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

Abstract

To provide a novel assay and / or platform for measuring functional C1-INH involved in HAE disease pathology.SOLUTION: A device and a lateral flow assay (LFA) method performed using such device is a device for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH), including: (i) a conjugate pad comprising a first zone and a second zone in which a first agent and a second agent are immobilized, respectively; and (ii) a membrane in communication with the conjugate pad, in which the membrane includes a third zone in which a third agent is immobilized.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 833,235, filed April 12, 2019, and U.S. Provisional Application No. 62 / 930,615, filed November 5, 2019, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] C1-esterase inhibitor (also known as C1-inhibitor or C1-INH) is a protease inhibitor belonging to the serpin 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 cause hereditary angioedema (HAE), including types I and II.

[0003] Currently available assays used to assess functional levels of C1-INH utilize either a chromogenic assay or a complex ELISA method to measure the inhibition of C1s in the complement cascade by C1-INH. Although chromogenic assays are generally considered preferable, both methods have limitations. Chromogenic assays are more likely to produce occasional false positives, while complex ELISAs have a negative predictive value of only 62%.

[0004] It is of interest to develop newer assays and / or platforms to measure functional C1-INH, which is involved in HAE disease pathology. Summary of the Invention

[0005] Provided herein are devices and methods for detecting functional C1-INH (fC1-INH) in a rapid, cost-effective, qualitative and / or quantitative manner. In some embodiments, the detection of fC1-INH is achieved using a device configured to detect and / or quantify fC1-INH by lateral flow immunoassay (LFA).

[0006] Thus, one aspect of the present disclosure provides a device for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH), comprising: (i) a conjugate pad including a first zone and a second zone in which a first agent and a second agent are immobilized, respectively; and (ii) a membrane in communication with the conjugate pad, the membrane including a third zone in which a third agent is immobilized. The first agent may be a functional C1-inhibitor (fC1-INH)-binding substance or a C1-inhibitor (C1-INH)-binding substance. The second agent and the third agent are one of a functional C1-inhibitor (fC1-INH)-binding substance, a C1-inhibitor (C1-INH)-binding substance, and a capture substance. The first agent, the second agent, and the third agent are different from each other. One of the fC1-INH binding substance, the C1-INH binding substance, and the capture substance is conjugated to a detectable label, and one of the fC1-INH binding substance and the C1-INH binding substance is conjugated to a docking agent. The detectable label and the docking agent are conjugated to different agents. The conjugate pad further includes a fourth zone for placing a biological sample that flows through the device in the order of the first zone, the second zone, and the third zone. In some examples, the fourth zone for placing a biological sample can overlap with the second zone where the fC1-INH binding substance can be placed.

[0007] In some embodiments, the first agent, the second agent, and the third agent are a C1-INH binding agent, a fC1-INH binding agent, and a capture agent, respectively. In other embodiments, the first agent, the second agent, and the third agent are a fC1-INH binding agent, a C1-INH binding agent, and a capture agent, respectively.

[0008] In some examples, the first agent is conjugated to a detectable label and the second agent is conjugated to a docking agent, hi other examples, the first agent is conjugated to a docking agent and the second agent is conjugated to a detectable label.

[0009] In some embodiments, the C1-INH binding agent can be the activated form of factor XII (FXIIa). Alternatively, or in addition, the C1-INH binding agent can be an antibody that binds to C1-INH. Furthermore, the docking agent and capture agent can be members of a receptor-ligand pair. For example, the receptor-ligand pair can include biotin and avidin (e.g., streptavidin or polystreptavidin).

[0010] 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 examples, the detectable label may be linked to a latex particle.

[0011] In one example, the first agent is a C1-INH binding agent disposed in the first zone, the second agent is a fC1-INH binding agent disposed in the second zone, and the third agent is a capture agent disposed in the third zone. The C1-INH binding agent may be an antibody that binds to C1-INH, which may be conjugated to a detectable label as disclosed herein. Alternatively, or in addition, the fC1-INH binding agent may be FXIIa, which may be conjugated to a docking agent (e.g., biotin). Furthermore, the capture agent may be avidin, such as streptavidin or polystreptavidin.

[0012] In some embodiments, the device further comprises an absorbent pad in communication with the membrane. The absorbent pad and the conjugate pad may be separated by a membrane. Alternatively, or in addition, the device may further comprise a support member to which the conjugate pad, membrane, and / or absorbent pad are attached.

