Lateral flow immunoassay for measuring functional C1-esterase inhibitor (C1-INH) in plasma samples

Abstract

An apparatus for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH), the apparatus comprising: (i) a conjugate pad including a first zone and a second zone, on which a first reagent and a second reagent are respectively immobilized; and (ii) a membrane in communication with the conjugate pad, wherein the membrane includes a third zone, on which a third reagent is immobilized. The conjugate pad may further include a fourth zone for placing a biological sample, and the biological sample flows through the apparatus in the order of the first zone, the second zone, and the third zone.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 833,235, filed on April 12, 2019, and U.S. Provisional Application No. 62 / 930,615, filed on November 5, 2019, under 35 U.S.C.§119(e), the entire contents of each of which are incorporated herein by reference. Background of the Invention

[0003] C1 - esterase inhibitor (also known as C1 - inhibitor or C1 - INH) is a protease inhibitor belonging to the serine protease inhibitor superfamily. Its main function is to inhibit the complement system to prevent spontaneous activation. C1 - INH is also an endogenous inhibitor of plasma kallikrein (pKal). Autosomal dominant mutations in C1 - INH result in hereditary angioedema (HAE), including type I and type II HAE.

[0004] Currently available assays for evaluating the functional level of C1 - INH utilize chromogenic assays or multiplex ELISA methods to measure the inhibition of C1 of the complement cascade by C1 - INH. The chromogenic assay is generally considered to be preferred, but both methods have limitations. The chromogenic assay is more likely to have occasional false positives, while the multiplex ELISA has a negative predictive value of only 62%.

[0005] There is interest in developing updated assays and / or platforms for measuring functional C1 - INH involved in the pathophysiology of HAE disease. Summary of the Invention

[0006] Provided herein are devices and methods for detecting functional C1 - INH (fC1 - INH) in a rapid and cost - effective qualitative and / or quantitative manner. In some embodiments, the detection of fC1 - INH is achieved by lateral flow immunoassay (LFA) using a device configured to detect and / or quantify fC1 - INH.

[0007] Accordingly, one aspect of the present disclosure provides an apparatus for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH). The apparatus includes (i) a conjugate pad including a first region and a second region, on which a first reagent and a second reagent are respectively immobilized; and (ii) a membrane in communication with the conjugate pad, wherein the membrane includes a third region, on which a third reagent is immobilized. The first reagent may be a functional C1 inhibitor (fC1-INH) binder or a C1 inhibitor (C1-INH) binder. The second reagent and the third reagent are one of a functional C1 inhibitor (fC1-INH) binder, a C1 inhibitor (C1-INH) binder, and a capture agent. The first reagent, the second reagent, and the third reagent are different from each other. One of the fC1-INH binder, the C1-INH binder, and the capture agent is conjugated to a detectable label, and one of the fC1-INH binder and the C1-INH binder is conjugated to a docking agent. The detectable label and the docking agent are conjugated to different reagents. The conjugate pad further includes a fourth region for placing a biological sample, and the biological sample flows through the apparatus in the order of the first region, the second region, and the third region. In some cases, the fourth region for placing the biological sample may overlap with the second region, and the fC1-INH binder may be located on the second region.

[0008] In some embodiments, the first reagent, the second reagent, and the third reagent are respectively a C1-INH binder, an fC1-INH binder, and a capture agent. In other embodiments, the first reagent, the second reagent, and the third reagent are respectively an fC1-INH binder, a C1-INH binder, and a capture agent.

[0009] In some examples, the first reagent is conjugated to a detectable label and the second reagent is conjugated to a docking agent. In other examples, the first reagent is conjugated to a docking agent and the second reagent is conjugated to a detectable label.

[0010] In some embodiments, the fC1-INH binder may be the active form of factor XII (FXIIa). Alternatively or additionally, the C1-INH binder may be an antibody that binds C1-INH. Further, the docking agent and the capture agent may be members of a receptor-ligand pair. For example, the receptor-ligand pair may include biotin and avidin (e.g., streptavidin or neutravidin).

[0011] In some embodiments, the detectable label may be europium, colloidal gold, phycoerythrin, fluorescein, rhodamine, green fluorescent protein, quantum dots, and chromophores. In some embodiments, the detectable label is europium. In some cases, the detectable label may be attached to latex particles.

[0012] In one example, the first reagent is a C1-INH binder located at a first zone, the second reagent is an fC1-INH binder located at a second zone, and the third reagent is a capture agent located at a third zone. The C1-INH binder can be an antibody that binds to C1-INH, which can be conjugated to a detectable label as disclosed herein. Alternatively or additionally, the fC1-INH binder can be FXIIa, which can be conjugated to a docking agent (e.g., biotin). Further, the capture agent can be avidin, such as streptavidin or neutravidin.

[0013] In some embodiments, the device further includes an absorbent pad in communication with the membrane. The absorbent pad and the binding pad can be separated by the membrane. Alternatively or additionally, the device can further include a support member on which the binding pad, the membrane, and / or the absorbent pad are mounted.

[0014] Further, the device can also include a housing. In some embodiments, the housing can include a first opening that forms a buffer port, a second opening that forms a sample port, and a third opening that forms a test window. The sample port can be located between the buffer port and the test window. In some examples, the buffer port can be aligned with the first zone on which the C1-INH binder is located. In some examples, the sample port can be aligned with the second zone on which the fC1-INH binder is located. In some examples, the test window is aligned with the third zone on which the capture agent is located.

[0015] In another aspect, the present disclosure provides a method of detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample using any of the LFA devices disclosed herein. Such method can include: (i) placing the sample in the sample port of the device described herein, (ii) placing a buffer in the buffer port of the device, wherein the buffer flows in a direction from the first zone to the third zone; (iii) examining a signal at the test window in the device, and (iv) determining the presence of fC1-INH in the sample or measuring the level of fC1-INH in the sample based on the presence or intensity of the signal at the test window. In some embodiments, step (ii) is performed at least 5 minutes after step (i). The fC1-INH binder can be immobilized at the second zone, which can be aligned with the sample port.

[0016] The present disclosure also provides methods for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample, the methods comprising (i) contacting the sample with an fC1-INH binder, a C1-INH binder, and a capture agent to form a complex, wherein one of the fC1-INH binder and the C1-INH reagent is conjugated to a docking agent that binds the capture agent, and wherein one of the fC1-INH binder, the C1-INH reagent, and the capture agent is conjugated to a detectable label, and the detectable label and the docking agent are conjugated to different reagents; and (ii) detecting a signal released from the detectable label from the complex; wherein the presence of a signal released from the detectable label from the complex indicates the presence of fC1-INH in the sample. Any fC1-INH binder, C1-INH binder, docking agent, detectable label, and capture agent disclosed herein can be used in the methods disclosed herein.

[0017] In some embodiments, step (i) can be carried out by: (a) incubating the sample with the fC1-INH binder for at least 5 minutes, and (b) contacting the sample with the C1-INH binder and the capture agent. In other embodiments, step (i) is carried out by: (a) incubating the sample with the fC1-INH binder for at least 5 minutes to form a first complex, (b) contacting the first complex with the C1-INH binder to form a second complex, and (c) contacting the second complex with the capture agent to form a complex, and wherein the capture agent is immobilized on a support member.

[0018] In any of the assay methods disclosed herein, the sample to be analyzed can be a biological sample obtained from a subject, e.g., a serum sample, a plasma sample, or a blood sample (e.g., whole blood). In some embodiments, the subject can be a human patient suspected of having an fC1-INH deficiency-mediated disorder or at risk of an fC1-INH deficiency-mediated disorder, the fC1-INH deficiency-mediated disorders including but not limited to hereditary angioedema (HAE), acquired angioedema (AAE), and C1-INH-related immune diseases. In some embodiments, the subject has symptoms of HAE. In some embodiments, the HAE is type I HAE or type II HAE. In other embodiments, the subject does not have symptoms of HAE, does not have a history of symptoms of HAE, or does not have a history of HAE. In some embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0019] Details of several embodiments of the devices and methods described herein are set forth in the accompanying drawings and the detailed description. Other features, objects, and advantages of the devices and methods described herein will be apparent from the description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various aspects and embodiments will be described with reference to the following figures. The figures are not necessarily drawn to scale.

[0021] Figure 1 Is a schematic description of an exemplary lateral flow assay (LFA) device 100 for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH). According to some embodiments of the techniques described herein, the LFA device 100 includes a conjugate pad 200, a membrane 300, and optionally an absorbent pad 400 and a support member 500.

[0022] Figure 2 Is a schematic description of an exemplary LFA device 100, showing exemplary dimensions of each component and exemplary dimensions of the overlap between two adjacent components, according to some embodiments of the techniques described herein.

[0023] Figure 3A Is a schematic top view description of an exemplary LFA device 100 for detecting and / or quantifying fC1-INH. According to some embodiments of the techniques described herein, the LFA device 100 includes a conjugate pad 200 that includes a first region 210 and a second region 220; and a membrane 300 that includes a third region 230.

[0024] Figure 3B Is a schematic top view description of an exemplary LFA device 100 for detecting and / or quantifying fC1-INH. According to some embodiments of the techniques described herein, the LFA device 100 includes a conjugate pad 200 that includes a first region 210 that overlaps a fifth region 250 for placing a sample, and a second region 220 that overlaps a fourth region 240 for placing a buffer; and a membrane 300 that includes a third region 230.

[0025] Figure 4 Is a schematic top view description of an exemplary LFA device 100 for detecting and / or quantifying fC1-INH. According to some embodiments of the techniques described herein, the LFA device 100 further includes a housing 600 that forms a buffer port 610, a sample port 620, and a test window 630. Also indicated are the first region 210, the second region 220, the third region 230, the conjugate pad 200, the membrane 300, and the absorbent pad 400.

[0026] Figure 5 Is an image of an exemplary LFA device according to some embodiments of the techniques described herein.

[0027] Figure 6 Is a graph showing a calibration curve generated from functional C1-esterase inhibitor (fC1-INH) in plasma diluted to C1-INH depletion using the LFA device disclosed herein.

[0028] Figure 7 It is a graph of the receiver operating characteristic curve (ROC) for diagnostic performance using the LFA device disclosed herein, based on samples from control subjects and subjects with hereditary angioedema (HAE).

[0029] Figure 8 It is a graph showing the levels of functional C1-INH in normal subjects (Normal) and subjects with hereditary angioedema (HAE) using the chromogenic assay (Chrom) or LFA device (LFA) described herein.

