Assay for detection of an A2E-Saposin B complex
Inventors
Assignees
Publication Number
US-12360121-B2
Publication Date
2025-07-15
Expiration Date
2037-10-25
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Abstract
An assay and kit for detecting N-retinylidene-N-retinylethanolamine (A2E) in a sample that uses a lipid binding protein, such as Saposin B, to capture A2E in the sample and assist in the extraction and measurement of A2E via mass spectroscopy. A2E thus serves as a marker for macular degeneration so that the assay and kit of the invention can be used to detect the presence or severity of macular degeneration.
Core Innovation
The invention provides an assay and kit for detecting N-retinylidene-N-retinylethanolamine (A2E) in a biological sample such as urine or blood. The assay uses a lipid binding protein, specifically Saposin B, to capture A2E present in the sample. After binding, the A2E can be extracted via immunoprecipitation using antibodies that target Saposin B and subsequently measured by mass spectroscopy. This method enables the detection and quantification of A2E, a marker for macular degeneration.
Macular degeneration is an age-related eye disease characterized by the loss of central vision and the progressive deterioration of the macula. Current methods lack an assay capable of detecting the presence or severity of macular degeneration by measuring biomarkers in biological fluids. A2E, a major lipofuscin constituent accumulating in retinal pigmented epithelial cells, correlates with the progression of macular degeneration but cannot be readily detected with existing assays.
The invention addresses this need by using Saposin B to bind A2E in samples such as urine, enabling its immunoprecipitation and detection through mass spectroscopy. This approach facilitates an easy and rapid quantification of A2E levels, allowing diagnosis or monitoring of macular degeneration severity. The kit includes components such as lipid binding proteins, antibodies against Saposin B, and protein G beads to assist in extraction and measurement.
Claims Coverage
The patent contains one independent claim with main inventive features regarding a method to prepare an A2E-Saposin B complex from a urine sample for detecting A2E.
Preparation of A2E-saposin B complex in urine sample
The method comprises obtaining a urine sample from a human subject and adding a quantity of saposin B to the urine sample to form an A2E-saposin B complex.
Immobilization of the A2E-saposin B complex via immunoprecipitation
The urine sample is incubated with protein G beads pre-incubated with anti-saposin B antibodies to immobilize the A2E-saposin B complex.
Washing and extraction steps
The method includes washing the protein G beads comprising the immobilized complex and extracting bound A2E from the protein G beads for detection.
These inventive features together provide a method for preparing and isolating an A2E-Saposin B complex from urine for subsequent detection of A2E, with particular emphasis on the complex formation, immunoprecipitation with specific antibodies, and extraction procedures.
Stated Advantages
Enables detection and quantification of A2E as a marker for macular degeneration.
Provides an easy and rapid assay for diagnosing or monitoring macular degeneration severity.
Facilitates early detection by relating measured A2E levels in biological fluids to clinical diagnosis.
Documented Applications
Diagnosis and monitoring of macular degeneration through detection of A2E levels in urine or blood.
Use of the assay and kit for the extraction and measurement of A2E to correlate urine A2E levels with disease progression.
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