[0013] The device may further include a housing. In some embodiments, the housing may include a first opening for forming a buffer port, a second opening for forming a sample port, and a third opening for forming a test window. The sample port may be disposed between the buffer port and the test window. In some examples, the buffer port may be aligned with a first zone in which the C1-INH binding substance is disposed. In some examples, the sample port may be aligned with a second zone in which the fC1-INH binding substance is disposed. In some examples, the test window is aligned with a third zone in which the capture substance is disposed.

[0014] In another aspect, the present disclosure provides a method for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample using any of the LFA devices disclosed herein. Such a method may include: (i) placing a sample in a sample port of a device described herein; (ii) placing a buffer in a buffer port of the device, wherein the buffer flows from the first zone to the third zone; (iii) examining a signal in a test window of the device; and (iv) determining the presence or intensity of the signal in the test window based on the presence or intensity of the signal. In some embodiments, step (ii) is performed at least 5 minutes after step (i). An fC1-INH binding substance may be immobilized in a second zone that may be aligned with the sample port.

[0015] Also provided herein is a method for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample, the method comprising: (i) contacting the sample with a fC1-INH binding agent, a C1-INH binding agent, and a capture agent to form a complex, wherein one of the fC1-INH binding agent and the C1-INH agent is conjugated to a docking agent that binds to the capture agent, and one of the fC1-INH binding agent, the C1-INH agent, and the capture agent is conjugated to a detectable label, wherein the detectable label and the docking agent are conjugated to separate agents; and (ii) detecting a signal emitted from the detectable label in the complex, wherein the presence of a signal emitted from the detectable label in the complex indicates the presence of fC1-INH in the sample. Any of the fC1-INH binding agents, C1-INH binding agents, docking agents, detectable labels, and capture agents as disclosed herein can be used in the methods disclosed herein.

[0016] In some embodiments, step (i) may be performed by (a) incubating the sample with the fC1-INH binding agent for at least 5 minutes, and (b) contacting the sample with the C1-INH binding agent and the capture agent. In other embodiments, step (i) is performed by (a) incubating the sample with the fC1-INH binding agent for at least 5 minutes to form a first complex, (b) contacting the first complex with the C1-INH binding agent to form a second complex, and (c) contacting the second complex with the capture agent to form a complex, wherein the capture agent is immobilized on a support member.

[0017] In any of the assay methods disclosed herein, the sample analyzed may 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 may be a human patient suspected of having or at risk for a fC1-INH deficiency-mediated disorder, including, but not limited to, hereditary angioedema (HAE), acquired angioedema (AAE), and C1-INH-associated immune disorders. 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.

[0018] The details of several embodiments of the apparatus and methods described herein are set forth in the accompanying drawings and detailed description. Other features, objects, and advantages of the apparatus and methods described herein will be apparent from the description and claims.