[0030] Figure 9 It is a graph showing the correlation between the levels of functional C1-INH in subjects with hereditary angioedema (HAE) determined by chromogenic assay compared to the LFA device described herein.

[0031] Figure 10A It is a schematic diagram showing the collection of blood samples using a sample loop and fingerstick.

[0032] Figure 10B It shows the addition of a sample loop filled with Figure 10A blood to a bottle.

[0033] Figure 11A It is a graph showing the use of anti-C1-INH Fab conjugated to europium nanoparticles under specified conditions.

[0034] Figure 11B It is a graph showing the use of anti-C1-INH Fab conjugated to red gold nanoparticles under specified conditions.

[0035] Figure 12 It is a graph showing the results of comparing streptavidin-poly with streptavidin as a capture agent in the test line. R 2 The value indicates the fit to the model line (dashed line).

[0036] Figure 13 It is a graph showing the results from using the specified reagent as a capture agent in the test line.

[0037] Figure 14 It is a graph showing the results from the specified anti-C1-INH Fab or antibody clone as a detection agent. For each antibody, the columns refer to the concentration of the detection agent, from left to right, 1200 mU / mL, 600 mU / mL, 100 mU / mL, 0 mU / mL.

[0038] Figure 15It is a figure showing the results of using buffers containing an inorganic blocker (IBA) or an organic blocker (OBA) at different pH values.

[0039] Figure 16 It is a figure showing the results from using a streptavidin test line (0.75 mg / mL streptavidin) and a specified concentration of the results.

[0040] Figure 17 It is a schematic diagram of the exemplary method described herein. A capture protein, such as streptavidin, interacts with its pair (e.g., biotin) conjugated to a capture agent (e.g., biotinylated FXIIa), where the capture agent binds to functional C1-INH (fC1-INH, ). The bound FC1-INH is detected using a detection agent, such as an anti-C1-INH antibody conjugated to gold particles (anti-C1-INH Au conjugate).

[0041] Detailed Description

[0042] The present disclosure is at least partially based on the development of a lateral flow assay (LFA) method and device for measuring functional C1-esterase inhibitor (fC1-INH). The LFA method and device are designed to specifically detect the presence and / or measure the level of fC1-INH in, for example, biological samples. The presence and / or level of fC1-INH typically indicates a disease condition related to the biological pathway in which C1-INH functions. Similarly, the LFA method and device will be particularly useful for the diagnosis and prognosis of diseases mediated by C1-INH deficiency, such as diseases mediated by the plasma kallikrein pathway (e.g., diseases such as hereditary angioedema), since FC1-INH is an inhibitor of the pKal pathway.

[0043] As used herein, "functional C1-INH" or "fC1-INH" refers to the C1-INH protein in a form capable of binding to protein factors, where C1-INH substantially binds to the protein factors and exerts its biological activity. Such protein factors include, but are not limited to, C1s, factor XIIa (FXIIa), and plasma kallikrein (pKal). Detecting this subset of fC1-INH is particularly helpful for assessing the functional level of C1-INH in diseases such as HAE.

[0044] HAE is a very rare and potentially life-threatening genetic disorder that occurs in approximately 1 in 10,000 to 1 in 50,000 people. Symptoms include swelling (edema) in various parts of the body, including the hands, feet, face, and airway (throat). Patients typically suffer from excruciating abdominal pain, nausea, and vomiting caused by swelling of the intestinal wall. Swelling of the airway or throat is particularly dangerous; it can lead to death by asphyxiation. The three specific blood tests required to confirm HAE types I & II are C1INH antigen, fC1INH, and C4. Many diagnostic assays use outdated technology and are not rapid or standardized or available worldwide.

[0045] The rapid and sensitive LFA methods and devices disclosed herein can be performed in a doctor's office laboratory for the rapid diagnosis of HAE (e.g., types I & II) based on fC1INH levels. Such methods and devices will result in low-cost consumables for Medicare reimbursement, a high confidence level of quantitative results, easy data interpretation by doctors, and / or a low level of need for confirmatory analysis. Such rapid analysis for diagnosing type I or type II HAE in a doctor's office can expand HAE screening and more quickly identify new HAE patients. Currently, the global diagnosis rate of HAE is only 40%; thus, there is a high unmet need for undiagnosed patients. The availability of rapid tests on common device platforms can expand HAE identification. Further, the rapid tests for fC1INH disclosed herein can help monitor HAE disease progression or response to therapy in a timely manner in a clinical setting.

[0046] The LFA methods and devices disclosed herein relate to a first binder specific to fC1-INH (fC1-INH binder), a second binder specific to C1-INH (specific to functional C1-INH, non-functional C1-INH, or both), and a capture agent. Either the fC1-INH binder or the C1-INH binder can be conjugated to a docking agent capable of binding to the capture agent. One of the FC1-INH binder, C1-INH binder, and capture agent can be conjugated to a detectable label. Accordingly, fC1-INH in a sample (e.g., a biological sample) can form a complex with the fC1-INH binder and the C1-INH binder. Such a complex can be bound to the capture agent via the interaction between the capture agent and a docking agent conjugated to one of the C1-INH binder and the fC1-INH binder. After detecting a signal (e.g., presence or intensity) released from the detectable label, the presence or level of fC1-INH in the sample can be determined and / or quantified based on standards tested therewith. For example, different levels of Purified human plasma-derived C1INH can be used as a standard to generate a standard curve for inferring and determining the level of fC1INH in a sample measured by any method disclosed herein. Based on the standard, the signal intensity released from the detectable label can be quantified as U / ml of fC1INH in the sample and indicates the level of fC1-INH in the sample.

[0047] I. Components for Lateral Flow Assay (LFA)

[0048] The LFA methods and devices disclosed herein relate to (i) an fC1-INH binder, (ii) a C1-INH binder, one of which is conjugated to a docking agent, and (iii) a capture agent that binds the docking agent. One of (i)-(iii) is conjugated to a detectable label.

[0049] (a) fC1-INH binder

[0050] An fC1-INH binder is a molecule that specifically binds functional C1-INH (e.g., a protein or a fragment thereof that binds fC1-INH). A molecule is considered to exhibit "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with a specific target (e.g., those disclosed herein) than with an alternative target, which may be a modified form of the specific target. For example, a molecule that specifically binds fC1-INH reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity with fC1-INH than with an alternative target, such as non-functional C1-INH. "Specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding.

[0051] In some cases, a protein or polypeptide or a binding fragment thereof that is capable of binding to fC1-INH in nature can be used as an fC1-INH binder. In some examples, the fC1-INH binder is FXII, for example, the active form of FXII (FXIIa). Factor XII is a serum glycoprotein involved in blood coagulation, the initiation of fibrinolysis, and the generation of bradykinin and angiotensin. Prekallikrein is cleaved by factor XII to form kallikrein, which then activates factor XII, resulting in the formation of factor XIIa and factor XII fragments (factor XIIf) ("Histidine-rich glycoprotein binds factor XIIa with high affinity and inhibits contact-initiated coagulation" Macquarrie et al., Blood 117:4134-4141 2011). C1 inhibitor (C1-INH) has been shown to be an important plasma inhibitor of both factor XIIa and factor XIIf ("Effect of negatively charged activating compounds on inactivation of factor XIIa by C1 inhibitor" Pixley et al., Arch Biochem Biophys 256(2):490-81987).

[0052] FXII proteins, including their precursor forms, mature forms, and active forms, are well known in the art. For example, the precursor protein sequences of human factor XII and its active form are provided under GenBank accession number: NP_000496.2. FXII proteins of other species, such as non-human mammals, are also known in the art. Their structural information can be found in the art, for example, identified from publicly available gene databases using the human FXII sequence as a query.

[0053] "Active" or "functional" factor XII refers to a factor XII polypeptide or a factor XII polypeptide fragment that retains biological activity similar to, but not necessarily equivalent to, the naturally occurring factor XII counterpart, including the mature form. In some embodiments, the active or functional factor XII is a factor XII polypeptide or a factor XII polypeptide fragment that binds to fC1-INH. In some embodiments, the active or functional factor XII is a factor XIIa polypeptide or a factor XIIa polypeptide fragment that binds to fC1-INH. In some embodiments, the active or functional factor XII is a factor XIIf polypeptide or a factor XIIf polypeptide fragment that binds to fC1-INH.

[0054] In other examples, the fC1-INH binder is plasma kallikrein (pKal), e.g., a catalytic fragment of the naturally occurring pKal protein. Plasma kallikrein is a serine protease component of the contact system (Sainz I.M. et al., Thromb Haemost 98, 77-83, 2007). The contact system is activated by factor XIIa upon exposure to foreign or negatively charged surfaces or by proline carboxypeptidase on the endothelial cell surface (Sainz I.M. et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein enhances intrinsic coagulation via its feedback activation of factor XII and inflammation via the production of the pro-inflammatory nonapeptide bradykinin. As the major kininogenase in circulation, plasma kallikrein is largely responsible for the production of bradykinin in blood vessels.

[0055] The pKal protein is well-known in the art. Exemplary plasma kallikrein sequences can include the human (Accession No.: NP_000883.2), mouse (Accession No.: NP_032481.1), or rat (Accession No.: NP_036857.2) plasma kallikrein amino acid sequences.

[0056] "Active" or "functional" plasma kallikrein refers to a plasma kallikrein polypeptide or a plasma kallikrein polypeptide fragment that retains biological activity (e.g., protease activity) similar to, but not necessarily equivalent to, the naturally occurring plasma kallikrein counterpart, including the mature form. In some embodiments, the active or functional plasma kallikrein is a plasma kallikrein polypeptide or a plasma kallikrein polypeptide fragment that binds to fC1-INH.

[0057] In still other examples, the fC1-INH binder disclosed herein can be C1s and C1r, or their active / functional fragments. C1s and C1r are activated homologous serine proteases of the first component of complement (C1). Both C1s and C1r can form a complex with C1-INH. Arlaud et al., (1993) Methods Enzymol. 223, 61–82. C1s is a modular serine protease that performs the catalytic function of the C1 complex. C1r is the enzyme that activates C1s to its active form by proteolytic cleavage.

[0058] The C1s and C1r proteins are also well-known in the art. For example, the precursor protein sequence of human C1s is provided at GenBank Accession No.: NP_001725.1 and human C1r is provided at GenBank Accession No.: NP_001724.4.

[0059] "Active" or "functional" C1s and C1r refer to counterparts that retain, respectively, a likeness to, but not necessarily an identity with, naturally occurring C1s and C1r, including biologically active C1s and C1r polypeptide fragments in their mature forms. In some embodiments, the active or functional C1s or C1r fragment is a C1s polypeptide or a portion of a C1s polypeptide that binds to fC1-INH.

