WO2021205433A1

WO2021205433A1 - Lateral flow test utensils

Info

Publication number: WO2021205433A1
Application number: PCT/IL2021/050374
Authority: WO
Inventors: Dekel Tzidon; Amos SOMMER; Aviv Tzidon
Original assignee: Rapid Bio Pass Ltd.
Priority date: 2020-04-05
Filing date: 2021-04-05
Publication date: 2021-10-14

Abstract

Lateral flow test utensils, testing kits and diagnostic methods are provided. The test utensils comprise, on one or more interconnected pad(s) made of capillary material and consecutively along a flow path of a sample: a sample-receiving region, optionally an antibodies-separation region configured to separate different types of immune components in the sample, a marking region comprising marked pathogen parts, a fixation region comprising fixated pathogen parts, and a control region. Marked pathogen parts (pathogen part(s) attached to indicator particles) are bonded by antibodies and/or T-cells in the sample, flow along pad(s) of the utensil and are then immobilized by fixated pathogen parts (rather than binding to anti-human antibodies as in the prior art) - to enhance the analytical sensitivity of the test. Disclosed lateral flow tests enable wide range, possibly home -use sensitive serological testing, allowing population-wide epidemiological monitoring.

Description

LATERAL FLOW TEST UTENSILS

BACKGROUND OF THE INVENTION

1. TECHNICAL FIELD

[0001] The present invention relates to the field of serological testing, and more particularly, to lateral flow immunochromatography.

2. DISCUSSION OF RELATED ART

[0002] Lateral flow tests (or lateral flow immunochromatographic assays) are configured to allow a liquid sample (e.g., containing bodily fluids such as blood, urine, or saliva) to run along a pad or interconnected pads made of capillary material(s) (e.g., porous paper, or polymeric material, e.g., microporous nitrocellulose) and interact with conjugates (reactive molecules and/or tags) along the flow direction. Typically, conjugates are present along the path of the flow and may interact with specific (target) components in the sample, such as certain antibodies or other proteins. The marked components flow on and are fixed at specific test lines, while other sample components are indicated by attachment to affinity ligands at a control line - to indicate that the sample indeed flowed along the pad(s) of the test. The indications are typically provided by colored particles (e.g., gold nanoparticles or latex particles), or possibly by fluorescent or magnetic particles.

[0003] Management of novel pandemics, such as the COVID-19 (Coronavirus disease 2019, also termed SARS-CoV-2 for severe acute respiratory syndrome - SARS -coronavirus 2) pandemic requires efficient tests to indicate the immunity status of many individuals at the population level.

SUMMARY OF THE INVENTION

[0004] The following is a simplified summary providing an initial understanding of the invention. The summary does not necessarily identify key elements nor limit the scope of the invention, but merely serves as an introduction to the following description.

[0005] One aspect of the present invention provides a lateral flow test utensil comprising, on one or more interconnected pad(s) made of capillary material and consecutively along a flow path of a sample: a sample-receiving region, an antibodies-separation region configured to separate different types of immune components in the sample, a marking region comprising marked pathogen parts, a fixation region comprising pathogen parts that are anchored to the capillary material, the fixation region providing a test line, and a control region, wherein, in operation, immune components in the sample flowing along the utensil are separated by type, bind to the marked pathogen parts and are then fixated at the test line by further binding to the anchored pathogen parts.

[0006] One aspect of the present invention provides a lateral flow test utensil comprising, on one or more interconnected pad(s) made of capillary material and consecutively along a flow path of a sample: a sample-receiving region, a marking region comprising marked pathogen parts, a fixation region comprising pathogen parts that are anchored to the capillary material, the fixation region providing a test line, and a control region, wherein, in operation, any pathogen-recognizing immune components in the sample flowing along the utensil may bind to the marked pathogen parts and may then be fixated at the test line by further binding to the anchored pathogen parts. For example, disclosed embodiments broaden the range of possible virus-recognizing immune components that can be detected by the disclosed tests.

[0007] One aspect of the present invention provides a diagnostic method comprising: configuring a lateral flow test to receive a sample, separating pre -defined types of immune components in the sample, leaving specified types of immune components to flow along the lateral flow test, using marked pathogen parts for binding the specified types of pathogen-recognizing immune components, and fixating the specified types of immune components to anchored pathogen parts at a test line of the lateral flow test, wherein the separating and the marking enhance an analytical sensitivity of the lateral flow test.

[0008] One aspect of the present invention provides a diagnostic method comprising using marked pathogen parts and anchored pathogen parts in a lateral flow test to enhance an analytical sensitivity of the test, wherein immune components in a sample applied to the lateral flow test bind to the marked pathogen parts and are then fixated by further binding to the anchored pathogen parts. [0009] These, additional, and/or other aspects and/or advantages of the present invention are set forth in the detailed description which follows; possibly inferable from the detailed description; and/or learnable by practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] For a better understanding of embodiments of the invention and to show how the same may be carried into effect, reference will now be made, purely by way of example, to the accompanying drawings in which like numerals designate corresponding elements or sections throughout.

[0011] In the accompanying drawings: [0012] Figures 1A-1C are high-level schematic illustrations of a prior art test utensil (illustrated in Figure 1A) compared with test utensils (illustrated in Figures IB and 1C) according to some embodiments of the invention.

[0013] Figure ID is a high-level schematic illustration of the separation and/or saturation of some immunoglobulin types, according to some embodiments of the invention.

[0014] Figure IE is a high-level schematic illustration of a testing kit comprising a plurality of test utensils, according to some embodiments of the invention.

[0015] Figures 2A-2D illustrate schematically different types of marking and of fixation in test utensils, according to some embodiments of the invention.

[0016] Figure 3 provides experimental data illustrating the sensitivity of disclosed test utensils, according to some embodiments of the invention.