[0019] Various aspects and embodiments are described with reference to the following drawings, which are not necessarily drawn to scale. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a schematic diagram of an exemplary lateral flow assay (LFA) device 100 for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH). The LFA device 100 includes a conjugate pad 200, a membrane 300, an optional absorbent pad 400, and a support member 500, in accordance with some embodiments of the technology described herein. [Figure 2] FIG. 2 is a schematic diagram of an example LFA device 100 showing example sizes of each component and an example size of overlap between two adjacent components, in accordance with some embodiments of the techniques described herein. [Figure 3A] 3A is a schematic diagram of a top view of an exemplary LFA device 100 for detecting and / or quantifying fCl-INH. The LFA device 100 includes a conjugate pad 200 including a first zone 210 and a second zone 220, and a membrane 300 including a third zone 230, in accordance with some embodiments of the technology described herein. [Figure 3B] 3B is a schematic diagram of a top view of an exemplary LFA device 100 for detecting and / or quantifying fCl-INH. The LFA device 100 includes a conjugate pad 200 including a first zone 210 overlapping a fifth zone 250 for depositing a sample and a second zone 220 overlapping a fourth zone 240 for depositing a buffer, and a membrane 300 including a third zone 230, in accordance with some embodiments of the technology described herein. [Figure 4]4 is a schematic diagram of a top view of an exemplary LFA device 100 for detecting and / or quantifying fCl-INH. LFA device 100 further includes a housing 600 forming a buffer port 610, a sample port 620, and a test window 630, in accordance with some embodiments of the technology described herein. Also shown are first zone 210, second zone 220, third zone 230, conjugate pad 200, membrane 300, and absorbent pad 400. [Figure 5] FIG. 5 is an image of an exemplary LFA device in accordance with some embodiments of the techniques described herein. [Figure 6] FIG. 6 is a graph showing a calibration curve generated from functional C1-esterase inhibitor (fC1-INH) diluted in C1-INH-depleted plasma using the LFA device disclosed herein. [Figure 7] FIG. 7 is a graph of the receiver operating curve (ROC) for diagnostic performance based on samples from control subjects and subjects with hereditary angioedema (HAE) using the LFA device disclosed herein. [Figure 8] FIG. 8 is a graph showing levels of functional C1-INH in normal subjects (Normal) and subjects with hereditary angioedema (HAE) using a chromogenic assay (Chromogenic) or the LFA device described herein (LFA). [Figure 9] FIG. 9 is a graph showing the correlation between levels of functional C1-INH in subjects with hereditary angioedema (HAE) determined by a chromogenic assay compared to the LFA device described herein. [Figure 10A] FIG. 10A is a schematic diagram showing collection of a blood sample using a sample loop and fingerstick. [Figure 10B] FIG. 10B is a schematic diagram showing the addition of the blood-filled sample loop of FIG. 10A to a SampleTainer® bottle. [Figure 11A] FIG. 11A is a graph showing the use of anti-C1-INH Fab conjugated to europium nanoparticles under the conditions shown. [Figure 11B] FIG. 11B is a graph showing the use of anti-C1-INH Fab conjugated to red gold nanoparticles under the conditions shown. [Figure 12] Figure 12 is a graph showing the results of polystreptavidin R compared to streptavidin as a capture agent at the test line. The R2 values ​​indicate the fit to the model line (dotted line). [Figure 13] FIG. 13 is a graph showing the results of using the indicated reagents as capture agents in a test line. [Figure 14] Figure 14 is a graph showing the results of the indicated anti-C1-INH Fab or antibody clones as detection agents. For each antibody, the bars correspond to detection agent concentrations from left to right: 1200 mU / mL, 600 mU / mL, 100 mU / mL, and 0 mU / mL. [Figure 15] FIG. 15 is a graph showing the results using buffers of various pHs containing inorganic blocking agents (IBA) or organic blocking agents (OBA). [Figure 16] FIG. 16 is a graph showing results using a streptavidin test line (0.75 mg / mL streptavidin) and the indicated concentrations of C1-INH / CINRYZE®. [Figure 17] Figure 17 is a schematic diagram of an exemplary method described herein. A capture protein such as streptavidin interacts with its counterpart (e.g., biotin) conjugated to a capture agent (e.g., biotinylated FXIIa), which binds to functional C1-INH (fC1-INH, Synlize®). 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). DETAILED DESCRIPTION OF THE INVENTION

[0021] The present disclosure is based, at least in part, on the development of lateral flow assay (LFA) methods and devices for measuring functional C1-esterase inhibitor (fC1-INH). These LFA methods and devices are designed to specifically detect the presence of fC1-INH and / or measure levels of fC1-INH, for example, in biological samples. The presence and / or levels of fC1-INH often indicate disease states related to biological pathways in which C1-INH plays a role. Thus, these LFA methods and devices will be particularly useful in the diagnosis and prognosis of diseases mediated by defects in C1-INH (e.g., diseases mediated by the plasma kallikrein pathway, such as hereditary angioedema, because fC1-INH is an inhibitor of the pKal pathway).

[0022] As used herein, "functional C1-INH" or "fC1-INH" refers to a form of C1-INH protein that is capable of binding to the protein factors to which C1-INH naturally binds and exerts its biological activity. Such protein factors include, but are not limited to, C1s, factor XIIa (FXIIa), and plasma kallikrein (pKal). Detection of this subpopulation of fC1-INH is particularly useful in assessing the functional levels of C1-INH in diseases such as HAE.

[0023] HAE is a very rare, potentially life-threatening genetic disease that occurs in approximately 1 in 10,000 to 1 in 50,000 people. Symptoms include edema (swelling) in various parts of the body, including the hands, feet, face, and airway (throat). Patients often suffer from extreme abdominal pain, nausea, and vomiting caused by swelling in the intestinal wall. Swelling of the airway or throat is particularly dangerous and can lead to death by suffocation. Three specific blood tests required to confirm type I and type II HAE are C1INH antigen, fC1INH, and C4. Many diagnostic assays use outdated technology and are not rapid, standardized, or available worldwide.

