Microfluidic device and leucocyte antigen mediated microfluidic assay
Inventors
Rodriguez, Rodolfo R. • Darr, Douglas J.
Assignees
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Publication Number
US-10620203-B2
Publication Date
2020-04-14
Expiration Date
Abstract
The present invention relates to an leucocyte antigen mediated microfluidic assay and a microfluidic device for analyzing a subjects' body fluids containing leucocytes to determine if the subject has been previously exposed to a predetermined antigen.
Core Innovation
The invention provides a leucocyte antigen mediated microfluidic assay (LAMMA) method and a microfluidic device for testing a leucocyte-containing body fluid from a subject for prior exposure to a predetermined antigen that would have caused leucocytes to become presensitized to the antigen. The method performs two parallel tests on microvolumes in microchambers, followed by optical scanning and comparison of results to detect whether differences are due to a leucocyte-antigen mediated response.
In a first test, a microvolume first sample of the subject’s leucocyte-containing body fluid is placed in a first sample microchamber and transported to a first reaction microchamber containing the predetermined antigen/antigen accelerator complex, including the predetermined antigen. The antigen accelerator is a composition that stimulates, amplifies and/or accelerates the production of measurement factors in leucocytes upon contact with the antigen, and the antigen accelerator is β2-macroglobulin.
In a second test, a microvolume second sample is placed in a second sample microchamber and transported to an antigen-free second reaction microchamber containing the same antigen accelerator but not containing the predetermined antigen. The reacted first and second samples are transported to observation microchambers, mixed with leucocyte observation colorants, and optically scanned using field-by-field XYZ scanning to measure at least one leucocyte measurement factor, including morphological and/or spectral measurement factors from multi-wavelength fluorescence image analysis.
Differences between the first optical scan and the second optical scan are compared to determine if they are due to a leucocyte-antigen mediated (presensitized) response.
Claims Coverage
The disclosed claim set centers on one independent method claim that includes two inventive test tracks, antigen-present reaction versus antigen-free control, combined with microfluidic sample, reaction, and observation microchambers and optically scanned measurement-factor readouts using leucocyte observation colorants, followed by scan comparison to determine a leucocyte-antigen mediated response. The independent claim includes the specific antigen accelerator β2-macroglobulin and field-by-field XYZ optical scanning for measurement factors; dependent claims refine the leucocyte types, sample matrices, microchamber geometry, and readout and analysis constraints.
Two-test antigen reaction with antigen-free control in microchambers
Perform a first test reaction of a microvolume leucocyte-containing body fluid with a predetermined antigen/antigen accelerator complex, then a second test reaction using the same antigen accelerator in an antigen-free reaction microchamber, with both samples routed to observation microchambers, colorant mixing, optical scanning, and comparison to determine if differences are due to a leucocyte-antigen mediated response.
Antigen accelerator stimulating measurement-factor production
Use an antigen accelerator in the antigen/antigen accelerator complex, wherein the antigen accelerator is a composition that stimulates, amplifies and/or accelerates production of measurement factors in leucocytes upon contact with the antigen; the antigen accelerator is β2-macroglobulin.
Optical field-by-field XYZ scanning of leucocyte measurement factors
Optically perform a first optical scan and a second optical scan in respective observation microchambers, each including a field-by-field XYZ scan under conditions that allow measurement of at least one leucocyte measurement factor, and measure the same at least one leucocyte measurement factor in both scans for direct comparison.
Colorant-mixed observation with measurement-factor based scan comparison
Mix one or more leucocyte observation colorants with the reacted first sample and with the reacted second sample, optically scan to measure leucocyte measurement factors, and compare differences between the first and second optical scans to determine whether differences are due to a leucocyte-antigen mediated response.
Across the independent claim, the inventive core is the combination of two microfluidic reaction tracks with antigen-present versus antigen-free control using the same antigen accelerator, colorant-mixed observation in microchambers, and field-by-field XYZ optical scanning to obtain comparable leucocyte measurement factors whose differences are attributed to a leucocyte-antigen mediated response.
Stated Advantages
Documented Applications
Testing MAP latent Johne’s disease in cattle by using T lymphocytes and quantifying T-cell responses via morphological and/or spectral factor changes versus an antigen-free control, using Astrozone Orange (Basic Orange 21).
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