[0017] Figure 4 is a high-level flowchart illustrating a diagnostic method, according to some embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION [0018] In the following description, various aspects of the present invention are described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the present invention. However, it will also be apparent to one skilled in the art that the present invention may be practiced without the specific details presented herein. Furthermore, well known features may have been omitted or simplified in order not to obscure the present invention. With specific reference to the drawings, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0019] Before at least one embodiment of the invention is explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is applicable to other embodiments that may be practiced or carried out in various ways as well as to combinations of the disclosed embodiments. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.

[0020] Embodiments of the present invention provide efficient and economical methods and mechanisms for testing with lateral flow test utensils, e.g., home serological testing, and thereby provide improvements to the technological field of epidemiological monitoring such as improving the sensitivity and specificity of the tests. Lateral flow test utensils, testing kits and diagnostic methods are provided. The test utensils comprise, on one or more interconnected pad(s) made of capillary material and consecutively along a flow path of a sample: a sample-receiving region, a marking region comprising marked pathogen parts, a fixation region comprising fixated pathogen parts, and a control region.

[0021] Marked particles comprise pathogen part(s) attached to indicator (e.g., colored) particles. Antibodies and/or T-cells in the sample that are specific to the marked pathogen part(s) are bonded to them and thus become marked as well. The sample, which now includes the immune components that bind to the marked particles flows along pad(s) of the utensil. The marked immune components are then anchored by fixated pathogen parts to which the marked immune components (e.g., antibodies and/or T-cells) bind - to enhance the analytical sensitivity of the test. Optionally, antibodies of one or more types may be removed before the anchoring to reach Ig-type-specific results or even T-cell-specific results.

[0022] Figures 1A-1C are high-level schematic illustrations of a prior art test utensil 20 (illustrated in Figure 1A) compared with test utensil 120 (illustrated in Figures IB and 1C), according to some embodiments of the invention.

[0023] Figure 1A illustrated schematically a prior art test utensil 20 comprising a lateral flow test for identifying human antibodies against a target molecule (e.g., a part of a pathogen such as a virus). Prior art test utensil 20 comprises a pad 28 or interconnected pads 28 made of capillary material(s) (e.g., porous paper, or polymeric material, e.g., microporous nitrocellulose) that receive the sample and let it flow in a flow direction 29 along pad(s) 28, through multiple regions that mark and/or fixate proteins in the sample. The sample typically comprises a drop of blood/plasma/serum, which is placed on a sample area 21 on utensil 20, and may optionally be followed by a drop of running buffer. The sample typically contains antibodies that include IgM and IgG type antibodies and possibly additional types of antibodies. Conjugates comprise viral parts attached to colored particles, set at identification area 22. During the flow of the plasma (along flow direction 29), antibodies in the plasma (IgM and/or IgG) may attach to the viral particles, bind them, and continue to flow along utensil 20. The flowing antibodies are then caught and fixated by respective proteins, such as anti-human IgM antibodies and/or anti-human IgG antibodies anchored at test lines 23 and/or 24, respectively. Test reading may be carried out optically by identifying the colored particles that are bonded to the respective fixated antibodies. A control line 25 includes an indication of sample flow to verify the test was carried out correctly.

[0024] The inventors note that a significant drawback of prior art test utensil 20 and related testing methods is that the signal of specific antibodies which bind to the pathogen (e.g., virus) parts is mixed or diluted within the nonspecific antibodies in human blood, which are also fixated on the respective test line. Therefore, in the prior art, non-colored bonded antibodies weaken the signal of the colored bonded antibodies. Moreover, the intensity of the test results (signals) depends on the ratio between colored and non-colored antibodies, potentially masking or making invisible weak positive test results. This disadvantage may prevent early-stage infection detection, when a small minority of the respective antibodies are able to bind the pathogen (e.g., virus) particles.

[0025] In the following, modified lateral flow tests and utensils 120 are disclosed to overcome this disadvantage and increase the analytical sensitivity of lateral flow test utensils 120, especially to weak signals.

[0026] Figure IB illustrated schematically a test utensil 120 comprising a lateral flow test for identifying human immune response (e.g., identified antibodies and/or activated T-cells) against parts of a target pathogen (e.g., virus), according to some embodiments of the invention. Test utensil 120 comprises pad 128 or interconnected pads 128 made of capillary material(s) (e.g., porous paper, or polymeric material, e.g., microporous nitrocellulose, pads 128 may be made of interconnected multiple types of materials) that receive the sample and let it flow in a flow direction 129 along pad(s) 128, through multiple regions that mark and/or fixate proteins in the sample. Lateral flow test utensil 120 may comprise, on one or more interconnected pad(s) made of capillary material and consecutively along flow path 129 of the sample, a sample-receiving region 121, a marking region 123 comprising marked pathogen parts (e.g., comprising pathogen, e.g., virus parts 113A attached to colored, or otherwise detectable particles 112), a fixation region 124 providing a test line and comprising fixated pathogen parts 114B, e.g., anchored to the capillary material, further along flowing path 129, and a control region 125.

[0027] In operation, any pathogen-recognizing immune components in the sample flowing along the utensil may bind to the marked pathogen parts and may then be fixated at the test line by further binding to the anchored pathogen parts. For example, disclosed embodiments broaden the range of possible virus-recognizing immune components that can be detected by the disclosed tests.

[0028] The sample, typically comprising a drop of blood, plasma and/or serum (with IgM, IgG and possibly other Ig types of antibodies and/or T cells and/or possibly other types of immune components) may be placed on sample area 121 of utensil 120. Marked pathogen parts comprise pathogen/viral parts 113A attached to colored, or otherwise detectible particles 112 (used as indicator particles, e.g., gold nanoparticles of latex particles, possibly fluorescent or magnetic particles) that are set at marking region 123. During the flow of the sample (along flow direction 129), immune components in the sample may attach to the marked pathogen/viral particles, bind them, and continue to flow along flow path 129 on utensil 120. The flowing marked complexes may then be caught and be fixated by fixated pathogen parts 114B at fixation (identification) region 124 - leaving unmarked antibodies 111 to continue their flow along flow direction 129 and thereby intensifying the signal with respect to prior art tests 20. Moreover, it is noted that disclosed tests 120, by virtue of using fixated (anchored) pathogen parts, accumulates all types of immune components at identification region 124 - further enhancing the resulting signal even beyond the avoidance of the dilution effect discussed above. Control region 125 may be configured to indicate the occurrence of sample flow over utensil 120, possibly by immobilizing remaining, non-bonding antibodies 111.

