Method and device for detecting antigen-specific antibodies in a biological fluid sample by using neodymium magnets
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-10564152-B2
Publication Date
2020-02-18
Expiration Date
2036-08-08
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Abstract
Methods for detecting antigen-specific antibodies in a biological sample are described. The disclosed methods can be used for the diagnosis of a variety of autoimmune and infectious diseases. The methods use a neodymium magnet to efficiently isolate immune complexes. The disclosed methods are rapid, highly specific and sensitive, require very small volumes of biological sample, and do not require the use of radioactivity. With these advantageous features, the disclosed methods are amendable for point-of-care testing (POCT), which is currently not available for the detection of autoantibodies associated with autoimmune disease or for the detection of many pathogen-specific antibodies.
Core Innovation
The invention provides a method and device for detecting antigen-specific antibodies in biological fluid samples using a neodymium magnet to rapidly isolate immune complexes formed by antigen-antibody binding. The method involves providing a fusion protein comprising an antigen fused to a light-emitting protein, contacting it with a biological sample to form immune complexes if specific antibodies are present, binding these complexes to magnetic beads coated with immunoglobulin-binding proteins, isolating the bead-bound complexes by directly contacting them with a neodymium magnet, and detecting light emission from the isolated complexes. This approach enables highly specific and sensitive detection with very small sample volumes and does not require radioactivity.
The problem solved by this invention is the lack of rapid, point-of-care testing (POCT) for the detection of autoantibodies associated with autoimmune diseases and many pathogen-specific antibodies. Current fluid-phase immunoprecipitation assays necessary to detect conformation-specific antibodies often involve radioactivity and are time-consuming, rendering them impractical for POCT. The disclosed method provides a rapid, non-radioactive, highly specific and sensitive assay format amenable to POCT for autoimmune and infectious diseases, thus satisfying an important clinical need.
The disclosed assay, termed LIPSTICK, integrates luciferase immunoprecipitation systems (LIPS) with the use of neodymium magnetic sticks to capture paramagnetic beads bound to antigen-specific antibodies. This format simplifies and accelerates the antibody detection process, requiring only a brief incubation and a single wash, resulting in assay completion times of less than five minutes. The method accommodates various biological fluids such as serum, plasma, blood, urine, saliva, and bronchoalveolar lavage fluid and uses different light-emitting proteins and immunoglobulin-binding proteins to tailor detection.
Claims Coverage
The patent claims cover one independent method claim that encompasses the core innovative method steps for detecting antigen-specific antibodies using fusion proteins, magnetic bead capture, and neodymium magnets, along with light emission detection.
Use of fusion protein comprising antigen fused to luciferase protein
Providing a fusion protein that contains an antigen linked to a luciferase protein to enable light-based detection of antigen-specific antibodies upon binding in the biological fluid sample.
Formation and capture of immune complexes with magnetic beads coated with immunoglobulin-binding proteins
Contacting the biological fluid sample with the fusion protein to form immune complexes if antigen-specific antibodies are present, then capturing these complexes using magnetic beads coated with immunoglobulin-binding proteins to form bead-bound immune complexes.
Isolation of bead-bound immune complexes by direct contact with a neodymium magnet
Isolating the bead-bound immune complexes by directly contacting them with a neodymium magnet, enhancing assay rapidity compared to indirect magnetic separation methods.
Detection of light emission from isolated bead-bound immune complexes
Measuring emitted light from the isolated bead-bound immune complexes to detect the presence of antigen-specific antibodies, typically using a luminometer.
Collectively, the claims define a rapid, sensitive, and specific method for antigen-specific antibody detection employing a fusion protein with luciferase, magnetic bead capture, direct magnetic isolation using neodymium magnets, and luminescence-based detection, suitable for small sample volumes and various biological fluids.
Stated Advantages
The method is rapid, allowing antibody detection within minutes, with some assays completing in less than five minutes.
It achieves high specificity and sensitivity for detecting antigen-specific antibodies in biological fluids.
The assay requires very small volumes of biological samples, such as serum or saliva.
It avoids the use of radioactivity, making it suitable for point-of-care testing environments.
The magnetic capture system uses direct contact with neodymium magnets, simplifying washing steps and reducing assay complexity and time.
The assay can be used with handheld luminometers, facilitating portability and on-site diagnostics.
Documented Applications
Diagnosis of autoimmune diseases by detecting autoantibodies specific to diseases such as Sjögren's syndrome, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, and others.
Diagnosis of infectious diseases by detecting pathogen-specific antibodies against viral pathogens including HIV, Epstein-Barr virus, equine non-primate hepacivirus and others.
Use in veterinary diagnostics, exemplified by detecting antibodies against equine non-primate hepacivirus in horse serum samples.
Point-of-care testing applications for rapid diagnosis directly from serum or saliva samples.
Monitoring changes in antibody levels over time, such as assessing treatment efficacy in HIV-infected individuals.
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