[0060] Any fC1-INH binder can be produced by recombinant techniques or isolated from a suitable natural source.

[0061] (b) C1-INH binder

[0062] The C1-INH binder for use in the LFA methods and devices disclosed herein can be any molecule (e.g., a protein or polypeptide) capable of binding to C1-INH. In some cases, the C1-INH binder is specific for fC1-INH. In other cases, the C1-INH binder cross-reacts with both functional and non-functional C1-INH.

[0063] The C1-INH binder can be an antibody that binds C1-INH. As used herein, the term "antibody" refers to a protein comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments (de Wildt et al., Eur J Immunol. 1996;26(3):629-39)) as well as intact antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes).

[0064] The VH and VL regions can be further subdivided into hypervariable regions, called "complementary determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The boundaries of the framework regions and CDRs have been precisely defined (see, Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al., (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk). The Kabat definitions are used herein. Each VH and VL typically includes three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0065] The VH or VL chain of an antibody can further include all or part of the heavy or light chain constant region, thereby forming a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are linked to each other, for example, by disulfide bonds. In IgG, the heavy chain constant region includes three immunoglobulin domains, CH1, CH2, and CH3. The light chain constant region includes the CL domain. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The light chain of an immunoglobulin can have a κ or λ chain. In one embodiment, the antibody is glycosylated. The antibody can be functional for antibody-dependent cell cytotoxicity and / or complement-mediated cytotoxicity.

[0066] In some embodiments, an antibody that binds C1-INH can specifically bind to Cl-INH, for example, an epitope of fC1-INH or an epitope shared by fC1-INH and non-functional C1-INH. An antibody that "specifically binds" to an antigen or epitope is a term well known in the art, and methods for determining such specific binding are also well known in the art. An antibody is considered to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, for a longer duration, and / or with greater affinity with a particular target antigen than with an alternative target. An antibody "specifically binds" to a target antigen or epitope if it binds to the target antigen or epitope with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., C1-INH) or an antigenic epitope therein is an antibody that binds to that target antigen with greater affinity, avidity, more readily, and / or more persistently than it binds to other antigens or other epitopes in the same antigen. It can also be understood from this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. For this reason, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding. In some examples, an antibody that "specifically binds" to a target antigen or its epitope may not bind to other antigens or other epitopes in the same antigen.

[0067] An antibody that binds C1-INH for use in the LFA methods and devices disclosed herein can have a suitable binding affinity for C1-INH or a suitable epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or KA. KA is the reciprocal of the dissociation constant (KD). The antibodies described herein can have a binding affinity (KD) of at least 10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 M or lower. An increase in binding affinity corresponds to a decrease in KD. A higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical KD) for binding the first antigen than for binding the second antigen. In this case, the antibody is specific for the first antigen relative to the second antigen. The difference in binding affinity (e.g., specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 10000, or 10 5times. The binding affinity (or binding specificity) can be determined by conventional methods.

[0068] An antibody that binds to C1-INH can be a full-length antibody. Alternatively, the antibody is an antigen-binding fragment of a full-length antibody. The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed by the term "antigen-binding fragment" of a full-length antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of the VH domain; and (vi) isolated complementarity-determining regions (CDRs) that retain functionality. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined together using recombinant methods by a synthetic linker that enables them to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule, called a single-chain Fv (scFv). See, e.g., U.S. Patents 5,260,203, 4,946,778, and 4,881,175; Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Antibody fragments can be obtained using any suitable technique including conventional techniques known to those skilled in the art.

[0069] Any antibody that binds to C1-INH described herein can be monoclonal or polyclonal. "Monoclonal antibody" refers to a population of homologous antibodies and "polyclonal antibody" refers to a population of heterologous antibodies. These two terms do not limit the source of the antibody or the manner in which it is prepared.

[0070] Antibodies that bind to C1-INH can be prepared by any method known in the art. See, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, antibodies specific for C1-INH (e.g., human C1-INH) can be prepared by conventional hybridoma techniques. In other embodiments, antibodies specific for C1-INH can be isolated from an antibody library in accordance with conventional antibody library screening techniques.

[0071] In some embodiments, the antibody is an antibody that specifically binds to human C1-INH. In some embodiments, the antibody is a monoclonal antibody that specifically binds to human C1-INH. In some embodiments, the antibody is a murine monoclonal antibody that specifically binds to human C1-INH, such as antibody clones MM06 or MM03 (also known as 10995-MM06 and 10995-MM03, respectively, from Sino Biological Inc.). In some embodiments, the antibody is a polyclonal antibody that specifically binds to human C1-INH. In some embodiments, the antibody is a rabbit polyclonal antibody that specifically binds to human C1-INH, such as antibody clones RP01 or RP02 (also known as 10995-RP01 and 10995-RP02, respectively, from Sino Biological Inc.). In some embodiments, the antibody is RP02.

[0072] (c) Docking - capture agent

[0073] One of the fC1-INH binder and the C1-INH binder is conjugated to a docking agent that is capable of binding to a capture agent also used in the LFA methods and devices disclosed herein. In one embodiment, the docking agent is conjugated to the fC1-INH binder (e.g., FXIIa). In other embodiments, the docking agent may be conjugated to C1-INH (e.g., an antibody that binds to C1-INH).

[0074] The docking agent and the capture agent are members of a receptor - ligand pair, which refers to any pair of molecules that are capable of binding to each other to form a complex. In one example, the docking agent and the capture agent are biotin and avidin, respectively, or vice versa. For example, the docking agent can be biotin, and the capture agent can be streptavidin or neutravidin.

[0075] (d) Detectable label

[0076] One of the fC1-INH binder, the C1-INH binder, and the capture agent for the LFA methods and devices disclosed herein may be conjugated to a detectable label. In some embodiments, the fC1-INH binder is conjugated to the detectable label. In other embodiments, the C1-INH binder is conjugated to the detectable label. Alternatively, the capture agent is conjugated to the detectable label.

[0077] As used herein, "detectable label" refers to any molecule that is capable of directly or indirectly emitting a detectable signal. In some embodiments, the detectable label can be a fluorophore (e.g., fluorescein). As used herein, the term "fluorophore" (also referred to as "fluorescent label" or "fluorescent dye") refers to a moiety that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength.

[0078] Examples of fluorophores include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, and Texas red), cyanine derivatives (e.g., cyanine, indocyanine, oxacarbocyanine, thiocyanine, and phthalocyanine), naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives (e.g., pyridooxazole, nitrobenzoxadiazole, and benzoxadiazole), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, and oxazine 170), acridine derivatives (e.g., proflavine, acridine orange, and acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, and malachite green), tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, and bilirubin), or fluorescent proteins (e.g., green fluorescent protein).

[0079] In some embodiments, the detectable label is phycoerythrin.

[0080] In some embodiments, the detectable label is a chromophore (e.g., anthracene). In some embodiments, the detectable label is a semiconductor particle (e.g., quantum dot). In some embodiments, the detectable label is attached to a semiconductor particle (e.g., quantum dot). In some embodiments, the detectable label is europium. In some embodiments, the detectable label is colloidal gold. In some embodiments, the detectable label is attached to gold particles. In some embodiments, the detectable label is attached to red gold particles. In some embodiments, the detectable label is attached to latex particles. In some embodiments, the C1-INH binder is antibody RP02 conjugated to europium. In some embodiments, the C1-INH binder is antibody RP02 conjugated to gold particles. In some embodiments, the C1-INH binder is antibody RP02 conjugated to red gold particles. In some embodiments, the C1-INH binder is antibody RP02 conjugated to latex particles.

[0081] II. LFA device

[0082] In some aspects, the present disclosure provides a lateral flow assay (LFA) device for measuring fC1-INH in a sample containing such fC1-INH. Now refer to Figures 1 - 4 , which depicts and illustrates various embodiments of the exemplary LFA device described herein.

[0083] As Figure 1As shown, device 100, in some embodiments, includes a conjugate pad 200, a membrane 300, and optionally an absorbent pad 400 and a support member 500. The conjugate pad 200, the membrane 300, and optionally the absorbent pad 400 are mounted on the support member 500.

[0084] The conjugate pad 200 communicates with the membrane 300 directly or via a linker. When the device contains an absorbent pad 400, the conjugate pad 200 and the absorbent pad 400 are separated by the membrane 300, and the membrane 300 communicates with the absorbent pad 400 directly or indirectly. In some embodiments, the conjugate pad 200 overlaps the membrane 300, for example, by 2 - 6 mm, such as 3 mm, as Figure 2 shown. Alternatively or additionally, the membrane 300 overlaps the absorbent pad 400, for example, by 2 - 6 mm, such as 3 mm, as Figure 2 shown.

[0085] The specific characteristics and dimensions of the conjugate pad 200, the membrane 300, the absorbent pad 400, and the support member 500 can be modified as needed to achieve the desired results. As Figure 2 shown, in some embodiments, the support membrane has a length of 80 mm, which is the combined length of the conjugate pad (42 mm), the membrane (25 mm), and the absorbent pad (19 mm) minus the overlaps of the sample pad and the absorbent pad on the membrane (3 mm, 3 mm).

[0086] As Figure 3A shown, a top view of device 100, the device may include various zones (210, 220, 230) for immobilizing, in some embodiments, those fC1-INH binders, C1-INH binders, and capture agents as described herein. In some embodiments, device 100 includes a first zone 210 and a second zone 220, which may be on the conjugate pad 200; and a third zone 230, which may be on the membrane 300.

[0087] The fC1-INH binder, the C1-INH binder, and the capture agent can each be immobilized on one of the zones 210, 220, and 230 (which can be in any order). Any of these reagents can be immobilized using any method known in the art. The reagent can be immobilized directly or indirectly, or conjugated to, the surface of the conjugate pad 200 and / or the membrane 300. In some embodiments, the binder is immobilized to the surface via a covalent bond. In some embodiments, the binder is immobilized to the surface via a non-covalent bond. In some embodiments, the binder is immobilized to the surface via a linker. Examples of linkers include, but are not limited to, carbon-containing chains, polyethylene glycol (PEG), nucleic acids, monosaccharide units, biotin, avidin, and peptides.

[0088] In some embodiments, an fC1-INH binder such as FXIIa is immobilized on the first region 210. The fC1-INH binder may be conjugated to a docking agent such as biotin. A C1-INH binder such as an antibody that binds C1-INH may be immobilized on the second region 220. The C1-INH binder may be conjugated to a detectable label such as those disclosed herein. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the third region 230.