[0029] It is noted that marked pathogen parts may be used in utensil 120 at an amount sufficient to bind most or all of the expected amount of antibodies in the sample, to reach a strong identification signal. It is further noted that the inventors have found out that even though antibodies bind to marked pathogen parts at marking region 123, they are still able to further bind to fixated pathogen parts at fixation region 124, singly and/or in complexes, at the same and/or at different binding sites, to enable signal identification.

[0030] In certain embodiments, illustrated schematically in Figure 1C and ID below, lateral flow test utensil 120 may further comprise an antibodies-separation region 122 configured to separate different types of immune components in the sample, to further enhance the analytical sensitivity of disclosed tests. Antibodies-separation region 122 may be set upstream of fixation region 124, possibly upstream of marking region 123, and possibly at least partly at sample area 121 or even as part of a running buffer or a solution mixed with the sample. Antibodies-separation region 122 may be configured to immobilize, mark and/or saturate binding sites (e.g., light chains) of one or more types of immunoglobulin and allow the further flowing of other types of immunoglobulin and/or other types of immune components, as disclosed in more detail below.

[0031] It is noted that while ELISA (enzyme -linked immunosorbent assay) serological tests may be designed to utilize pathogen parts, disclosed lateral flow tests are usable for home testing due to their simplicity of use and lower price, enabling quick and simple population wide testing that may provide epidemiological monitoring, e.g., in case of new pandemics. Moreover, disclosed lateral flow tests may be used as indicators of the state of immunity without need for elaborate sampling and laboratory procedures, simplifying significantly and practically enabling wide range sensitive serological testing.

[0032] Advantageously, the resulting analytical sensitivity for the test results may be significantly higher than in prior art tests 20, because the marked antibodies are separated from the diluting unmarked antibodies (reducing the extent of dilution), and furthermore, antibodies of different types are fixated at the same region (increasing the signal at the test line). It is noted that as the typical fraction of antigen-specific antibodies out of the total immunoglobulins ranges from 2% to 25% depending on the immunization effectivity - the enhancement of test sensitivity in disclosed tests by preventing Ig dilution may reach 4 to 6 -folds.

[0033] It is noted that disclosed lateral flow test utensil 120 may be used for serological testing at home, allowing large scale serology -based epidemiological monitoring of a population, that would be beneficial in coping with outbreaks of pandemics.

[0034] It is further noted that while the color indication is generally qualitative, various methods may be applied to derive a quantitative signal from the parameters of the resulting test lines, such as color density, or other properties, e.g., when other types of particles are used. For example, image processing may be used to derive quantitative data from the resulting signals at fixation region 124. In such cases, the disclosed improvement of the analytical sensitivity (reducing sample dilution) of tests 120 not only lowers the detection threshold but also provides a better signal to noise ratio as well as higher resolution and accuracy of the quantitative test results. For example, color signals may be more salient and/or background noise in fluorescent or magnetic measurements may be less significant and more easily removable compared to the improved signal achieved by disclosed utensils 120.

[0035] It is further noted that the disclosed lower detection threshold is particularly beneficial for detection of immunologic response to novel vims outbreaks as well as for epidemiological population monitoring - e.g., earlier detection and earlier management activities (e.g., immediate follow-up testing and isolation) may be enabled by disclosed tests 120. Earlier detection is provided both by the high analytical sensitivity of disclosed utensils 120 as explained above, as well as by the ability of utensils 120 to identify early immune response by detecting IgM over background IgG. As explained in embodiments below, disclosed lateral flow test utensil 120 may be configured to specifically detect IgM which indicates early stages of the immune response by introducing an additional antibody separation region for separating different types of Ig antibodies before fixation region 124, further enhancing the ability of disclosed test to provide early detection. Additionally, disclosed tests 120 with enhanced accuracy and sensitivity may also be used to reduce the error and safety margins in applying population control measures to the necessary minimum (e.g., by identifying early immunity), and therefore further improve the coping with new epidemics.

[0036] Figure 1C illustrated schematically a test utensil 120 comprising a lateral flow test for identifying human antibodies against a target pathogen (e.g., virus), according to some embodiments of the invention. Figure ID is a high-level schematic illustration of the separation and/or saturation of some immunoglobulin types (denoted schematically as “IgX”), according to some embodiments of the invention.

[0037] Test utensil 120 comprises pad 128 or interconnected pads 128 made of capillary material(s) (e.g., porous paper, or polymeric material, e.g., microporous nitrocellulose, pads 128 may be made of interconnected multiple types of materials) that receive the sample and let it flow in a flow direction 129 along pad(s) 128, through multiple regions that may mark and/or fixate proteins in the sample. Lateral flow test utensil 120 may comprise, on one or more interconnected pad(s) made of capillary material and consecutively along flow path 129 of the sample, a sample-receiving region 121, antibodies-separation region 122 configured to separate different types of antibodies in the sample, marking region 123 comprising marked pathogen parts (e.g., comprising pathogen, e.g., virus parts 113A attached to colored, or otherwise detectable particles 112), fixation region 124 comprising fixated pathogen parts 114B e.g., anchored to the capillary material, further along flowing path 129, and control region 125.

[0038] Antibodies-separation region 122 may be set upstream of (before) marking region 123, and may be configured in various ways to separate different types of antibodies, denoted schematically as IgX (e.g., one or more of IgG, IgM, IgA etc.) and are separated by anti-IgX antibodies of human and/or animal source. Separation may be carried out, e.g., by binding and removing certain types of antibodies (“IgX”), saturating the binding sites (e.g., the light chains) on certain types of antibodies (“IgX”) to prevent their marking at marking region 123 and/or their fixation at fixation region 124, marking of certain types of antibodies (“IgX”), e.g., to allow for optical distinction between types of antibodies, or otherwise providing a distinction between different types of antibodies in the sample. For example, certain immune components may be prevented from binding to marked pathogen parts and to anchored pathogen parts by binding their epitopes with corresponding antibodies.

