from the group consisting of: a medical apparatus a filtration device, a membrane, a diagnostic strip, a dipstick, and an extracorporeal device.

14. The method of claim 1 , wherein the solid substrate is selected from the group consisting of: a microparticle, a microbead, a nanotube, a microtiter plate, an implant, a microchip, a mixing element, a microscopic slide, a hollow fiber, and a hollow fiber cartridge.

Assignees

• Harvard College

Inventors

• Super Michael
• Cartwright Mark J
• Rottman Martin M
• Tomolonis Julie A

Classifications

• G01N33/5306

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N33/54393 Primary

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title

• G01N2469/10

  Detection of antigens from microorganism in sample from host · CPC title

• G01N2400/00

  Assays, e.g. immunoassays or enzyme assays, involving carbohydrates · CPC title

• G01N33/569 Primary

  for microorganisms, e.g. protozoa, bacteria, viruses · CPC title