[0089] In some embodiments, an fC1-INH binder such as FXIIa is immobilized on the first region 210. The fC1-INH binder may be conjugated to the detectable labels described herein. A C1-INH binder such as an antibody that binds C1-INH may be immobilized on the second region 220. The C1-INH binder may be conjugated to a docking agent such as biotin. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the third region 230.

[0090] In some embodiments, a C1-INH binder such as an antibody that binds C1-INH is immobilized on the first region 210. The C1-INH binder may be conjugated to a docking agent such as biotin. An fC1-INH binder such as FXIIa is immobilized on the second region 220. The fC1-INH binder may be conjugated to a detectable label such as those described herein. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the third region 230.

[0091] In some embodiments, a C1-INH binder such as an antibody that binds C1-INH is immobilized on the first region 210. The C1-INH binder may be conjugated to a detectable label such as those described herein. An fC1-INH binder such as FXIIa is immobilized on the second region 220. The fC1-INH binder may be conjugated to a docking agent such as biotin. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the third region 230.

[0092] In some embodiments, an fC1-INH binder such as FXIIa is immobilized on the first region 210. The fC1-INH binder may be conjugated to a docking agent such as biotin. A capture agent such as avidin (e.g., streptavidin) is immobilized on the second region 220. The capture agent may be conjugated to a detectable label such as those disclosed herein. A C1-INH binder such as an antibody that binds C1-INH is immobilized on the third region 230.

[0093] In some embodiments, an fC1-INH binder such as FXIIa is immobilized on the first region 210. The fC1-INH binder may be conjugated to a detectable label such as those described herein. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the second region 220. A C1-INH binder such as an antibody that binds to C1-INH may be immobilized on the third region 230. The C1-INH binder may be conjugated to a docking agent such as biotin.

[0094] In some embodiments, a C1-INH binder such as an antibody that binds to C1-INH is immobilized on the first region 210. The C1-INH binder may be conjugated to a docking agent such as biotin. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the second region 220. The capture agent may be conjugated to a detectable label such as those disclosed herein. An fC1-INH binder such as FXIIa is immobilized on the third region 230.

[0095] In some embodiments, a C1-INH binder such as an antibody that binds to 1-INH is immobilized on the first region 210. The C1-INH binder may be conjugated to a detectable label such as those described herein. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the second region 220. An fC1-INH binder such as FXIIa is immobilized on the third region 230. The fC1-INH binder may be conjugated to a docking agent such as biotin.

[0096] Any LFA device disclosed herein may further include a fourth region 240, which may be used for placing samples such as those described herein, and optionally a fifth region 250, which may be used for placing a buffer solution.

[0097] The fifth region 250 may be located at one end of the device such that when the buffer solution is placed on the fifth region 250, the buffer solution may flow through the device from the first region 210 to the third region 230. In some examples, the fifth region 250 may overlap with the first region 210. See Figure 3B In this case, a C1-INH binder such as an antibody that binds to C1-INH may be immobilized on the first region 210, which overlaps with the fifth region 250. The C1-INH binder may be conjugated to a detectable label.

[0098] Alternatively or additionally, the fourth region 240 may be located between the fifth region 250 and the second region 220. In some cases, the fourth region 240 and the second region 220 may overlap. See Figure 3B An fC1-INH binder such as FXIIa, which may be conjugated to a docking agent such as biotin, may be immobilized on the second region 220.

[0099] As Figure 4As shown in, the apparatus 100, in some embodiments, may further include a housing 600, which may be detachable. In Figure 5 An image of the apparatus in the housing as described herein is shown. The housing 600 may be configured to expose at least a portion of the binding pads 200 and the membrane 300 of the apparatus 100. In some embodiments, the housing 600 includes a first opening forming a buffer port 610, which may be aligned with the first region 210. The housing 600 may further include a second opening 620 forming a sample port, which may be aligned with the second region 220. Further, the housing 600 may include a third opening 630 forming a test window, which may be aligned with the third region 230.

[0100] In some embodiments, a C1-INH binder such as an antibody bound to C1-INH is immobilized on the first region 210, which is aligned with the buffer port 610. Figure 4 . The C1-INH binder may be conjugated to a detectable label such as those described herein. An fC1-INH binder such as FXIIa may be immobilized on the second region 220, which may be aligned with the sample port 620. The fC1-INH binder may be conjugated to a docking agent such as biotin. A capture agent such as avidin (e.g., streptavidin) may be immobilized on the third region 230, which may be aligned with the test window 630.

[0101] In some examples, a sample may be placed in the sample port 620, allowing the fC1-INH therein to bind to the FXIIa-biotin conjugate. A buffer solution may be placed in the buffer port 610, allowing the C1-INH binder on the first region 210 to migrate together with the buffer solution towards the second region 220. When the C1-INH binder contacts the fC1-INH-FXIIa complex at the second region 220, a C1-INH binder / fC1-INH / FXIIa-biotin complex is formed. The complex will migrate together with the buffer solution towards the third region 230 and be captured at the third region 230 via the interaction between biotin and streptavidin at the third region 230. The signal released by the detectable label conjugated to the C1-INH binder at the third region 230 (which is aligned with the test window 630) may be measured, which indicates the presence or level of fC1-INH in the sample.

[0102] Alternatively or additionally, the housing 600 may be clear to facilitate visualization of the sample port 610 and / or the buffer port 620 and / or the test window 630. In some embodiments, a portion of the housing is clear. In some embodiments, the entire housing 600 is clear.

[0103] The housing 600, in some embodiments, includes a label to facilitate the identification of the sample or result. In some embodiments, the housing 600 includes one or more labels to facilitate the identification of the result in the test window 630. In some embodiments, one or more labels identify the sample result.

[0104] It should be understood that the various embodiments of the device, including the various components in the device as described herein (e.g., conjugate pad, membrane, absorbent pad, and support member), can be formed of suitable materials, e.g., with any suitable conjugate pad, with any suitable membrane, with any suitable absorbent pad, with any suitable support member, with any suitable binder, and with any suitable combination thereof.

[0105] For example, the membrane 300 in the LFA device as disclosed herein can be any suitable membrane, including but not limited to nitrocellulose membrane, nylon membrane, cellulose membrane, polyvinylidene fluoride membrane, polycarbonate membrane, polypropylene membrane, polyethylene membrane, polytetrafluoroethylene membrane, and poly-paraphenylene terephthalamide membrane. In some embodiments, the membrane is a nitrocellulose membrane.

[0106] Any suitable support member can be used in the device described herein. In some embodiments, the support member includes metal. In some embodiments, the support member includes plastic. In some embodiments, the support member includes plastic and is selected from the group consisting of styrene, polycarbonate, polypropylene, polyethylene, and polyvinyl chloride.

[0107] Any suitable pad can be used as the conjugate pad in the device described herein. In some embodiments, the conjugate pad includes cellulose or glass fiber. In some embodiments, the absorbent pad includes cellulose or glass fiber.

[0108] The device provided herein may further include a sample pad. In some embodiments, the sample pad includes cellulose or glass fiber.

[0109] It should be recognized that the various embodiments of the present invention can be formed with one or more of the above features. The above aspects and features of the present invention can be employed in any suitable combination as the present invention is not limited in this regard. It should also be recognized that the drawings illustrate the various components and features of the various embodiments that can be incorporated into the present invention. For simplicity, some of the drawings may illustrate more than one optional feature or component. However, the present invention is not limited to the specific embodiments disclosed in the drawings. It should be recognized that the embodiments covered by the present invention may include only some of the components illustrated in any one of the drawings, and / or the embodiments covered by the present invention may also cover the combination of components illustrated in different drawings.

[0110] III.Measurement of functional C1-INH

[0111] The present disclosure also provides methods for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample. The assays disclosed herein involve the use of an fC1-INH binder, a C1-INH binder, and a capture agent, all of which are disclosed herein. One of the FC1-INH binder and the C1-INH reagent is conjugated to a docking agent that binds the capture agent. One of the FC1-INH binder, the C1-INH reagent, and the capture agent is conjugated to a detectable label. The detectable label and the docking agent are conjugated to different reagents.

[0112] To perform the assays disclosed herein, a sample suspected of containing fC1-INH can be contacted with an fC1-INH binder, a C1-INH binder, and a capture agent under conditions that permit the formation of a complex comprising fC1-INH, the fC1-INH binder, the C1-INH binder, and the capture agent (by interaction with a docking agent conjugated to the fC1-INH binder or the C1-INH binder). The presence or level of fC1-INH in the sample can be detected and / or quantified by measuring a signal released from the detectable label, which can be conjugated to any one of the fC1-INH binder, the C1-INH binder, and the capture agent.

[0113] In some examples, the sample and the fC1-INH binder (e.g., FXIIa) can first be incubated for an appropriate period of time (e.g., at least 5 minutes, such as 5 - 10 minutes) to allow the formation of an fC1-INH / FXIIa complex. The complex can then be incubated with a C1-INH binder such as an antibody that binds to C1-INH to form a three-component complex, which can then be contacted with a capture agent that binds the docking agent conjugated to the fC1-INH binder or the C1-INH binder. The signal released from the detectable label conjugated to one of the components of the final complex can be measured to determine the presence / absence and / or level of fC1-INH in the sample.

[0114] The methods for detecting and / or quantifying fC1-INH provided herein, in some embodiments, comprise (i) contacting a sample with an fC1-INH binder, a C1-INH binder, and a capture agent to form a complex, wherein one of the fC1-INH binder and the C1-INH reagent is conjugated to a docking agent that binds the capture agent, and wherein one of the fC1-INH binder, the C1-INH reagent, and the capture agent is conjugated to a detectable label, and the detectable label and the docking agent are conjugated to different reagents; and (ii) detecting a signal released from the detectable label from the complex; wherein the presence of a signal released from the detectable label in the complex indicates the presence of fC1-INH in the sample.

[0115] The methods described herein encompass capture agents immobilized on any suitable substrate in any suitable manner. Examples of substrates include, but are not limited to, beads, particles, slides, and microtiter plates. In some embodiments, the capture agent is covalently bound to the substrate. In some embodiments, the capture agent is non-covalently bound to the substrate. In some embodiments, the capture agent is indirectly bound to the substrate, e.g., via a linker.