[0039] For example, separation of specific immunoglobulin type(s) may be carried out using large amounts of antibodies affixed to the respective pad region 122. Antibodies-separation region 122 may thus be configured to bind and immobilize the specific immunoglobulin type(s) IgX. In certain embodiments, the specificity of separation of different types of Ig’s may be achieved by specific molecules (e.g., human or animal antibodies) specifically binding the respective different heavy chains a, d, e, g, and m of IgA, IgD, IgE, IgG, and IgM, respectively. In certain embodiments, the specific immunoglobulin type(s) IgX may be marked differently than other immune components to yield a test which detects both types of IgX simultaneously, e.g., at different regions on utensil 120.

[0040] In certain embodiments, antibodies-separation region 122 may be set (at least partly) at sample-receiving area 121 or even set (at least partly) within buffer fluid 121A or a solution added to the sample (illustrated schematically in Figure ID). Separation and/or removing (e.g., by saturation of the binding sites) of one or more types of immunoglobulins may be carried out at one or more location prior upstream of fixation region 124, as illustrated schematically in Figure ID. For example, specific molecules selected to bind the light chains of one or more types of immunoglobulins IgX may be added to the buffer fluid, to a solution and/or to sample-receiving area 121 and use to saturate these types of immunoglobulins IgX - to block and/or prevent their anchoring at fixation region 124 and make the detected signal more specific to un-saturated types. For example, IgG’s may be prevented this way from binding at fixation region 124, allowing mainly IgM’ s (denoting early immune response) to bind. In certain embodiments, saturation of the binding sites (e.g., the light chains) on certain types of IgX may be used merely to prevent their marking (e.g., to prevent their attachment to gold nanoparticles) and not necessarily to prevent their fixation. Un-colored IgX then are not visible optically even if they are fixated.

[0041] In certain embodiments, all Ig’s may be saturated or separated away before fixation region 124 - allowing detection of immune factors other than immunoglobulins to be detected, even if they appear at a much lower concentration in the sample than the Ig’s. For example, T cells or other immune factors may be detected this way, possibly indicating an even earlier stage of the immune response than IgM development. It is notes that by removing immunoglobulins and allowing remaining immune components to bind to the marked pathogen parts - disclosed test may be configured to detect immune components other than Ig’s that react to the pathogen, even if these are present at a low concentration, enhancing testing sensitivity specifically with respect to these immune components. In certain embodiments, these components may be further marked in various ways to enhance detection, e.g., by binding colored particles to T-cells or marking them in other ways. Advantageously, disclosed tests may enable detection of non-Ig immune components that are typically ignored in prior art lateral flow tests, possibly allowing for detection of early immune reactions. Specifically, using pathogen parts in test utensils 120 rather than specific antibodies as in the prior art does not limit the type of immune components that can be detected by test utensils 120.

[0042] In certain embodiments, following the introduction of the sample at sample -receiving region 121, antibodies-separation region 122 may be configured to include a substantial amount of human and/or animal antibodies to IgG type antibodies - removing IgG type antibodies and leaving IgM antibodies in the sample flowing towards marking region 123. The anti-IgG type antibodies may be fixed at region 122 and remove the IgG molecules from the sample. The remaining IgM type antibodies may then bind the marked pathogen/viral parts at marking region 123 and move on to be detected at fixation region 124 as disclosed above, providing the disclosed improved analytical sensitivity of the test, as well as specificity to the type of antibody provided by the introduction of antibodies-separation region 122.

[0043] Advantageously, disclosed IgM-specific tests 120 may be used for early detection of immunological response, for distinguishing early immunological response characterized by a large amount of IgM from late or past immunological response characterized by a large amount of IgG and/or to identify and distinguish second from first infection, which may be beneficial for various viral diseases (e.g., Flaviviruses like the Dengue and the Zika viruses), as the second infection may put the patient at risk for more severe symptoms, that, given disclosed test 120, may be handled earlier and more efficiently. It is noted that disclosed IgM-specific tests 120 may be particularly beneficial as the blood levels of IgM are typically much lower than the blood levels of IgG, so that IgG’s typically mask IgM’s by their abundance. However, detection of early immune response may be carried out most reliably by detecting the level of IgM, providing an important benefit of disclosed IgM-specific tests 120. [0044] Along similar lines, disclosed tests for IgG may be provided. In certain embodiments, following the introduction of the sample at sample -receiving region 121, antibodies-separation region 122 may be configured to configured to include a substantial amount of human and/or animal antibodies to IgM type antibodies - removing IgM type antibodies and leaving IgG antibodies in the sample flowing towards marking region 123. The anti-IgM type antibodies may be fixed at region 122 and remove the IgM molecules from the sample. Examples for anti-IgM type antibodies include any of animal antibodies to human IgM light chain antibodies and/or to the whole molecules and/or to the Fc part (fragment crystallizable region) of the IgM type immunoglobulins. The remaining IgG type antibodies may then bind the marked pathogen/viral parts at marking region 123 and move on to be detected at fixation region 124 as disclosed above, providing the disclosed improved analytical sensitivity of the test, as well as specificity to the type of antibody provided by the introduction of antibodies-separation region 122.

