Multiplex lateral flow immunoassay device and method for contextual immune profiling

Abstract

The present invention relates to multiplex lateral flow immunochromatographic diagnostic devices configured to detect immune activity biomarkers in biological samples. The device is configured to detect granulysin as a biomarker indicative of cytotoxic immune effector activity and at least one secondary biomarker indicative of tissue-associated immune injury. In certain embodiments, the device comprises a sample pad, a conjugate pad containing labeled detection antibodies, a porous membrane comprising spatially distinct capture zones corresponding to granulysin and the secondary biomarker, a control zone, and an absorbent pad configured to facilitate capillary flow. Detection of granulysin and the secondary biomarker within a single multiplex device enables determination of immune activation state and tissue-associated immune context from a single biological sample. The device is suitable for rapid diagnostic use, including point-of-care and laboratory environments, using whole blood, plasma, serum, or related biological fluids.

Description

Multiplex lateral flow immunoassay device and method for contextual immune profilingTECHNICAL FIELD

[0001] The present invention relates to immunochromatographic diagnostic devices and, more particularly, to multiplex lateral flow immunoassay systems configured to simultaneously detect a cytotoxic immune activation biomarker and at least one contextual tissue-associated biomarker within a single biological sample.BACKGROUND ART

[0002] Clinical monitoring of immune-mediated conditions, including organ transplant rejection and graft-versus-host disease (GVHD), requires timely detection of immune activation to prevent irreversible tissue injury and graft loss. Acute cellular rejection (ACR) and antibody-mediated rejection (AMR) are commonly diagnosed through histopathological evaluation of tissue biopsy specimens.

[0003] Although biopsy-based diagnosis provides high specificity, it is invasive, associated with procedural risk, and subject to processing delays. As a result, substantial research efforts have focused on identifying circulating or urinary biomarkers capable of signaling immune activation through non-invasive means.

[0004] Among such biomarkers, granulysin (GNLY), including the 15 kDa isofonn, has been identified as a cytotoxic immune effector protein associated with immune-mediated tissue injury. Elevated granulysin expression has been reported in peripheral blood lymphocytes and graft biopsy samples during episodes of acute renal allograft rejection. Detection of GNLY mRNA in urinary cell sediments has also been proposed as a non-invasive indicator of emerging rejection. While GNLY reflects cytotoxic immune activation and immune effector engagement, measurement of GNLY alone may not, in certain clinical contexts, distinguish between generalized systemic immune activation and localized graft-specific immune injury.

[0005] To improve diagnostic specificity, additional biomarkers have been investigated for tissue-associated immune responses. For example, CXCL9 has been associated with T-cell mediated inflammation and transplant rejection. Soluble ST2 (sST2) has been associated with immune activation and tissue injury in transplant and inflammatory conditions. Soluble interleukin-2 receptor (sIL-2R) and soluble CD30 (sCD30) have been linked to T-cellactivation and transplant rejection in various organ systems. These biomarkers may provide contextual information regarding tissue-specific immune processes. However, these biomarkers are typically measured independently, and isolated measurement of either cytotoxic immune activation markers or tissue-associated biomarkers may not provide sufficient information to determine the immune context or clinical significance of the observed signal.

[0006] Despite the identification of both cytotoxic activation markers and tissue-associated immune markers, existing diagnostic methods primarily rely on centralized laboratory platforms, including enzyme-linked immunosorbent assays (ELISA), polymerase chain reaction (PCR), flow cytometry, and sequencing-based molecular assays. These approaches typically require specialized infrastructure, trained personnel, and extended processing time, limiting their utility for rapid clinical decision-making and routine outpatient monitoring.

[0007] In addition to protein-based biomarkers, molecular diagnostic approaches such as donor-derived cell-free DNA (dd-cfDNA) assays have been developed for transplant monitoring. These assays measure circulating fragments of donor DNA released during graft injury and may provide early indication of allograft damage. However, dd-cfDNA testing typically requires centralized laboratory processing, including next-generation sequencing or digital polymerase chain reaction platforms and associated bioinformatic analysis, and is not generally configured for rapid bedside or point-of-care deployment.

[0008] Lateral flow immunoassays (LFAs) are widely used immunochromatographic diagnostic devices capable of rapidly detecting specific analytes in biological samples such as whole blood, plasma, or serum. LFAs have been successfully applied in infectious disease testing, pregnancy detection, and cardiac biomarker monitoring due to their rapid turnaround time, ease of use, and suitability for decentralized and point-of-care environments.