[0116] In some embodiments, the assay methods disclosed herein can be performed using any of the LFA devices disclosed herein. For example, a sample can be placed in the sample port 620 ( Figure 4 ) and a buffer solution can be placed in the buffer port 610. The sample port 620 can be aligned with the second zone 220. The buffer port 610 can be aligned with the first zone 210. The buffer solution will flow from, e.g., the first zone 210 to the second zone 220 and the third zone 230 together with the sample and the fC1-INH binder, the C1-INH binder, and / or the capture agent immobilized on the first zone 210 and the second zone 220. When the buffer solution passes through the first zone 210, the second zone 220, and the third zone 230, this allows the sample to contact the fC1-INH binder, the C1-INH binder, and the capture agent, such that a complex comprising fC1-INH, the fC1-INH binder, the C1-INH binder, and the capture agent in the sample can be formed. The presence or level of fC1-INH in the sample can be determined by measuring the signal released from a detectable label conjugated to a component of the complex.

[0117] In one example, an LFA device comprising FXIIa-biotin and an anti-C1-INH antibody conjugated to europium particles, e.g., a device configured as shown in Figure 4 is described for illustrative purposes only. In this example, the anti-C1-INH antibody conjugated to europium particles serves as the C1-INH binder conjugated to a detection label, and the anti-C1-INH antibody conjugate is immobilized in the first zone 210. FXIIa conjugated to biotin serves as the fC1-INH binder conjugated to a docking agent, and FXIIa-biotin is immobilized in the second zone 220. To detect the presence of fC1-INH in a sample, the sample is placed at the second zone 220 on the conjugate pad 200 via the sample port 620. When the sample contacts the FXIIa-biotin in the second zone 220, the fC1-INH in the sample binds to the FXIIa, thereby forming an FXIIa-biotin:fC1-INH complex.

[0118] As used herein, the term "contact" refers to the exposure of a sample to one or more binders for a suitable period of time sufficient to form a complex with fC1-INH and / or C1-INH (if any) in the sample. In some embodiments, the sample and / or buffer contacts one or more binders by capillary action, wherein the sample and / or buffer moves through a conjugate pad or membrane.

[0119] The buffer can be placed at the first zone 210 on the conjugate pad 200 through the buffer port 610. After the sample is placed at the second zone 220, the buffer can be placed at the first zone 210 for any length of time, e.g., the buffer can be placed for at least 5 minutes after the sample is placed in the device. The buffer dissolves the anti-C1-INH antibody conjugate and moves it along the conjugate pad 200 from the first zone 210 to the membrane 300 by capillary action. When the buffer reaches the second zone 220, it contacts the FXIIa-biotin:fC1-INH complex, and the anti-C1-INH antibody conjugate in the buffer binds to the fC1-INH in the complex with FXIIa-biotin, thereby forming a "sandwich". In this example, the fC1-INH sandwich thus comprises FXIIa conjugated to biotin that is bound to fC1-INH, and the fC1-INH is bound by an anti-C1-INH antibody conjugated to europium particles.

[0120] The buffer comprising the fC1-INH sandwich continues to move forward on the conjugate pad 200 to the membrane 300, where streptavidin is immobilized at the third zone 230 (e.g., the test line). In this example, streptavidin serves as a capture agent for binding to a docking agent, specifically biotin. When the buffer contacts streptavidin at the third zone 230, the biotin in the fC1-INH sandwich binds to the streptavidin at the third zone 230, thereby capturing the fC1-INH sandwich. Then the presence of fC1-INH in the sample is detected through the test window 630 based on the presence of a signal from the europium particles at the third zone 230. The detection of the fC1-INH sandwich is not limited to detection via europium particles. For example, the presence of fC1-INH can be detected by a detectable change in color or pH. If fC1-INH is not present in the sample, no fC1-INH sandwich is formed and no signal is detected at the third zone 230.

[0121] After moving to the third zone 230, the sample continues to move forward from the membrane 300 to the absorbent pad 400, which acts as a wick to pull the sample up, thereby removing any background material from the third zone 230.

[0122] The methods provided herein encompass detecting and / or quantifying fC1-INH, or its deficiency, in various samples. In some embodiments, the sample is a biological sample obtained from a subject. In some embodiments, the biological sample is a serum sample, a plasma sample, or a blood sample. In some embodiments, the sample is obtained from a subject suspected of having an fC1-INH deficiency-mediated disorder (e.g., HAE) or at risk of an fC1-INH deficiency-mediated disorder (e.g., HAE).

[0123] In some embodiments, the biological sample is a blood sample, e.g., whole blood obtained from a subject. Whole blood includes red blood cells, white blood cells, platelets, and blood plasma. In some embodiments, the blood sample can be collected from a blood vessel (e.g., capillary, vein, and artery). In some embodiments, the blood sample can be obtained by finger prick to produce a few drops of blood. In some embodiments, after obtaining the blood sample, the blood sample is processed at 2-8 °C. For plasma or serum preparation, the plasma or serum can be prepared after collecting the blood by centrifugation. Plasma and serum samples can be stored at -80 °C before analysis.

[0124] In some embodiments, the methods and / or devices described herein may further include a control line. As will be understood by one of ordinary skill in the art, a control line can be used to ensure that the method and / or device operates as expected, e.g., to detect fC1-INH.

[0125] IV. Applications of LFA methods and devices

[0126] The methods and devices described herein can be used for the assessment of a disease, e.g., the diagnosis or prognosis of a disease. The assessment can include identifying that a subject is at risk of a disease described herein or has a disease described herein, e.g., an fC1-INH deficiency-mediated disorder. The assessment can also include monitoring the treatment of a disease, such as assessing the treatment efficacy of an fC1-INH deficiency-mediated disorder. Examples of fC1-INH deficiency-mediated disorders include, but are not limited to, hereditary angioedema (e.g., type I and / or type II HAE), acquired angioedema (e.g., type I and / or type II AAE), C1-INH deficiency-related immune diseases (e.g., systemic lupus erythematosus (SLE)), and C1-INH deficiency-related cancers (e.g., lymphoma).

[0127] In some embodiments, the methods and devices used herein are for assessing whether a subject has hereditary angioedema or is at risk of hereditary angioedema. Generally, there are different types of hereditary angioedema that exhibit similar inflammatory responses but differ in etiology. For example, type I HAE is associated with functional but low levels of C1-INH, while type II HAE is associated with non-functional C1-INH present at normal concentrations.

[0128] A. Diagnosis

[0129] In some embodiments, the methods and devices described herein are used to determine the fC1-INH level in a biological sample (e.g., a serum sample, a plasma sample, or a blood sample) collected from a subject (e.g., a human patient suspected of having an fC1-INH deficiency-mediated disorder such as HAE). The fC1-INH level is then compared to a reference value to determine whether the subject has an fC1-INH deficiency-mediated disorder or is at risk of an fC1-INH deficiency-mediated disorder. The reference value can be a control level of fC1-INH that is capable of binding to an fC1-INH binder (e.g., FXIIa) as described herein. In some embodiments, the control level is the level of fC1-INH in a control sample that is capable of binding to the fC1-INH binder. In some embodiments, the control sample is obtained from a healthy subject or a population of healthy subjects. As used herein, a healthy subject is a subject who is apparently free of an fC1-INH deficiency-mediated disorder or does not have a history of the disease at the time of measurement of the fC1-INH level.

[0130] The control level can also be a predetermined level. Such a predetermined level can represent the fC1-INH level in a population of subjects who do not have an fC1-INH deficiency-mediated disorder or are not at risk of an fC1-INH deficiency-mediated disorder. The predetermined level can take various forms. For example, it can be a single cut-off value, such as a median or an average. In some embodiments, such a predetermined level can be established based on a comparison group, such as one defined group known to have the target disease and another defined group known not to have the target disease. Alternatively, the predetermined level can be a range, e.g., a range representing the fC1-INH levels in a control population within a predetermined percentile.

[0131] The control level as described herein can be determined by various methods. In some embodiments, the control level can be obtained by performing a known method. In some embodiments, the control level can be obtained by performing the same assay used to determine the fC1-INH level in a sample from a subject. In some embodiments, the control level can be obtained by performing the methods described herein. In some embodiments, the control level can be obtained using the devices described herein. In some embodiments, the control level can be obtained from members of a control population and the results can be analyzed, e.g., by a computational program, to obtain a control level (predetermined level) representing the fC1-INH level in the control population.

[0132] By comparing the level of fC1-INH in a sample obtained from a subject that binds to an fC1-INH binder with a reference value as described herein, it can be determined whether the subject has an fC1-INH deficiency-mediated disease (e.g., HAE) or is at risk of an fC1-INH deficiency-mediated disease (e.g., HAE). For example, if the level of fC1-INH in the subject that binds to the fC1-INH binder deviates from the reference value (e.g., is reduced compared to the reference value), then the candidate subject can be identified as having an fC1-INH deficiency-mediated disease such as HAE or being at risk of an fC1-INH deficiency-mediated disease such as HAE. The assays disclosed herein can be used to predetermine a cut-off value for fC1-INH representative of a normal subject. Such a cut-off value can be used to determine whether a subject has an fC1-INH deficiency-mediated disease (e.g., HAE) or is at risk of an fC1-INH deficiency-mediated disease (e.g., HAE). In some cases, an fC1-INH level in a subject that is less than the cut-off value can indicate disease risk or occurrence.

[0133] As used herein, "a level below a reduced level or reference value" means that the level of fC1-INH that binds to an fC1-INH binder is less than the reference value, such as a threshold or level of a predetermined fC1-INH that binds to an fC1-INH binder in a control sample.

[0134] Reduced levels of fC1-INH that bind to an fC1-INH binder include fC1-INH levels that are, for example, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more lower than the reference value. Reduced levels of fC1-INH that bind to an fC1-INH binder also include a reduction in the phenomenon from a non-zero state (e.g., some or detectable fC1-INH that binds to an fC1-INH binder in a sample) to a zero state (e.g., no or undetectable fC1-INH that binds to an fC1-INH binder in a sample).

[0135] In some embodiments, the subject is a human patient having symptoms of an fC1-INH deficiency-mediated disease, such as those disclosed herein, such as HAE. For example, the subject has edema, swelling, wherein the swelling is entirely or predominantly peripheral; urticaria; redness, pain, and swelling without signs of infection; recurrent episodes of non-histamine-mediated edema, swelling, or a combination thereof. In some embodiments, the subject does not have symptoms of an fC1-INH deficiency-mediated disease at the time of sample collection, does not have a history of symptoms of an fC1-INH deficiency-mediated disease, or does not have a history of an fC1-INH deficiency-mediated disease such as HAE. In some embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0136] Examples of fC1-INH deficiency-mediated diseases include, but are not limited to, non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemia / reperfusion injury, brain edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina, edema, eye diseases, gout, bowel diseases, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis - degenerative spondylosis, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergies and respiratory diseases (e.g., allergies, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis), and tissue injury (e.g., burns or chemical injury).