[0045] Along similar lines, disclosed tests for IgA may be provided. In certain embodiments, following the introduction of the sample at sample -receiving region 121, antibodies-separation region 122 may be configured to configured to include a substantial amount of human and/or animal antibodies to IgG and IgM antibodies - removing both IgG and IgM antibodies and leaving IgA antibodies in the sample flowing towards marking region 123. In case of saliva or mucus samples, slgA (secretory IgA) may be detected by similarly removing IgG’s and IgM’s. The anti- IgG and anti-IgM antibodies may be fixed at region 122 and remove the IgG and IgM molecules from the sample. Examples for anti-IgG and anti-IgM antibodies include any of animal antibodies to human IgG or IgM light chain antibodies and/or to the whole molecules and/or to the Fc part of the IgG or IgM type immunoglobulins, respectively. The remaining IgA type antibodies may then bind the marked pathogen/viral parts at marking region 123 and move on to be detected at fixation region 124 as disclosed above, providing the disclosed improved analytical sensitivity of the test, as well as specificity to the type of antibody provided by the introduction of antibodies-separation region 122.

[0046] Advantageously, detecting IgA separately from IgG and IgM may be beneficial in serological tests of saliva or mucus, which typically include IgA secreted by mucous membranes, and may be used to indicate early stages of infection. It is noted that either IgA and/or its secreted form slgA may be detecting along these lines.

[0047] In various embodiments and depending on the type of the sample(s), any type of immunoglobulin may be separated from other types, e.g., IgE (e.g., in relation to allergy detection), IgD (e.g., as indication of a corresponding stage of the immune response related to B-cell maturation), etc., using similar configurations and respective anti-Ig antibodies. The target Ig may be left free to move and detected at marking region 123.

[0048] In certain embodiments, antibodies may be filtered or otherwise separated (e.g., by binding site saturation) to leave T cells (or other immune components other than immunoglobulins) to be detected at marking region 123, e.g., using blood samples.

[0049] In certain embodiments, antibodies-separation region 122 may be part of (or overlap) sample-receiving region 121, and/or possibly part of (or overlap) marking region 123 - to prevent the respective antibody type(s) from continuing to flow in the direction of fixation region 124. [0050] Figure IE is a high-level schematic illustration of a testing kit 110 comprising a plurality of test utensils 120, according to some embodiments of the invention. In a non-limiting manner, testing kit 110 may comprise any number of test utensils 120 of one or more types, to enable repeated testing and/or testing of different sample components such as different immunological components (e.g., different types of immunoglobulins and/or T-cells). For example, testing kit 110 may comprise one or more test utensil(s) 120 for measuring IgM (configured to remove IgG as disclosed above), one or more test utensil(s) 120 for measuring IgG (configured to remove IgM as disclosed above) and/or one or more test utensil(s) 120 for measuring T-cells (configured to remove immunoglobulins as disclosed above). Testing kits 110 may comprise any of the combinations of test utensils 120 disclosed herein (illustrated schematically in Figure IE), including various utensil types with respect to the type of separated Ig(s), the pathogen types used (see, e.g., Figures 2A-2D below), different methods of separation and/or different types of marking and fixation. Specifically, different utensils 120 in kit 100 may be different or same with respect to the type of separated immune components (the latter configured for reiterating testing). Multiple test utensils 120 of one or more test type may be included in kits 110, e.g., for enabling recurrent testing. Testing kit 110 may further comprise instructions for use with respect to repeated tests and/or testing of different immunological components.

[0051] In certain embodiments, disclosed test utensils 120 may be configured to include parts of any pathogen, such as viruses, bacteria, fungi, and/or eucaryotic (single cell or multiple cells) parasites. The following examples related to viruses, and specifically to coronaviruses is therefore not limiting.

[0052] Figures 2A-2D illustrate schematically different types of marking and of immobilization in test utensils 120, according to some embodiments of the invention. In the non-limiting case of virus detection, either marked viral parts (attached to indicator nanoparticles at marking region 123) and/or fixated viral parts (at fixation region 124) may comprise (protein) nucleocapsid and/or (glycoprotein) spike viral parts.

[0053] In homologous tests (illustrated schematically in Figures 2A and 2B), the same viral parts (113A and 113B in Figure 2A, e.g., nucleocapsids; 114A and 114B in Figure 2B, e.g., spikes) are used as marked viral parts bond to nanoparticles 112 and applied to marking region 123 of utensil and as fixated viral parts 113B, 114B (respectively) at fixation region 124. In heterologous tests (illustrated schematically in Figures 2C and 2D), different viral parts (114A and 113B in Figure 2C; 113A, 114A and 114B in Figure 2D) are used as marked viral parts bond to nanoparticles 112 and applied to marking region 123 of utensil and as fixated viral parts 113B, 114B (respectively) at fixation region 124.

[0054] The inventors have found out that in certain cases, a number of antibodies (or other immune components) may interact to form complexes 116 which may bind to more than one antigen. For example, as illustrated schematically by complex 116 in Figure 2C, antigens in complex 116 may bind to both marked pathogen parts 114A and to anchored pathogen parts 113B. Moreover, the inventors have found out that the antibodies (or other immune components) in complex 116 do not necessarily have to be antibodies against the same pathogen. For example, complex 116 may comprise antibodies against different pathogen parts (e.g., nucleocapsid and spike proteins) and/or possibly antibodies against pathogen parts in different pathogen (e.g., proteins from COVID 19, seasonal corona viruses, SARS, etc.). The inventors term this type of binding heterologous binding in the sense that given antibodies, when associated within complex 116, may bind multiple different pathogen parts. As a result, pathogen parts that are used for marking the immunological components and pathogen parts that are used for fixating the marked immunological components may comprise different pathogen parts or parts from different (related) pathogens, as disclosed in further detail below. Evidence for this type of interaction are provided in Table 1 and in Figure 3 discussed below.

[0055] The inventors further note that the heterologous approach may be applied also to ELISA tests. While present ELISA tests may employ homologous recognition of pathogen parts (with antibodies binding to the same pathogen parts used to mark the antibodies as well as to fixate the antibodies), disclosed heterologous ELISA tests may comprise heterologous assays as illustrated, e.g., in Figures 2C and 2D. Disclosed heterologous ELISA tests may thus comprise marked pathogen parts 113A, 114A configured to bind to immune components in a sample, to yield marked immune components, and anchored pathogen parts 113B, 114B configured to fixate the marked immune components, wherein marked pathogen parts 113A, 114A and anchored pathogen parts 113B, 114B are different from each other, e.g., different proteins from the same pathogen (e.g., a nucleocapsid protein and a spike glycoprotein of a virus) and/or proteins from different (related) pathogens.