[0009] Lateral flow assays have been developed for detection of individual immune-associated biomarkers, including chemokines and cytokines. However, single-analyte detection may not provide sufficient information to distinguish generalized immune activation from localized tissue-specific immune injury. Multiplex lateral flow assays have been developed in other diagnostic fields; however, simultaneous detection of a biomarker indicative of cytotoxic immune effector activity and a contextual tissue-associated biomarker within a single rapid immunochromatographic architecture presents technical challenges. Such challenges may include differences in analyte concentration ranges, binding kinetics, diffusion behaviorwithin porous membranes, potential high-dose hook effects, and competitive interactions between detection reagents within multiplex conjugate systems.

[0010] Accordingly, there remains a need for a rapid, point-of-care diagnostic system capable of simultaneously detecting a biomarker indicative of cytotoxic immune effector activity and a biomarker indicative of tissue-specific immune injury within a single biological sample, thereby enabling contextual interpretation of immune activation state without reliance on centralized laboratory infrastructure.SUMMARY OF INVENTION

[0011] The present invention provides a multiplex immunochromatographic diagnostic system configured to detect, within a single biological sample, (i) granulysin (GNLY) as a primary biomarker indicative of cytotoxic immune effector pathways and cytolytic immune engagement and (ii) at least one secondary biomarker indicative of tissue-specific immune injury associated with organ transplant rejection or related immune-mediated conditions.

[0012] The system is configured such that detection of GNLY establishes the presence of cytotoxic immune effector activation, and detection of the secondary biomarker is interpreted relative to the GNLY detection state to provide contextual determination of immune-mediated tissue injury. Simultaneous detection of GNLY and the secondary biomarker within a single multiplex diagnostic device enables differentiation between generalized immune activation and localized transplant-associated immune injury. This configuration establishes GNLY as a cytotoxic immune effector indicator within the multiplex system, such that interpretation of the secondary biomarker occurs within the defined context of GNLY-detected immune effector engagement.

[0013] In certain embodiments, the secondary biomarker may be selected from CXCL9, soluble ST2 (sST2), soluble CD30 (sCD30), soluble interleukin-2 receptor (sIL-2R), or other biomarkers associated with tissue-specific immune injury or transplant rejection.

[0014] In certain embodiments, the diagnostic system is implemented as a multiplex lateral flow immunoassay comprising a sample pad configured to receive the biological sample, a conjugate pad containing labeled detection antibodies specific for GNLY and the secondary biomarker, and a porous membrane comprising spatially distinct capture zonescorresponding to GNLY, the secondary biomarker, and a control zone configured to verify assay performance.

[0015] The multiplex configuration enables simultaneous detection of GNLY and the secondary biomarker within a single integrated device, generating a combined diagnostic output representing the immune activation and tissue-specific biomarker states of the sample. The diagnostic system is configured to provide rapid diagnostic results suitable for point-of-care or decentralized clinical use without reliance on centralized laboratory instrumentation.

[0016] In certain embodiments, the GNLY detected by the system comprises one or more isoforms of GNLY, including the 15 kDa and 9 kDa forms.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 Shows a top view of an example lateral flow immunoassay device (1) illustrating a multiplex configuration comprising a first test line (Tl) and a second test line (T2) (6), a sample application port (3), a cassette housing (4), a result viewing window (5), and a control line (7).

[0018] FIG. 2 Shows a schematic side view of a lateral flow assay strip illustrating the arrangement of a sample pad (8), a conjugate pad (9), a porous membrane (10), and an absorbent pad (11) disposed on a backing card (12).

[0019] FIG. 3 Shows an exploded view of a diagnostic cassette housing illustrating a lower housing portion (13) comprising alignment rails (14) configured to position a lateral flow test strip (15), and a corresponding upper housing portion (16).DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention provides a multiplex immunochromatographic diagnostic system configured for detection of granulysin (GNLY) and at least one secondary biomarker indicative of tissue-specific immune injury within a single biological sample. The system enables structured interpretation of cytotoxic immune activation in combination with contextual tissue-associated biomarker detection, thereby facilitating rapid assessment of immune-mediated pathology, including organ transplant rejection and related conditions.