[0137] B. Evaluating treatment efficacy

[0138] The methods and devices described herein can also be applied to evaluate the therapeutic efficacy of fC1-INH deficiency-mediated diseases (e.g., HAE). For example, before and after treatment or during the course of treatment, multiple biological samples (e.g., serum, plasma, or blood samples) can be collected from a subject undergoing treatment. The level of fC1-INH can be measured by any method described herein. If the fC1-INH level increases after treatment or during the course of treatment (the fC1-INH level in a later collected sample compared to the fC1-INH level in a previously collected sample), remains the same, or rises, this indicates that the treatment is effective.

[0139] If a subject is identified as not responding to treatment, a higher dose and / or dosing frequency of the therapeutic agent is administered to the identified subject. In some embodiments, for a subject identified as responding to treatment or not requiring further treatment, the dose or dosing frequency of the therapeutic agent is maintained, decreased, or stopped. Alternatively, a different treatment can be applied to a subject found not to respond to the first treatment.

[0140] Therapeutic agents include, but are not limited to, kallikrein binders, bradykinin B2 receptor antagonists, C1-INH substitutes, DX-2930, and DX88 (see, e.g., PCT Publication No. WO 2014 / 113701, which is incorporated herein by reference in its entirety). Examples

[0141] The following examples are provided to more fully understand the devices and methods described herein. The examples described in this application are for illustrative purposes of the methods and compositions provided herein and should not be construed in any way as limiting their scope.

[0142] Example 1 : Preparation of a lateral flow assay (LFA) device for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH)

[0143] Stripping of the membrane

[0144] FRONTLINE HR TM (BioDot) was used to stripe the membrane. The Front lines were washed with 10 cycles of wash buffer (0.05% in diH2O (Stepan Company)) and aligned so that the test line was 11 mm from the bottom of the membrane. The Front lines were emptied and primed with 0.5 mg / mL streptavidin in 10 mM phosphate, pH 7.3, 0.5% sucrose. The test line of streptavidin was striped onto the membrane (see, e.g., Figure 3A the third zone 230 on the middle membrane 300). The membrane was then labeled and dried at 40 °C for 30 minutes. The Front lines were washed with 10 cycles of wash buffer after stripping. The components used to stripe the membrane are shown in Table 1.

[0145] Table 1. Components used to stripe the membrane.

[0146]

[0147]

[0148] Stripping of the conjugate pad

[0149] FRONTLINE HR TM(BioDot) was used to stripe the anti-C1-INH Eu particle conjugate and FXIIa-biotin onto the conjugate pad (Ahlstrom). Before striping the conjugate pad, the anti-C1-INH Eu particle conjugate was diluted to 0.04% w / v in Eu latex diluent and FXIIa-biotin was diluted to 2.4 μM in FXIIa-biotin diluent. Then, the Front lines were washed with 10 cycles of wash buffer (0.05% in diH2O) (Stepan Company). The Front lines were aligned so that the anti-C1-INH Eu particle conjugate was striped from 15 mm from the bottom of the conjugate pad and the FXIIa-biotin conjugate was striped from 8 mm from the top of the conjugate pad at rates of 10 and 2.5 μL / cm, respectively. The positions of the anti-C1-INH Eu particle conjugate and FXIIa-biotin conjugate were aligned with the buffer port and sample port in the custom SLA housing cassette, respectively.

[0150] Empty the Front lines and perfuse with either conjugate. The conjugate was striped onto the conjugate pad, which was then labeled and dried at 40 °C for 30 minutes. The conjugate pad was sealed and dried. The Front lines were washed with 10 cycles of wash buffer after striping. The components used to stripe the conjugate pad are shown in Table 2. The components of the Eu latex diluent and FXIIa-biotin diluent are shown in Tables 3 and 4, respectively.

[0151] Table 2. Components for striping the conjugate pad

[0152]

[0153]

[0154] Table 3. Components of the Eu latex diluent

[0155]

[0156] Table 4. Components of the FXIIa-biotin diluent

[0157]

[0158] Preparation of the anti-C1-INH Eu particle conjugate

[0159] To prepare the anti-C1-INH Eu particle conjugate, using the manufacturer's protocol, 0.1 mg of anti-C1-INH antibody was exchanged into 50 mM borate, pH 8 via a ZEBA TM spin column (ThermoFisher). By absorbance (A 280, 1 mm, 1 OD = 1.4 mg / mL) to determine the concentration of the antibody. The stored latex was rotated for 10 minutes and then sonicated using a micro sonicator (set at 25) for 10 - 15 seconds. The stored latex solution (10%) was diluted to 1% in 0.1 M MES, pH 6.5 and microcentrifuged at 17,000 g for 10 minutes. The supernatant was removed and the pellet was resuspended in 0.1 M MES, pH 6.5 in which the volume of the buffer was equal to the starting volume of the latex. The resulting solution was sonicated, microcentrifuged and resuspended as previously described.

[0160] To prepare 15 mg / mL of EDC in 0.1 M MES buffer, the EDC was allowed to equilibrate to room temperature and weighed. The EDC solution was prepared within 10 minutes for use in the preparation of anti-C1-INH Eu particle conjugates. The EDC stock powder was dried and frozen at -20 °C for long-term storage.

[0161] To prepare 50 mg / mL of sulfo-NHS in 0.1 M MES buffer, the sulfo-NHS was allowed to equilibrate to room temperature and weighed. The sulfo-NHS solution was prepared within 10 minutes for use in the preparation of anti-C1-INH Eu particle conjugates. The sulfo-NHS solution was activated by incubation on a shaker at 1000 rpm for 30 minutes. The solution was microcentrifuged at 17,000 g for 8 minutes. The pellet was resuspended in 50 mM borate buffer, vortexed and sonicated. The volume of the buffer was equal to the starting volume of the latex solution (600 μL). The solution was then microcentrifuged as previously described, resuspended in borate buffer (300 μL), vortexed and sonicated. The activated particles were aliquoted into 50 μL / tube and an appropriate amount of buffer and protein (e.g., 20 μg of protein (i.e., antibody)) was added to obtain a 20:1 mass-to-mass ratio of particles to protein. The tubes were vortexed immediately after the addition of buffer and protein.

[0162] The tubes were incubated on a shaker at 1,000 rpm at room temperature for 2 hours. After incubation, 1 M ethanolamine was added at 10 μL / mL and the tubes were incubated on a shaker at 1,000 rpm at room temperature for 30 minutes. The tubes were microcentrifuged at 17,000 g for 10 minutes and the pellet was resuspended in 7-day aged 1% casein and incubated with shaking overnight. After overnight incubation, the tubes were microcentrifuged and the pellet was resuspended in 7-day aged 1% casein, vortexed and sonicated. The particle conjugates were then streaked onto the binding pads as described herein. The components for the anti-C1-INH Eu particle conjugates are shown in Table 5.

[0163] Table 5. Anti-C1-INH Eu particle conjugates

[0164]

[0165]

[0166] Laminating components onto a backing card

[0167] To laminate the components onto an 80 mm long backing card (DCN), the backing sticker was cut off at 39 mm from the bottom of the card using a ruler razor. The backing sticker was removed from the position 39 mm from the top of the card. The film was attached to the card at 39 mm from the bottom of the card. The absorbent pad (Ahlstrom) was attached to the top of the card so that the pad overlapped the top of the film by 3 mm. The remaining backing sticker was removed, and the binding pad was attached to the bottom of the card so that the pad overlapped the bottom of the film by 3 mm. Tables 6 and 7 show the components and the component order, respectively.

[0168] Table 6. Card components

[0169]

[0170] Table 7. Laminating component order, orientation, and position

[0171]

[0172] Preparing test strips

[0173] The card was placed into a Kinematic cutter (Kinematic), and the card was cut into 5.0 mm wide strips. Strips shorter than 5.0 mm or strips marked with a pen during strip cutting were discarded. The cut strips were placed into a foil bag with a desiccant. The foil bag was sealed and stored in a drying oven until use.

[0174] Example 2 : Detecting functional C1-esterase inhibitor (fC1-INH) from a patient plasma sample using an LFA device.

[0175] To perform a lateral flow assay (LFA) for determining the fC1-INH concentration, the test strips prepared as described in Example 1 above were placed into a custom stereolithography (SLA) cassette (see, for example, Figure 4) Standards and quality controls were prepared by spiking the C1-INH reference standard (lot #TCP103, Shire, Takeda Company) into C1-INH depleted human K3-EDTA plasma (prepared by Shire, Takeda Company). Control samples containing 0 mU / mL to 800 mU / mL of purified C1-INH were used to generate a calibration curve. The control samples were diluted 1:20 in C1-INH depleted media. Samples were added to the sample port and incubated for 5 minutes. Running buffer (30 μL) was added to the sample port to allow the sample to run onto the membrane and up to the test window. Then running buffer (150 μL) was added to the buffer port and the cassette was incubated for an additional 20 minutes. The test strip was removed from the cassette and the intensity of the test line region was measured using an Axxin AX-2X fluorescence reader (Axxin). Figure 5 An image of the test strip in the cassette is shown. The intensity of the measured test line region was plotted against the C1-INH concentration to generate Figure 6 the calibration curve shown in 2 .

[0176] Fifty normal K3-EDTA plasma samples were obtained commercially and fifty HAE plasma samples were obtained from patients who consented to participate in SAHARA, a Phase III, randomized, double-blind, placebo-controlled, two-period, three-sequence, partially-crossed study that evaluated the efficacy and safety of subcutaneous administration of 2000 IU of C1-esterase inhibitor [human] injection solution for the prevention of attacks of angioedema in adolescents and adults with HAE.

[0177] Then the calibration curve from Figure 6 was used to determine the fC1-INH concentration in plasma samples from control subjects and subjects with hereditary angioedema (HAE). Briefly, plasma samples from subjects were diluted 1:20 in C1-INH depleted plasma. Diluted plasma samples (20 μL) were added to the sample port and incubated for 5 minutes. The fC1-INH concentration determined using the test strip was within 20% of the concentration value determined by ELISA.