[0024] The rapid and sensitive LFA method and device disclosed herein can be implemented in physicians' offices for the rapid diagnosis of HAE (e.g., types I and II) based on fC1INH levels. Such methods and devices would offer low-cost consumables reimbursed by health insurance, a high level of confidence in quantitative results, ease of data interpretation by physicians, and / or a low level of need for confirmatory analysis. Such a rapid assay for diagnosing type I or type II HAE in the clinic could expand screening for HAE and more rapidly identify new HAE patients. Currently, the global diagnostic rate for HAE is only 40%, and therefore, undiagnosed patients represent a high unmet need. The availability of a rapid test on a common device platform could potentially expand HAE awareness. Furthermore, the rapid test for fC1INH disclosed herein could be useful for timely monitoring of HAE disease progression or response to treatment in clinical settings.

[0025] The LFA method and device disclosed herein involve a first binding substance specific for fC1-INH (fC1-INH binding substance), a second binding substance specific for C1-INH (specific for functional C1-INH, non-functional C1-INH, or both), and a capture substance. Either the fC1-INH binding substance or the C1-INH binding substance may be conjugated to a docking substance capable of binding to the capture substance. One of the fC1-INH binding substance, the C1-INH binding substance, and the capture substance may be conjugated to a detectable label. Thus, fC1-INH in a sample (e.g., a biological sample) can form a complex with the fC1-INH binding substance and the C1-INH binding substance. Such a complex can bind to the capture substance through interaction between the capture substance and a docking substance conjugated to one of the C1-INH binding substance and the fC1-INH binding substance. By detecting the signal (e.g., presence or intensity) emitted from the detectable label, the presence or level of fC1-INH in the sample can be determined and / or quantified based on a standard tested together. For example, various levels of Synlyse® (purified human plasma-derived C1INH) can be used as a standard to generate a standard curve for extrapolating and determining the level of fC1INH in a sample measured by any of the methods disclosed herein. The intensity of the signal emitted from the detectable label can be quantified based on the standard in U / ml of fC1INH in the sample, indicating the level of fC1INH in the sample.

[0026] I. Components for Use in Lateral Flow Assays (LFAs) The LFA method and apparatus disclosed herein involve (i) an fC1-INH binding agent, (ii) a C1-INH binding agent, and (iii) a capture agent, wherein one of the fC1-INH binding agent and the C1-INH binding agent is conjugated to a docking agent, and the capture agent binds to the docking agent. One of (i) to (iii) is conjugated to a detectable label.

[0027] (a) fC1-INH binding substance An fC1-INH binder is a molecule (e.g., a protein or fragment thereof that binds to fC1-INH) that specifically binds to functional C1-INH. A molecule is said to exhibit "specific binding" if it reacts with a particular target (e.g., one disclosed herein) more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it reacts with another target (which may be another form of the particular target). For example, a molecule that specifically binds to fC1-INH will react with fC1-INH more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it would with another target, such as non-functional C1-INH. "Specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding.

[0028] In some examples, a protein or polypeptide that can naturally bind to fC1-INH, or a binding fragment thereof, may be used as the fC1-INH binding substance. In some examples, the fC1-INH binding substance is FXII, for example, the activated form of FXII (FXIIa). Factor XII is a serum glycoprotein involved in the initiation of blood coagulation, fibrinolysis, and the production 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 fragment (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):490-8 1987).

[0029] FXII proteins, including their precursor, mature, and activated forms, are well known in the art. For example, the precursor protein sequence of human factor XII and its activated form are provided in GenBank accession number NP_000496.2. FXII proteins of other species (e.g., mammals other than humans) are also known in the art. Their structural information can be found in the art, for example, by using the sequence of human FXII as a query and identifying them from public gene databases.

[0030] "Activated" or "functional" factor XII refers to a factor XII polypeptide or a fragment of a factor XII polypeptide that retains similar, but not necessarily identical, biological activity to its naturally occurring counterpart, including mature forms of factor XII. In some embodiments, activated or functional factor XII is a factor XII polypeptide or a fragment of a factor XII polypeptide that binds to fC1-INH. In some embodiments, activated or functional factor XII is a factor XIIa polypeptide or a fragment of a factor XIIa polypeptide that binds to fC1-INH. In some embodiments, activated or functional factor XII is a factor XIIf polypeptide or a fragment of a factor XIIf polypeptide that binds to fC1-INH.

[0031] In another example, the fC1-INH binding agent 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 IM 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 prolylcarboxypeptidase on endothelial cell surfaces (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies intrinsic coagulation through its feedback activation of factor XII and enhances inflammation through the production of the pro-inflammatory nonapeptide bradykinin. As the first kininogenase in the circulation, plasma kallikrein is critically involved in the generation of bradykinin in the vascular system.