[0021] The diagnostic system is implemented in certain embodiments as a multiplex lateral flow immunoassay device configured to receive a biological sample, transport the sample through a porous membrane system by capillary action, and generate spatially distinct detection signals corresponding to GNLY, the secondary biomarker, and an internal procedural control.Overall Device Architecture

[0022] Referring to FIG. 1 through FIG. 3, the diagnostic system comprises a lateral flow immunoassay strip (15) positioned within a protective cassette housing (4) having a sample application port (3) and a result viewing window (5).

[0023] The lateral flow strip comprises sequentially overlapping functional membranes mounted on a backing card (12), including:• a sample pad (8)• a conjugate pad (9)• a porous detection membrane, such as a nitrocellulose membrane (10)• an absorbent pad (11)

[0024] These components are arranged in fluid communication such that a biological sample applied to the sample pad migrates sequentially through the conjugate pad and detection membrane and is drawn into the absorbent pad.

[0025] In certain embodiments, the cassette housing includes alignment rails (14) configured to maintain proper positioning of the strip and to ensure consistent visualization of detection zones.Biological Sample Compatibility

[0026] The diagnostic system is configured to operate using biological samples including, but not limited to:• whole blood• plasma• serumcapillary blood obtained by fingerstick

[0027] In certain embodiments, sample volumes between approximately 5 pL and 100 pL are sufficient for detection.

[0028] The sample pad may comprise glass fiber or other porous material treated with surfactants, buffering agents, or separation reagents configured to facilitate fluid transport and, in certain embodiments, partial separation of cellular components from plasma.Detection Reagent and Conjugate System

[0029] The conjugate pad (9) contains labeled detection antibodies configured to bind granulysin and at least one secondary biomarker.

[0030] In certain embodiments, antibodies are conjugated to visible reporter particles configured to produce a detectable signal upon immobilization at a capture zone.Suitable reporter particles include, but are not limited to:• gold nanoparticles• colloidal gold• colored latex particles• dyed polymer microspheres• carbon particles• selenium particles

[0031] In certain embodiments, reporter particles comprise fluorescent labels, including fluorescent nanoparticles, fluorophore-labeled particles, or quantum dots configured for optical detection.

[0032] In certain embodiments, reporter particles comprise magnetic particles configured for magnetic detection.

[0033] In certain embodiments, reporter particles comprise enzymatic labels configured to produce a detectable colorimetric signal.

[0034] In certain embodiments, reporter particles comprise chemiluminescent or electrochemical labels.

[0035] Detection signals may be visually interpreted directly or detected using optical, electronic, magnetic, or fluorescence-based readers.

[0036] The invention is not limited to any specific reporter particle type.Detection Membrane and Capture Zones

[0037] The detection membrane (10) comprises a porous material, such as nitrocellulose, configured to immobilize capture reagents and support capillary transport.

[0038] In certain embodiments, membrane pore size is between approximately 5 pm and 15 pm.

[0039] The membrane comprises spatially distinct capture zones including:a first capture zone configured to bind granulysin-associated detection complexes; a second capture zone configured to bind detection complexes associated with the secondary biomarker;and a control zone configured to capture excess labeled detection reagents to verify proper fluid migration and assay function.

[0040] Capture zones comprise immobilized antibodies or other binding agents specific to the corresponding target biomarker.

[0041] Upon migration of the sample-conjugate mixture through the membrane, biomarker-associated reporter complexes are captured at their corresponding zones, producing visually or instrument-detectable signals.Absorbent Pad and Fluid Transport

[0042] The absorbent pad (11) comprises a porous material configured to draw fluid through the detection membrane by capillary action.

[0043] The absorbent pad provides continuous fluid transport and helps ensure complete migration of the sample across the detection zones.

[0044] In certain embodiments, overlap between adjacent membrane components is approximately 1 mm to 3 mm to ensure uninterrupted capillary flow.Biomarker Detection Configuration

[0045] The diagnostic system is configured to detect granulysin as a primary biomarker indicative of cytotoxic immune effector activity.

[0046] Granulysin is a cytolytic protein released by activated cytotoxic T lymphocytes and natural killer cells during immune-mediated tissue injury.

[0047] In certain embodiments, detection antibodies recognize one or more granulysin isoforms, including:• the approximately 15 kDa isoform• the approximately 9 kDa isoform• fragments or processed forms thereof

[0048] The diagnostic system further detects at least one secondary biomarker indicative of tissue-specific immune injury.

