ASSAY FOR THE DIAGNOSIS OF MACULAR DEGENERATION

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.

Claims

1. A method of diagnosing a subject as having macular degeneration, comprising the steps of: obtaining a sample from the subject; adding a quantity of a lipid binding protein to the sample to bind any N-retinylidene-N-retinylethanolamine in the sample; and incubating the sample and the quantity of the lipid binding protein with an immunoprecipitation substrate having a plurality of antibodies to the lipid binding protein.

2. The method of claim 1, further comprising the step of collecting any bound lipid binding protein and N-retinylidene-N-retinylethanolamine.

3. The method of claim 2, further comprising the step of determining how much of any bound lipid binding protein and N-retinylidene-N-retinylethanolamine has been collected.

4. The method of claim 3, wherein the lipid binding protein is Saposin B.

5. The method of claim 4, wherein the immunoprecipitation substrate comprises protein G beads that have been incubated with antibodies targeting Saposin B.

6. The method of claim 5, wherein the antibodies are IgG anti-saposin B antibodies.

7. The method of claim 5, wherein the step of determining how much of any bound lipid binding protein and N-retinylidene-N-retinylethanolamine has been collected comprises the use of a mass spectrograph.

8. The method of claim 1, wherein the sample is a urine sample.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0008] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0009] FIG. 1A is a first fluorescence spectroscopy graph showing that Saposin B binds A2E;

[0010] FIG. 1B is a second fluorescence spectroscopy graphs showing that Saposin B binds A2E;

[0011] FIG. 2 is a mass spectrum of A2E; and

[0012] FIG. 3 is a mass spectrum of A2E recovered from SapB-A2E in urine.

[0013] FIG. 4 is a Western blot of SapB from urine, RPE cells, and E. coli expression where the lanes (from left to right) are: Lane 1, Gel Ladder; Lane 2, Saposin B from urine; Lane 3, Retinal Pigment Epithelium cell Saposin B; Lane 4, RPE cell Saposin B; and Lane 5, Saposin B expressed in BL21 E. coli.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Referring to the figures, wherein like numerals refer to like parts throughout, the present invention comprises an assay that employs saposin B (sapB) to bind A2E in human urine so that is may be detected as measured as an indicator for the presence and severity of macular degeneration. A2E is a compound produced as a part of the light cycle in the human eye and is associated with the development and/or progression of macular degeneration. As A2E builds up in the eye, it is possible it is removed from the body via the blood and then the kidneys in urine, with levels rising in the blood and urine as the disease progresses. Thus, the present invention uses a lipid binding protein, such as Saposin B, to bind A2E, for extraction and measurement.

Example

[0015] SapB bound A2E was pre-complexed in a 2:1 molar ratio of sapB:A2E in 50 mM phosphate buffer. A volume of this solution was then added to an aliquot of human urine. The sapB-A2E plus urine mixture was then added to magnetic Protein G Beads, which had been incubated with IgG anti-saposin B antibody. After one hour of incubation at 37° C., the sample was magnetized and the excess sample volume was discarded. The sample was then washed with 50 mM phosphate buffered saline solution three times. After each wash, the solution was re-magnetized and the PBS solution was discarded. Following the third wash, mass spectrometry grade methanol was added to the sample and incubated at 4° C. overnight. The sample was then re-magnetized and the MeOH fraction, containing the A2E, was collected for mass spectrometry analysis.

[0016] FIG. 2 depicts the mass spectrum for pure A2E and FIG. 3 shows the mass spectrum for sapB-A2E recovered from the urine sample. SapB binding to the Protein G Bead-anti SapB complex was confirmed via western blot analysis, as seen in FIG. 4.

[0017] The present invention may comprise a kit for the extraction of A2E from a urine sample so that it can be detected and quantified that includes a lipid binding protein, such as Saposin B, that can bind to A2E and enable its extraction. The kit may further include components to assist in the extraction and measurement of A2E. For example, immunoprecipitation substrates, such as protein G beads, that have been incubated with antibodies targeting the Saposin B lipid binding protein, and the associated washing compounds, may be provided in the kit. The present invention thus provides for easy and rapid quantification of a known marker of macular degeneration.

[0018] Using A2E levels measured in the blood or urine of healthy patients, patients with diagnosed early stage macular degeneration, patients with advancing and patients with late stage macular degeneration, a relationship between urine levels of A2E and clinical diagnosis can be developed. A classic diseases progression curve or line can thus be generated to enable physicians to track and, in early stage once validated, even diagnose earlier the presence of macular degeneration.