[0056] Figure 3 and Table 1 provide experimental data illustrating the sensitivity of disclosed test utensils, according to some embodiments of the invention. Test line intensity is provided (in arbitrary units) for use of nucleocapsid and spike proteins as the pathogen parts to detect corresponding immunoglobulins, after varying number of days passed since the appearance of symptoms. A threshold line is presented in Figure 3 to illustrate the detection threshold, and a 8- days line (from the appearance of symptoms) is presented to indicate that the test sensitivity up to the 8^th day was 82% while the test sensitivity after the 8^th day was 95% (excluding the false negative cases). It is noted that the high test sensitivity at early stage may be understood to indicate high test sensitivity even before symptoms appear. [0057] Table 1 provides experimental data comparing the signal intensity for multiple types of homologous and heterologous assay models represented schematically in Figures 2A-2D, with SARS CoV-2 (PCR confirmed) plasma samples. The respective viral parts were fixated to nitrocellulose (NC) as pad material 128 at fixation region 124. The nanoparticles to which the respective viral parts were bonded were gold nanoparticles.

Table 1: Comparison of signal intensity for homologous and heterologous tests.

Table 1 (continued): Comparison of signal intensity for homologous and heterologous tests.

[0058] In certain embodiments, ELISA (enzyme -linked immunosorbent assay) serological tests may also be configured as heterologous tests, along the lines disclosed above. [0059] In certain embodiments, disclosed test utensils 120 may be configured to provide dual identification of specific viruses. For example, dual identification of antibodies against SARS- CoV-2 may be carried out using segment(s) of viral nucleocapsid protein (113B) on the NC membrane as test line (fixation region 124) and viral spike protein (114A) bound to colored particles (nanoparticles 112), as illustrated schematically in Figure 2C (and see Table 1 for initial results).

[0060] In certain embodiments, disclosed test utensils 120 may be configured to provide dual identification of specific viruses. For example, dual identification of antibodies against SARS- CoV-2 may be carried out using segment(s) of viral spike protein (114B) on the NC membrane as test line (fixation region 124) and viral nucleocapsid protein (113A) bound to colored particles (nanoparticles 112), as illustrated schematically in Figure 2D, but without the additional viral spike protein 114A bound to nanoparticles 112 (and see Table 1 for initial results).

[0061] In certain embodiments, disclosed test utensils 120 may be configured to detect viral parts that are shared by several pathogens (e.g., viruses) of the same genus of family. Such configurations may be useful to indicate a level of immunity to a new pathogen in a population exposed to existing related pathogen(s). For example, protein(s) (or peptides) specific to COVID-19 as an example for a new pathogen may be detected in relation to more general proteins (or peptides) of coronaviruses, such as proteins (or peptides) known for other SARS viruses. Such configurations may be used to characterize potent immunity in the population that is continuously exposed to new emerging viruses from the same family.

[0062] In certain embodiments, marked pathogen parts and fixated pathogen parts may belong to pathogens at different taxonomic levels, to distinguish broader from narrower immunity, with respect to variants of specific pathogens.

[0063] For example, referring to Figure 2C as a non-limiting example, more general proteins may be used as marked pathogen parts represented by numeral 114A (attached to the nanoparticles) while more specific proteins may be used as fixated pathogen parts represented by numeral 113B (immobilizing the antibodies). It is noted that the specific proteins are specific to a virus taxon (e.g., subspecies) and the general proteins characterize a higher virus taxon (e.g., species, respectively) and/or multiple variants of the virus. For example, the more general proteins may comprise one or more epitopes, or epitope parts, which characterize an aggressive variant (e.g., SARS corona viruses, or aggressive flu viruses) as well as less aggressive variants (e.g., non-SARS corona viruses or less aggressive seasonal flu viruses), while the specific proteins may comprise one or more epitopes, or epitope parts, which characterize only the aggressive variant. In such away, a partial immunity, resulting to exposure to less aggressive strains (variants), may be identified. The respective antibodies may bind these epitopes with their light chains, singly or as complexes 116 (see above).

[0064] In certain embodiments, referring to Figure 2D as an example, a mix of specific and general proteins may be used as marked pathogen parts represented by numerals 113A and 114A (attached to the nanoparticles) while more general proteins may be used as fixated pathogen parts represented by numeral 114B (fixating the antibodies). Such assay design may be used to detect the correct peptides representing shared epitopes between a specific pathogen and other species or subtypes sharing the same genus or family.

[0065] In certain embodiments, homologous tests using more general proteins may be used to detect potential immunity to specific new pathogens (e.g., viruses) - by testing asymptomatic patients and identifying the most prominent proteins belonging to the same virus family to which immunity is exhibited within the group of asymptomatic patients. It is noted that antibodies and/or T cells of the checked patients may be used as indicators of some level of immunity. [0066] It is noted that elements from Figures IB- IE and 2A-2D may be combined in any operable combination, and the illustration of certain elements in certain figures and not in others merely serves an explanatory purpose and is non-limiting.

[0067] Figure 4 is a high-level flowchart illustrating a diagnostic method 200, according to some embodiments of the invention. The method stages may be carried out with respect to test utensils 120 described above, which may optionally be configured to implement method 200. Method 200 may comprise any of the following stages, irrespective of their order.

[0068] Diagnostic method 200 comprises using marked pathogen parts and fixated pathogen parts (e.g., anchored to the substrate) in a lateral flow test to enhance the analytical sensitivity of the test (stage 210). Immune components in a sample applied to the lateral flow test bind to the marked pathogen parts and are then fixated by further binding to the anchored pathogen parts.