[0049] In certain embodiments, secondary biomarkers include, but are not limited to:• CXCL9• soluble ST2 (sST2)• soluble interleukin-2 receptor (sIL-2R)• soluble CD30 (sCD30)

[0050] Additional biomarkers associated with transplant rejection, immune-mediated tissue injury, or inflammatory conditions may also be used.

[0051] In certain embodiments, the secondary biomarker detection configuration is modular, permitting substitution of capture and detection reagents to support monitoring of different organ systems or disease conditions.Multiplex Detection and Interpretation Framework

[0052] The diagnostic system generates a combined diagnostic output representing detection states of granulysin and the secondary biomarker.

[0053] In certain embodiments, interpretation of detection states includes:presence of granulysin detection indicating cytotoxic immune activation; presence of both granulysin and the secondary biomarker indicating cytotoxic immune activity associated with tissue-specific injury;presence of the secondary biomarker in the absence of granulysin indicating tissue-associated biomarker elevation without detectable cytotoxic activation;absence of both biomarkers indicating no detectable activation within assay sensitivity.

[0054] Interpretation may be performed visually by a user or by automated electronic or optical detection systems.

[0055] In certain embodiments, the diagnostic system may be used repeatedly over time to monitor changes in biomarker levels.Reporter Stabilization and Release

[0056] The conjugate pad may include stabilizing agents including:• sugars such as sucrose or trehalose• proteins such as bovine serum albumin• buffering agents• surfactants

[0057] These agents facilitate preservation and controlled release of reporter-conjugated antibodies.Manufacturing and Assembly

[0058] Membrane components are mounted onto a backing card and cut into strips of defined width, such as between approximately 3 mm and 6 mm.

[0059] The strips may be enclosed within a cassette housing to protect the detection system from environmental exposure.

[0060] In certain embodiments, the device is packaged in moisture-resistant packaging.Alternative Embodiments

[0061] Although certain embodiments utilize a lateral flow immunoassay format, the diagnostic system may also be implemented using alternative rapid immunoassay formats capable of detecting granulysin and at least one secondary biomarker within a single sample.

[0062] Alternative detection formats may include:• microfluidic devices• cartridge-based immunoassays• fluorescence-based immunoassay systems• electrochemical detection systems

[0063] The invention is not limited to a specific assay format.Operation

[0064] A biological sample is applied to the sample pad and migrates through the conjugate pad, where labeled detection antibodies bind to granulysin and the secondary biomarker, if present.

[0065] The resulting complexes migrate to the detection membrane, where they are captured at their respective capture zones.

[0066] Reporter particles accumulate at capture zones, producing detectable signals.

[0067] The presence or absence of signals at the capture zones indicates detection of the corresponding biomarkers.

[0068] The diagnostic process is completed within approximately 5 minutes to 30 minutes in certain embodiments.Scope

[0069] The embodiments described herein are illustrative and not limiting.

[0070] Various modifications may be made without departing from the scope of the invention as defined by the claims.

Claims

CLAIMS1. A diagnostic system configured to determine an immune context in a biological sample, comprising:a detection arrangement configured to detect, within the biological sample:(a) granulysin (GNLY) as a biomarker indicative of cytotoxic immune effector activity; and(b) at least one secondary biomarker indicative of tissue-associated immune activity or tissue injury; andan output configured to generate a diagnostic result based on a combined detection state defined by presence, absence, or level of GNLY and presence, absence, or level of the secondary biomarker,wherein detection of GNLY establishes a cytotoxic immune activation context for interpretation of the secondary biomarker, andwherein the combined detection state enables differentiation between distinct immune conditions including cytotoxic immune activation, localized tissue-associated immune activity, and absence of immune-mediated tissue injury.

2. The diagnostic system of claim 1, wherein the detection arrangement comprises a multiplex lateral flow immunoassay device.

3. The diagnostic system of claim 2, wherein the multiplex lateral flow immunoassay device comprises:a sample pad configured to receive the biological sample;a conjugate pad comprising labeled detection antibodies specific for GNLY and the secondary biomarker;a porous membrane comprising:a first capture zone configured to capture GNLY;a second capture zone configured to capture the secondary biomarker; and a control zone configured to verify assay performance; andan absorbent pad configured to promote capillary flow of the biological sample.

