Method for detecting the presence of a phospholipid
Inventors
Gilbert, Gary E. • Shi, Jialan • Heegaard, Christian W. • Rasmussen, Jan T.
Assignees
Brigham and Womens Hospital Inc • US Department of Veterans Affairs
Publication Number
US-7771956-B2
Publication Date
2010-08-10
Expiration Date
2024-06-30
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Abstract
A method, kit and probe for detecting the presence of a phospholipid, such as phosphatidylserine, in a biological material is provided. A binding agent including lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin, is used to detect the presence of any phospholipid.
Core Innovation
The invention provides a method, kit, and probe for detecting the presence of a phospholipid, such as phosphatidylserine (PS), in biological material using a binding agent that includes lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, or a functional equivalent of a fragment of lactadherin. This agent binds specifically and with high affinity to phospholipid membranes containing phosphatidylserine, enabling detection of any phospholipid present in the biological sample.
The problem being solved arises from the need to detect phospholipids, particularly phosphatidylserine, on cell membranes, which play significant roles in physiological and pathological processes. Prior detection methods, such as annexin V binding, were limited by dependence on calcium, restricted affinity at low PS concentrations, and sensitivity to membrane curvature. The invention addresses these limitations by providing an agent that binds independently of calcium and phosphatidylethanolamine (PE), exhibits stereoselective preference for the phospho-L-serine moiety of phosphatidylserine, and binds preferentially to highly curved membranes, enabling detection at low phosphatidylserine levels not accessible by previously known agents.
The invention also overcomes challenges related to the specific detection and inhibition of phospholipid-mediated processes such as blood coagulation. Lactadherin competes with blood coagulation factors VIII and V for membrane binding sites and inhibits procoagulant activity more effectively and over a wider range of phosphatidylserine content and membrane curvature than annexin V. Further, lactadherin's binding properties allow for detection of reversible and limited phosphatidylserine exposure on stimulated platelets, which traditional probes could not reliably detect.
Claims Coverage
The patent includes one independent claim covering a method for detecting the presence of a phospholipid in biological material using a specified binding agent. The claim encompasses multiple inventive features related to the composition of the binding agent and its binding characteristics.
Use of lactadherin or functional equivalents as binding agent
The method employs at least one binding agent selected from lactadherin, a fragment of lactadherin, a functional equivalent of lactadherin, and a functional equivalent of a fragment of lactadherin to detect phospholipids in biological material.
Detection of phospholipid binding
The method includes steps of contacting the biological material with the binding agent, allowing binding between the biological material and the agent, and detecting the presence of phospholipid bound to the binding agent or the agent bound to the biological material.
Specificity for phosphatidylserine and phospho-L-serine moiety
The method detects phospholipids comprising phosphatidylserine or the phospho-L-serine moiety of phosphatidylserine within the biological material.
Applicable biological materials
The biological material can include cells, cell membranes, cell appendages, cell fragments, lipoproteins, or cellular particles.
Calcium and phosphatidylethanolamine independence
The binding of the agent to the biological material is independent of calcium ions (Ca++) or phosphatidylethanolamine content.
Preference for binding to curved membranes
The binding of the agent is increased with increasing cell membrane curvature and is specifically directed to curved regions of the cell membrane.
Proportional binding to phosphatidylserine content
The binding increases proportionally to the content of phosphatidylserine in a range of about 0-2%.
Overall, the independent claim describes a novel method of detecting phospholipids, especially phosphatidylserine, in biological samples by employing lactadherin or its equivalents, utilizing their unique binding properties including calcium independence, stereoselectivity, and preference for highly curved phospholipid membranes.
Stated Advantages
The agent detects phosphatidylserine exposure at physiologically relevant low levels not detectable by annexin V.
Binding is independent of calcium ions and phosphatidylethanolamine, simplifying detection conditions.
The agent preferentially binds to highly curved membranes, allowing detection of phospholipid exposure in specialized membrane regions.
Lactadherin competes effectively with coagulation factors for binding sites and inhibits procoagulant enzyme complex activity more completely than annexin V.
Allows detection of reversible and low-level phosphatidylserine exposure on stimulated platelets, relevant for physiological hemostasis regulation.
Documented Applications
Detecting the presence of phosphatidylserine on cells, including platelets stimulated by agonists such as A23187 ionophore or thrombin receptor activation peptide (TRAP).
Blocking or reducing binding of coagulation proteins by occupying phospholipid binding sites to inhibit procoagulant activity on membranes.
Protecting biological materials from enzyme action by binding of lactadherin to phospholipid regions.
Pharmacological bridge ligand to target drug-laden phospholipid vesicles to cells expressing lactadherin-binding integrins.
Providing compositions and kits for detecting phospholipids in biological samples using lactadherin-based binding agents.
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