[0069] In certain embodiments, diagnostic method 200 may comprise configuring a lateral flow test to receive a sample (stage 205), optionally separating pre-defined types of immune components in the sample, leaving specified types of immune components to flow along the lateral flow test (stage 212), using marked pathogen parts for binding the specified types of immune components (stage 214) and fixating the specified types of immune components to anchored pathogen parts at a test line of the lateral flow test (stage 216). Separating 212 and marking 214 may be configured to enhance the clinical sensitivity of the lateral flow test, e.g., by accumulating multiple types of immune components that bind to the specific pathogen parts, by isolating one type of immune component that binds to the specific pathogen parts (e.g., IgM which indicates early immune response) and/or by measuring non-Ig immune components that may bind to the specific pathogen parts.

[0070] In certain embodiments, separating antibodies by type 212 may be carried out e.g., prior to marking 214 and/or prior to fixating 216 to yield type-specific testing, such as, for example, removing IgG antibodies to yield an IgM test (stage 222), or removing IgM antibodies to yield an IgG test (stage 224), or removing IgG and IgM antibodies to yield an IgA and/or slgA test (stage 225), and/or removing all antibodies to yield a T-cell test (stage 226), optionally marking the T cells, or other immune components remaining after the Ig’s have been removed. Separation 212 may comprise any of the following: (i) immobilizing or anchoring removed molecules, (ii) marking removed molecules in a way that enables distinguishing them from molecules bonded to the marked pathogen parts, and/or (iii) saturating the binding light chains of removed antibody molecules (or respective binding locations of other immune components) to prevent them from binding the marked pathogen parts and/or from binding the anchored pathogen parts.

[0071] In certain embodiments, diagnostic method 200 may comprise homologous testing - using same marked pathogen parts and fixated pathogen parts (stage 230), as disclosed above (see, e.g., Figures 2A-2B and the related disclosure). In certain embodiments, diagnostic method 200 may comprise heterologous testing - using different marked pathogen parts and fixated pathogen parts (stage 240), as disclosed above (see, e.g., Figures 2C-2D and the related disclosure).

[0072] In certain embodiments, diagnostic method 200 may comprise using marked pathogen parts and fixated pathogen parts of pathogens at different taxonomic levels, to distinguish general from specific immunity (stage 250) - such as partial immunity resulting to exposure to less aggressive strains and a more complete immunity against aggressive strains, respectively.

[0073] In certain embodiments, diagnostic method 200 may comprise combining two or more same test types and/or two or more different test types in a test kit (stage 260), wherein the test types refer to any of the following: different separated immunoglobulin, leading to different specificity of the test (e.g., IgM or IgA), (ii) use of different types of pathogen parts (e.g., homologous or heterologous tests as described above), (iii) different methods of separation or removed molecules (e.g., anchoring or saturation), and/or (iv) different types of marking of the marked pathogen parts (e.g., gold nanoparticles or polymer particles). Test kits 110 may comprise any of these combinations, optionally with multiple test utensils 120 per test type for enabling recurrent testing, e.g., every few days or upon requirement.

[0074] In the above description, an embodiment is an example or implementation of the invention. The various appearances of "one embodiment”, "an embodiment", "certain embodiments" or "some embodiments" do not necessarily all refer to the same embodiments. Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment. Certain embodiments of the invention may include features from different embodiments disclosed above, and certain embodiments may incorporate elements from other embodiments disclosed above. The disclosure of elements of the invention in the context of a specific embodiment is not to be taken as limiting their use in the specific embodiment alone. Furthermore, it is to be understood that the invention can be carried out or practiced in various ways and that the invention can be implemented in certain embodiments other than the ones outlined in the description above.

[0075] The invention is not limited to those diagrams or to the corresponding descriptions. For example, flow need not move through each illustrated box or state, or in exactly the same order as illustrated and described. Meanings of technical and scientific terms used herein are to be commonly understood as by one of ordinary skill in the art to which the invention belongs, unless otherwise defined.

[0076] While the invention has been described with respect to a limited number of embodiments, these should not be construed as limitations on the scope of the invention, but rather as exemplifications of some of the preferred embodiments. Other possible variations, modifications, and applications are also within the scope of the invention. Accordingly, the scope of the invention should not be limited by what has thus far been described, but by the appended claims and their legal equivalents.

Claims

CLAIMS What is claimed is:

1. A lateral flow test utensil comprising, on one or more interconnected pad(s) made of capillary material and consecutively along a flow path of a sample: a sample -receiving region, an antibodies-separation region configured to separate different types of immune components in the sample, a marking region comprising marked pathogen parts, a fixation region comprising pathogen parts that are anchored to the capillary material, the fixation region providing a test line, and a control region, wherein, in operation, immune components in the sample flowing along the utensil are separated by type, bind to the marked pathogen parts and are then fixated at the test line by further binding to the anchored pathogen parts.

2. A lateral flow test utensil comprising, on one or more interconnected pad(s) made of capillary material and consecutively along a flow path of a sample: a sample -receiving region, a marking region comprising marked pathogen parts, a fixation region comprising pathogen parts that are anchored to the capillary material, the fixation region providing a test line, and a control region, wherein, in operation, immune components in the sample flowing along the utensil bind to the marked pathogen parts and are then fixated at the test line by further binding to the anchored pathogen parts.

3. The lateral flow test utensil of claim 2, further comprising an antibodies-separation region set upstream of the marking region, wherein the antibodies-separation region is configured to separate different types of antibodies.

4. The lateral flow test utensil of claim 1 or 3, wherein the antibodies-separation region is configured to immobilize, mark and/or saturate binding sites of at least one first type of immunoglobulin and allow the further flowing of at least one second type of immunoglobulin.

5. The lateral flow test utensil of claim 4, wherein the at least one first type comprises IgG and the at least one second type comprises IgM.

6. The lateral flow test utensil of claim 4, wherein the at least one first type comprises IgG and IgM and the at least one second type comprises serum IgA or secretory IgA.

7. The lateral flow test utensil of claim 6, wherein the sample comprises saliva or mucus.

8. The lateral flow test utensil of claim 4, wherein the at least one first type comprises IgM and the at least one second type comprises IgG.