4. The diagnostic system of claim 1, wherein GNLY comprises one or more isoforms of granulysin.

5. The diagnostic system of claim 4, wherein the granulysin comprises a 15 kDa isoform, a 9 kDa isoform, or both.

6. The diagnostic system of claim 1, wherein the secondary biomarker is selected from the group consisting of:CXCL9;soluble ST2 (sST2);soluble CD30 (sCD30);soluble interleukin-2 receptor (sIL-2R);and combinations thereof.

7. The diagnostic system of claim 1, wherein the secondary biomarker comprises a biomarker associated with tissue-specific immune activity, inflammation, or immune-mediated tissue injury.

8. The diagnostic system of claim 1, wherein the diagnostic result is determined based on a plurality of possible combined detection states of GNLY and the secondary biomarker.

9. The diagnostic system of claim 1, wherein interpretation of the secondary biomarker is performed relative to the detection state of GNLY.

10. The diagnostic system of claim 1, wherein the detection arrangement is configurable to detect different secondary biomarkers while maintaining detection capability for GNLY.

11. The diagnostic system of claim 3, wherein the detection antibodies are conjugated to detectable labels selected from the group consisting of:gold nanoparticles;colored particles;fluorescent particles;latex particles;magnetic particles;enzymatic labels;and combinations thereof.

12. The diagnostic system of claim 3, wherein the conjugate pad comprises one or more stabilizing agents.

13. The diagnostic system of claim 12, wherein the stabilizing agents comprise sugars, proteins, surfactants, or combinations thereof.

14. The diagnostic system of claim 13, wherein the stabilizing agents comprise sucrose, trehalose, bovine serum albumin, or combinations thereof.

15. The diagnostic system of claim 3, wherein the porous membrane comprises nitrocellulose.

16. The diagnostic system of claim 15, wherein the porous membrane has a pore size between 5 pm and 15 pm.

17. The diagnostic system of claim 1, wherein the biological sample is selected from the group consisting of:whole blood;plasma;serum;and capillary blood derived therefrom.

18. The diagnostic system of claim 1, configured to produce a diagnostic result within 1 to 30 minutes.

19. The diagnostic system of claim 3, further comprising a housing containing the multiplex lateral flow immunoassay device.

20. The diagnostic system of claim 1, configured for point-of-care use without laboratory instrumentation.

21. The diagnostic system of claim 1, configured for at-home monitoring.

22. The diagnostic system of claim 1, configured to monitor transplant recipients.

23. A method of determining immune context in a biological sample, comprising:detecting granulysin in the biological sample;detecting at least one secondary biomarker in the biological sample;and determining an immune condition based on a combined detection state defined by presence, absence, or level of granulysin and presence, absence, or level of the secondary biomarker.

24. The method of claim 23, further comprising repeating detection at multiple time points.

25. A diagnostic kit comprising the diagnostic system of claim 1 and instructions for determining immune context based on combined biomarker detection.

26. A multiplex immune monitoring platform comprising:a primary detection channel configured to detect granulysin;and a secondary detection channel configured to detect a selectable tissue-associated biomarker;wherein the platform generates a contextual immune interpretation based on combined detection states.

27. The platform of claim 26, wherein the secondary detection channel is interchangeable.

28. The diagnostic system of claim 1, wherein the output comprises visually detectable signals corresponding to biomarker detection states.

29. The diagnostic system of claim 1, further comprising an electronic reader configured to:detect signals corresponding to GNLY and the secondary biomarker;determine the combined detection state;and generate an interpreted immune context output.

30. The diagnostic system of claim 1, wherein the system is configured to detect immune activation and tissue-associated immune activity in a single assay.

31. The diagnostic system of claim 1, wherein the diagnostic result is determined based on whether GNLY exceeds a defined concentration threshold indicative of cytotoxic immune activation.

32. The diagnostic system of claim 31, wherein the defined concentration threshold corresponds to a clinically relevant cutoff.

33. The diagnostic system of claim 29, wherein the electronic reader comprises a portable reader, cartridge reader, or smartphone-based optical detection system.

34. The diagnostic system of claim 1, configured for monitoring transplant rejection.

35. The diagnostic system of claim 34, wherein the transplanted organ is selected from the group consisting of: kidney, heart, lung, liver, pancreas, and composite tissue grafts.

36. The method of claim 23, further comprising determining whether GNLY exceeds a predefined concentration threshold.

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