[0178] As Figure 8As shown, in both methods, C1-INH levels were lower in HAE subjects compared to healthy controls. In the chromogenic and LFA methods, the mean C1-INH concentrations measured in healthy controls were 1345 and 1089 mU / mL, respectively, and the mean C1-INH concentrations measured in HAE subjects were 275 and 163 mU / mL, respectively (Table 8). The SEM and 95% confidence intervals measured are provided in Table 8. The C1-INH data for all HAE subjects obtained from the two methods were correlated with each other, where R 2 was 0.86, including the C1-INH concentrations of two HAE subjects within the healthy control range ( Figure 9 ). The mean ratios between fC1INH measured in normal controls and HAE subjects in the chromogenic and LFA methods were 4.9 and 6.7, respectively.

[0179] The receiver operating characteristic curve (ROC) for the diagnostic performance based on samples from control subjects and subjects with HAE was 0.98, indicating that the C1-IHN concentrations determined by LFA precisely discriminated between control and HAE subjects ( Figure 7 ). The ROC curve indicated that a C1-INH cut-off point of 496 mU / mL yielded 94% sensitivity (true positive rate) and 96% specificity (false positive rate) ( Figure 7 ); false negatives and false positives are listed in Table 9.

[0180] As shown in Table 8 and Figures 8 - 9As shown, the results obtained using LFA were compared with the chromogenic assay of ELISA. Briefly, the chromogenic method directly measures fC1-INH levels and involves cleaving C1s of a synthetic substrate to form a colored compound, where a decrease in color intensity indicates inhibition of C1s enzyme activity. The precision, accuracy, linearity, and upper and lower limits of quantification of the chromogenic assay were all acceptable. C1-INH protein (2000 IU of C1 esterase inhibitor [human] injection liquid, Shire, Takeda Company) was used to prepare three quality controls and a standard curve with 10 standard points ranging from 1000 - 1.95 mU / mL. The highest and lowest points of the standard curve were used as anchor points. Briefly, K3-EDTA plasma samples and reference protein were pre-incubated with recombinant human complement component C1s protein (R&D Systems) in a polypropylene plate for 30 minutes at room temperature (RT). The formed C1-INH and C1s complex was diluted 1:5 in assay buffer and mixed with substrate solution (synthetic substrate with a thiobenzyl ester group, M-1300, Bachem) and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) #D-8000, Biosynth), and the reaction was allowed to incubate for 40 minutes at RT. Absorbance was recorded at 405 nm using a SpectraMax M5 plate reader with SoftMax Pro software.

[0181] Table 8. Results comparing the chromogenic assay and LFA to determine fC1-INH concentration

[0182]

[0183]

[0184] Table 9. Results showing false positives and negatives for control and HAE samples.

[0185]

[0186] In summary, these results indicate that similar FC1-INH concentrations were detected in patient plasma samples by LFA and ELISA (referred to as the chromogenic assay). Thus, the LFA and test strips described herein may be effective tools for identifying patients with HAE based on fC1-INH levels in plasma samples from patients. The results obtained using the LFA described herein are correlated with the results from the chromogenic assay for assessing FC1-INH.

[0187] The rapid and sensitive LFA methods and devices disclosed herein can be performed in a physician's office laboratory for the rapid diagnosis of HAE (e.g., types I & II) based on fC1INH levels. Such methods and devices can result in low-cost consumables for Medicare reimbursement, a high confidence level for quantitative results, easy interpretation of data by physicians, and / or a low level of need for confirmatory analysis. This rapid assay for diagnosing type I or type II HAE in the physician's office can expand HAE screening and more quickly identify new HAE patients. Currently, the global diagnostic rate of HAE is only 40%; thus, undiagnosed patients have a high unmet need. The availability of rapid tests on common device platforms can expand the recognition of HAE. In addition, the rapid test for fC1INH disclosed herein can help clinically monitor the progression of HAE disease or the response to therapy in a timely manner.

[0188] References

[0189] 1. Maurer, M. et al., (2018) The international WAO / EAACI guideline for the management of hereditary angioedema - the 2017 revision and update. World Allergy Organization Journal

[0190] 2. Aabom, A. et al., (2017) Complement factor C4 activation in patients with hereditary angioedema. Clinical Biochemistry 50(15), 816 - 821.

[0191] 3. Bork, K. and Davis - Lorton, M. (2013) Overview of hereditary angioedema caused by C1 - inhibitor deficiency: assessment and clinical management. Eur Ann Allergy Clin Immunol 45(1), 7 - 16.

[0192] 4. Csuka, D. et al., (2017) The role of the complement system in hereditary angioedema. Mol Immunol 89, 59 - 68.

[0193] 5. Li, H. H., et al. (2015). Comparison of chromogenic and ELISA functional C1 inhibitor tests in diagnosing hereditary angioedema. J Allergy Clin Immunol Pract 3(2), 200 - 5.

[0194] 6. Campbell, R. L., Wagner, D. B., and O’Connel, J. P. (1987). Solid phase assay with visual readout. U.S. Patent No. 4,703,017.

[0195] 7. Rosenstein, R. W., and Bloomster, T. G. (1989). Solid phase assay employing capillary flow. U.S. Patent No. 4,855,240.

[0196] 8. May, K., Prior, M. E., and Richards, I. (1997). Capillary immunoassay and device therefore comprising mobilizable particulate labelled reagents. U.S. Patent No. 5,622,871.

[0197] 9. O’Farrell, B. (2009). Evolution in Lateral Flow - Based Immunoassay Systems. In: Wong, R. C. and Tse, H. Y. (eds.). Lateral Flow Immunoassay . Humana Press New York (NY).

[0198] 10. Zahedi R, Aulak KS, Eldering E, Davis AE 3rd (1996). Characterization of C1 inhibitor - Ta. A dysfunctional C1INH with deletion of lysine 251. J Biol Chem. 1996 Sep 27; 271(39):24307 - 12

[0199] Example 3: Competitive binding assays demonstrated the specificity of the lateral flow assay (LFA) device for detecting functional C1-esterase inhibitor (fC1-INH).

[0200] The specificity of the lateral flow assay (LFA) device, as described herein, for determining fC1-INH concentration was examined by performing the assay in the presence of different competing binding proteins. Samples contained 100 mU / mL of purified C1-INH. No competing protein was added to the control sample. The intensity of the test line region was measured and the percent reduction of the signal from the control reaction was calculated for each sample. For samples with competing proteins, the intensity of the test line (TL) decreased between 40% and 60% compared to the control sample (Table 10). By adding biotinylated BSA, the TL intensity decreased by 55%, indicating that non-related proteins were not detected in the assay (Table 10). The TL intensities of unlabeled FXIIa and unlabeled antibody decreased by 61% and 48%, respectively, indicating the specificity of FXIIa and antibody with C1-INH for signal generation (Table 10).

[0201] Table 10. Results of specificity tests

[0202]

[0203] The specificity of detection was further tested using C1-INH protein that had been heat-denatured. When heated at 40 °C, the TL intensity did not decrease, but when heated at 53 °C, the TL intensity decreased to the TL intensity of the background signal (Table 11).

[0204] Table 11. Results of specificity tests with heat-treated C1-INH

[0205]

[0206] In summary, these results demonstrate that the LFA and test strips described herein are specific for detecting FC1-INH in plasma samples.

[0207] Example 4: Blood samples from patients were used to detect functional C1-esterase inhibitor (fC1-INH) in the LFA device.

[0208] Briefly, whole blood samples were collected by finger prick according to laboratory practices that are obvious to those of ordinary skill in the art. After wiping away the first blood drop, a second blood drop was formed on the finger. A sample loop was used to contact the second blood drop to fill the loop with blood ( Figure 10A ). The loop was then added to a container, such as a bottle ( Figure 10B)。When the ring touches the bottom of the bottle, snap and twist the bottle so that the bottom of the shaft enters the bottle. Finally, replace the bottle cap and shake the bottle to mix. Whole blood samples can be added to any device for analysis.

[0209] Example 5: Selection of Reagents

[0210] Reagents were selected for the methods and / or devices described herein. Two initial detection agents were developed: one was europium nanoparticles conjugated to anti-fC1-INH Fab ( Figure 11A ), and one was red gold nanoparticles conjugated to anti-fC1-INH Fab ( Figure 11B ).

[0211] A sharp decline in signal was observed at the lower end of the dynamic range, indicating that the system reached its maximum signal. To reduce the slope of this relationship, different amounts of different reagents were evaluated. Two reagents were evaluated as test lines: streptavidin and a polymeric form of streptavidin (poly-streptavidin R), which were printed at a concentration of 0.5 mg / mL. Streptavidin was found to have a higher signal compared to poly-streptavidin, while poly-streptavidin was found to have a higher linearity ( Figure 12 ). The europium conjugate was adjusted from 0.1% to 0.05% solids. The concentration of FXIIa used in the incubation step with was reduced from 1 pmol / μL to 0.5 pmol / μL to reduce the dynamic range of the assay. This resulted in an assay with a signal within the detectable range.

[0212] Different reagents were also evaluated as test lines. Briefly, three different test lines were generated: human biotinylated FXIIa (B-HFXIIa) mixed with streptavidin, B-HFXIIa mixed with poly-streptavidin, and HFXIIa (non-biotinylated) alone. Using HFXIIa provided a signal with a still low positive, while B-HFXIIa and streptavidin as well as B-HPFXIIa and streptavidin did not show any distinct positive signal ( Figure 13 ).

[0213] In addition, anti-C1-INH antibodies were also evaluated for the methods described herein. Four europium conjugates (anti-C1-INH Fab and antibodies RP01, RP02, MM03, and MM06 from Sino Biological Inc.) were evaluated at four concentrations (1200 mU / mL, 600 mU / mL, 100 mU / mL, and 9 mU / mL) ( Figure 14)。The signal obtained with each of the antibodies RP01, RP02, MM03, and MM06 was enhanced compared to the Fab conjugate. The polyclonal antibody RP02 was selected for further analysis.

[0214] Buffer conditions were also evaluated. For example, buffers with an inorganic buffer (IBA) or an organic buffer (OBA) were prepared at different pH values from 7.0 - 9.5. The buffer with IBA performed better (e.g., higher signal) at the higher end of the pH range (pH 9.5), while the buffer with OBA performed better (e.g., higher signal) in the physiological pH range (approx. 7 - 7.5)( Figure 15 ).

[0215] Finally, detectable reagents conjugated to anti-C1-INH binders were also evaluated. In particular, the antibody RP02 was evaluated with an europium conjugate or a red gold conjugate and compared to anti-C1-INH Fab. The Fab conjugate did not result in a positive signal, while the RP02 antibody with the red gold conjugate resulted in a positive signal( Figure 16 ).