9. The lateral flow test utensil of claim 4, wherein the antibodies-separation region is configured to immobilize immunoglobulins and allow further flowing of T-cells.

10. The lateral flow test utensil of any one of claims 1 or 3-9, wherein the antibodies-separation region at least partly overlaps the sample-receiving region.

11. The lateral flow test utensil of any one of claims 4-9, wherein the antibodies-separation region is configured to immobilize the at least one first type of immunoglobulin.

12. The lateral flow test utensil of any one of claims 4-9, wherein the antibodies-separation region is configured to mark the at least one first type of immunoglobulin to be distinguishable from the marked pathogen parts.

13. The lateral flow test utensil of any one of claims 4-9, wherein the antibodies-separation region is configured to saturate binding sites of the at least one first type of immunoglobulin to prevent them from binding to the anchored pathogen parts.

14. The lateral flow test utensil of any one of claims 10-13, wherein the antibodies-separation region is at least partly implemented in a solution applied to the sample.

15. The lateral flow test utensil of any one of claims 1-14, wherein the marked pathogen parts comprise pathogen parts attached to colored particles.

16. The lateral flow test utensil of any one of claims 1-15, configured as a homologous test, wherein the marked pathogen parts are the same pathogen parts as the anchored pathogen parts.

17. The lateral flow test utensil of claim 16, wherein the pathogen is a virus and the same virus part comprises a nucleocapsid protein or a spike glycoprotein.

18. The lateral flow test utensil of any one of claims 1-15, configured as a heterologous test, wherein the marked pathogen parts are different pathogen parts than the fixated pathogen parts.

19. The lateral flow test utensil of claim 18, wherein the pathogen is a virus, the marked virus parts comprise at least one nucleocapsid protein and the fixated virus parts comprise at least one spike glycoprotein.

20. The lateral flow test utensil of claim 18, wherein the pathogen is a virus, the marked virus parts comprise at least one spike glycoprotein and the fixated virus parts comprise at least one nucleocapsid protein.

21. The lateral flow test utensil of claim 18, wherein the pathogen is a virus, the marked virus parts comprise at least one specific protein and the fixated virus parts comprise at least one general protein, wherein the specific protein is specific to a virus taxon and the general protein characterizes a higher virus taxon.

22. The lateral flow test utensil of claim 18, wherein the pathogen is a virus, the marked virus parts comprise at least one specific protein and at least one general protein, and the fixated virus parts comprise at least one general protein, wherein the specific protein is specific to a virus taxon and the general protein characterizes a higher virus taxon.

23. The lateral flow test utensil of any one of claims 1 to 22, configured to receive a sample of at least one of blood, plasma, serum, saliva and/or musus, and optionally running buffer.

24. The lateral flow test utensil of any one of claims 1-23, wherein the marked pathogen parts and the fixated pathogen parts comprise virus parts.

25. The lateral flow test utensil of any one of claims 1-16 or 18, the marked pathogen parts and the fixated pathogen parts comprise parts of at least one of: bacteria, fungi, and/or eucaryotic parasites.

26. The lateral flow test utensil of claim 24 or 25, wherein the marked pathogen parts comprise gold nanoparticles and/or latex particles.

27. A testing kit comprising a plurality of lateral flow test utensils according to at least one of claims 1-26.

28. The testing kit of claim 27, comprising at least two lateral flow test utensils according to any one of claims 5, 6, 8 and 9.

29. The testing kit of claim 28, wherein at least two of the lateral flow test utensils are different with respect to the type of separated immune components.

30. The testing kit of claim 28, wherein at least two of the lateral flow test utensils are same with respect to the type of separated immune components and configured for reiterating testing.

31. A diagnostic method comprising: configuring a lateral flow test to receive a sample, separating pre-defined types of immune components in the sample, leaving specified types of immune components to flow along the lateral flow test, using marked pathogen parts for binding the specified types of immune components, and fixating the specified types of immune components to anchored pathogen parts at a test line of the lateral flow test, wherein the separating and the marking enhance a clinical sensitivity of the lateral flow test.

32. A diagnostic method comprising using marked pathogen parts and anchored pathogen parts in a lateral flow test to enhance an analytical sensitivity of the test, wherein immune components in a sample applied to the lateral flow test bind to the marked pathogen parts and are then fixated by further binding to the anchored pathogen parts.

33. The diagnostic method of claim 32, further comprising separating pre-defined types of immune components upstream of the fixation to yield type-specific testing.

34. The diagnostic method of claim 31 or 33, wherein the separating comprises removing IgG antibodies to yield an IgM test.

35. The diagnostic method of claim 31 or 33, wherein the separating comprises removing IgM antibodies to yield an IgG test.

36. The diagnostic method of claim 31 or 33, wherein the separating comprises removing IgG and IgM antibodies to yield an IgA test.

37. The diagnostic method of claim 31 or 33, wherein the separating comprises removing immunoglobulins to yield a T cell test.

38. The diagnostic method of any one of claims 34-37, further comprising combining two or more same test types and/or two or more different test types in a test kit.

39. The diagnostic method of any one of claims 31-38, further comprising homologous testing - using same marked pathogen parts and fixated pathogen parts.

40. The diagnostic method of any one of claims 31-38, further comprising heterologous testing - using different marked pathogen parts and fixated pathogen parts.

41. The diagnostic method of any one of claims 31-38, further comprising using marked pathogen parts and fixated pathogen parts of pathogens at different taxonomic levels, to distinguish general from specific immunity.

42. A heterologous ELISA test comprising: marked pathogen parts configured to bind to immune components in a sample, to yield marked immune components, and anchored pathogen parts configured to fixate the marked immune components, wherein the marked pathogen parts and the anchored pathogen parts are different from each other.

43. The heterologous ELISA test of claim 42, wherein the pathogen parts are virus parts that include at least a nucleocapsid protein and a spike glycoprotein.

44. The heterologous ELISA test of claim 42, wherein the pathogen parts are parts of different viruses.