[0216] Other embodiments

[0217] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features. From the above description, those skilled in the art can easily determine the basic features of the present disclosure and, without departing from its spirit and scope, can make various changes and modifications to adapt the present disclosure to various uses and conditions. Accordingly, other embodiments are within the scope of the claims.

[0218] Equivalents and scope

[0219] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather as set forth in the appended claims.

[0220] In a claim, the articles “a,” “an,” and “the” mean one or more than one, unless contrary indication or context indicates otherwise. A claim or description including “or” among one or more members of a group is satisfied if one, more than one, or all of the members of the group are present in, employed in, or otherwise related to a given product or process, unless contrary indication or context indicates otherwise. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise related to a given product or process. The present disclosure includes embodiments in which more than one or all of the members of the group are present in, employed in, or otherwise related to a given product or process.

[0221] In addition, the present disclosure also encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same basic claim. When elements are presented in a list form, such as in a Markush group, each sub-group of the elements is also disclosed, and any element can be removed from the group. It should be understood that, generally, where the present disclosure or an aspect of the present disclosure is referred to as including a particular element and / or feature, certain embodiments of the present disclosure or an aspect of the present disclosure consist of or consist essentially of such element and / or feature. For purposes of simplicity, those embodiments are not specifically stated in such words herein. It is also noted that the terms “comprising” and “including” are intended to be open and allow the inclusion of additional elements or steps. Where ranges are given, endpoints are included. In addition, unless otherwise indicated or can be seen from the context and the understanding of those of ordinary skill in the art, in different embodiments of the present disclosure, a numerical value expressed as a range can assume any specific value or sub-range within the said range, to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0222] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If any conflict exists between any of the incorporated references and this specification, this specification shall prevail. Additionally, any particular embodiments of the present disclosure that fall within the prior art may be expressly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if not explicitly stated as such herein. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0223] Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications may be made to this description without departing from the spirit or scope of the present disclosure as defined by the claims.

Claims

1. An apparatus for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH), the apparatus comprising: (i) a binding pad comprising a first zone and a second zone, wherein the first zone comprises a first reagent and the second zone comprises a second reagent, and (ii) a membrane in communication with the binding pad, wherein the membrane comprises a third zone, wherein the third zone comprises a third reagent, wherein the first reagent is a functional C1 inhibitor (fC1-INH) binder or a C1 inhibitor (C1-INH) binder and the second reagent is an fC1-INH binder or a C1-INH binder, and the first reagent and the second reagent are different from each other; wherein the third reagent is a capture agent capable of binding to a docking agent; wherein one of the fC1-INH binder and the C1-INH binder is conjugated to a detectable label, and one of the fC1-INH binder and the C1-INH binder is conjugated to a docking agent, and the detectable label and the docking agent are conjugated to different reagents; and wherein the binding pad further comprises a fourth zone for placing a biological sample and a fifth zone for placing a buffer, and wherein the buffer flows through the apparatus in the order of the first zone, the second zone, and the third zone.

2. The apparatus according to claim 1, wherein the first reagent, the second reagent, and the third reagent are a C1-INH binder, an fC1-INH binder, and a capture agent, respectively, or wherein the first reagent, the second reagent, and the third reagent are an fC1-INH binder, a C1-INH binder, and a capture agent, respectively.

3. The apparatus according to claim 1 or claim 2, wherein the first reagent is conjugated to the detectable label, the second reagent is conjugated to the docking agent, or wherein the first reagent is conjugated to the docking agent, and the second reagent is conjugated to the detectable label.

4. The apparatus according to claim 1 or claim 2, wherein the fC1-INH binder is the active form of factor XII (FXIIa).

5. The apparatus according to claim 1 or claim 2, wherein the C1-INH binder is an antibody that binds C1-INH.

6. The apparatus according to claim 1 or claim 2, wherein the docking agent and the capture agent are members of a receptor-ligand pair.

7. The apparatus according to claim 6, wherein the receptor-ligand pair comprises biotin and avidin.

8. The apparatus according to claim 7, wherein the docking agent is biotin and the capture agent is avidin.

9. The apparatus according to claim 8, wherein the avidin is streptavidin or polyavidin.

10. The apparatus according to claim 1 or claim 2, wherein the detectable label is selected from the group consisting of: europium, colloidal gold, phycoerythrin, fluorescein, rhodamine, green fluorescent protein, quantum dots, and chromophores.

11. The apparatus according to claim 10, wherein the detectable label is europium.

12. The apparatus according to claim 10, wherein the detectable label is colloidal gold.

13. The device according to claim 1 or claim 2, wherein the detectable tag is attached to latex particles.

14. The device according to claim 1 or claim 2, wherein the fourth region overlaps with the second region.

15. The device according to claim 1 or claim 2, wherein the first reagent is a C1-INH binder located in the first region, the second reagent is an fC1-INH binder located in the second region, and the third reagent is a capture agent located in the third region.

16. The device according to claim 15, wherein the C1-INH binder is an antibody that binds to C1-INH and is conjugated to a detectable tag, the fC1-INH binder is FXIIa conjugated to a docking agent, wherein the docking agent is biotin, and the capture agent is avidin.

17. The device according to claim 16, wherein the avidin is streptavidin or polyavidin.

18. The device according to claim 1 or claim 2, further comprising an absorbent pad in communication with the membrane, wherein the absorbent pad and the binding pad are separated by the membrane.

19. The device according to claim 18, further comprising a support member on which the binding pad, the membrane, and / or the absorbent pad are mounted.

20. The device according to claim 1 or claim 2, further comprising a housing.

21. The device according to claim 20, wherein the housing includes a first opening that forms a buffer port, a second opening that forms a sample port, and a third opening that forms a test window.

22. The device according to claim 21, wherein the sample port is located between the buffer port and the test window.

23. The device according to claim 21 or claim 22, wherein the buffer port is aligned with the first region, and the C1-INH binder is located on the first region.

24. The device according to claim 21 or claim 22, wherein the sample port is aligned with the second region, and the fC1-INH binder is located on the second region.

25. The device according to claim 21 or claim 22, wherein the test window is aligned with the third region, and the capture agent is located on the third region.

26. An in vitro non-diagnostic method for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample, the method comprising: (i) placing the sample in the sample port of the device according to any one of claims 21-25; (ii) placing a buffer in the buffer port of the device, wherein the buffer flows in the direction from the first region to the third region; (iii) detecting a signal at the test window of the device; and (iv) determining the presence of fC1-INH in the sample or measuring the level of fC1-INH in the sample based on the presence or intensity of the signal at the test window.

27. The method according to claim 26, wherein step (ii) is performed at least 5 minutes after step (i), and wherein the second region includes an fC1-INH binder and is aligned with the sample port.

28. The method according to claim 26 or claim 27, wherein: (a) The sample is a biological sample obtained from a subject; and / or (b) The subject is a human patient suspected of having an fC1-INH deficiency-mediated disorder or at risk of an fC1-INH deficiency-mediated disorder.

29. The method according to claim 28, wherein the biological sample is a serum sample, a plasma sample or a blood sample.

30. The method according to claim 28, wherein the fC1-INH deficiency-mediated disorder is selected from the group consisting of: hereditary angioedema (HAE), acquired angioedema (AAE) and C1-INH-related immune diseases.

31. The method according to claim 30, wherein (A) the subject has symptoms of HAE and / or HAE is type I HAE or type II HAE, or (B) the subject does not have symptoms of HAE, does not have a history of symptoms of HAE, or does not have a history of HAE.

32. An in vitro non-diagnostic method for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample, the method comprising: (i) contacting the sample with an fC1-INH binder and a C1-INH binder to form a complex, wherein one of the fC1-INH binder and the C1-INH binder is conjugated to a docking agent, wherein one of the fC1-INH binder and the C1-INH binder is conjugated to a detectable label, and the detectable label and the docking agent are conjugated to different binders; (ii) contacting the complex of (i) with a capture agent, wherein the capture agent binds to the docking agent; and (iii) detecting a signal released from the detectable label from the complex; wherein the presence of a signal released from the detectable label from the complex indicates the presence of fC1-INH in the sample.

33. The method according to claim 32, wherein step (i) is carried out by: (a) incubating the sample with the fC1-INH binder for at least 5 minutes.

34. The method according to claim 32 or claim 33, wherein the fC1-INH binder is the active form of factor XII (FXIIa).

35. The method according to claim 32 or claim 33, wherein the docking agent and the capture agent are members of a receptor-ligand pair.

36. The method according to claim 35, wherein the docking agent is biotin and the capture agent is avidin; or the docking agent is avidin and the capture agent is biotin.

37. The method according to claim 36, wherein the avidin is streptavidin or polyavidin.

38. The method according to claim 32 or claim 33, wherein the C1-INH binder is an antibody that binds C1-INH.

39. The method according to claim 32 or claim 33, wherein the detectable label is selected from the group consisting of: europium, colloidal gold, phycoerythrin, fluorescein, rhodamine, green fluorescent protein, quantum dots and chromophores.

40. The method according to claim 39, wherein the detectable label is europium.

41. The method according to claim 39, wherein the detectable label is colloidal gold.

42. The method according to claim 32 or claim 33, wherein the detectable label is attached to the latex particles.

43. The method according to claim 32 or claim 33, wherein the fC1-INH binder is the active form of factor XII (FXIIa) conjugated to biotin, wherein the C1-INH binder is an antibody that binds C1-INH, the antibody being conjugated to a detectable label, and wherein the capture agent is streptavidin.

44. The method according to claim 43, wherein step (i) is carried out by: (a) incubating the sample with the fC1-INH binder for at least 5 minutes to form a first complex, (b) contacting the first complex with the C1-INH binder to form a second complex, and wherein step (ii) is carried out by contacting the second complex with the capture agent to form a complex, and wherein the capture agent is immobilized on a support member.

45. The method according to claim 32 or claim 33, wherein: (a) the sample is a biological sample obtained from a subject; and / or (b) the subject is a human patient suspected of having an fC1-INH deficiency-mediated disorder or at risk of an fC1-INH deficiency-mediated disorder; and / or (c) the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

46. The method according to claim 45, wherein the biological sample is a serum sample, a plasma sample, or a blood sample.

47. The method according to claim 45, wherein the fC1-INH deficiency-mediated disorder is selected from the group consisting of: hereditary angioedema (HAE), acquired angioedema (AAE), and C1-INH-related immune diseases.

48. The method according to claim 47, wherein (A) the subject has symptoms of HAE and / or HAE is type I HAE or type II HAE, or (B) the subject does not have symptoms of HAE, does not have a history of symptoms of HAE, or does not have a history of HAE.

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