[0032] pKal proteins are well known in the art. Exemplary plasma kallikrein sequences may include the amino acid sequences of human (accession number: NP_000883.2), mouse (accession number: NP_032481.1), or rat (accession number: NP_036857.2) plasma kallikrein.

[0033] "Active" or "functional" plasma kallikrein refers to a plasma kallikrein polypeptide or a fragment of a plasma kallikrein polypeptide that retains a biological activity (e.g., protease activity) similar, but not necessarily identical, to that of its naturally occurring counterpart, including mature forms of plasma kallikrein. In some embodiments, active or functional plasma kallikrein is a plasma kallikrein polypeptide or a fragment of a plasma kallikrein polypeptide that binds to fCl-INH.

[0034] In yet another example, the fC1-INH binding substance disclosed herein may be C1s and C1r, or an active / functional fragment thereof. 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 an enzyme that activates C1s to its active form by proteolytic cleavage.

[0035] The C1s and C1r proteins are also well known in the art. For example, the precursor protein sequence of human C1s is provided under GenBank Accession No. NP_001725.1, and human C1r is provided under GenBank Accession No. NP_001724.4.

[0036] "Active" or "functional" C1s and C1r refer to fragments of C1s and C1r polypeptides that retain similar, but not necessarily identical, biological activity to their naturally occurring counterparts, including mature forms, respectively. In some embodiments, an active or functional C1s or C1r fragment is a C1s polypeptide or a portion of a C1s polypeptide that binds to fC1-INH.

[0037] Any of the fCl-INH binding agents may be produced by recombinant techniques or isolated from a suitable natural source.

[0038] (b) C1-INH binding substance The C1-INH binding substance 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 examples, the C1-INH binding substance is specific for fC1-INH. In other examples, the C1-INH binding substance cross-reacts with both functional and non-functional C1-INH.

[0039] The C1-INH binder may be an antibody that binds to 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 may 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 complete antibodies. Antibodies may have the structural characteristics of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof).

[0040] The VH and VL regions can be further subdivided into regions of hypervariability termed "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions termed "framework regions" ("FRs"). The extent of the framework regions and CDRs has been precisely defined (see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US 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). Kabat's definitions are used herein. VH and VL each typically consist of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0041] The VH or VL chain of an antibody may further comprise all or a portion of a heavy or light chain constant region to form a heavy or light immunoglobulin chain, respectively. In one embodiment, an antibody is a tetramer of two heavy and two light immunoglobulin chains, where the heavy and light immunoglobulin chains are interconnected, for example, by disulfide bonds. In IgG, the heavy chain constant region comprises three immunoglobulin domains, CH1, CH2, and CH3. The light chain constant region comprises a CL domain. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody typically mediates antibody binding 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 the immunoglobulin may be a kappa or lambda chain. In one embodiment, the antibody is glycosylated. The antibody may be functional for antibody-dependent cellular cytotoxicity and / or complement-mediated cytotoxicity.

[0042] In some embodiments, an antibody that binds to C1-INH may specifically bind to an epitope of C1-INH, e.g., 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 understood in the art, and methods for determining such specific binding are well known in the art. An antibody is said to exhibit "specific binding" if it reacts or binds to a particular target antigen more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it reacts or binds to another target. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, with higher avidity, more readily, and / or with 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 one that binds to the target antigen with higher affinity, higher avidity, more readily, and / or with longer duration than it binds to other antigens or other epitopes on the same antigen. It is also understood by reading 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. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding implies preferential binding. In some instances, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes on the same antigen.

[0043] Antibodies that bind to C1-INH for use in the LFA methods and devices disclosed herein may have suitable binding affinity for C1-INH or a suitable epitope thereof. As used herein, "binding affinity" refers to the apparent binding constant or KA. KA is the reciprocal of the dissociation constant (KD). The antibodies described herein have a binding affinity of at least 10 -5, 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 An antibody may have a binding affinity (KD) of or lower than M. An increase in binding affinity corresponds to a decrease in KD. An antibody that binds with higher affinity to a first antigen relative to a second antigen can be indicated by a higher KA (or lower numerical KD) for binding to the first antigen than the KA (or numerical KD) for binding to the second antigen. In such cases, the antibody has specificity for the first antigen over the second antigen. The difference in binding affinity (e.g., for specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 10,000, or 10 5 The binding affinity (or binding specificity) may be determined by conventional methods.

[0044] The antibody that binds to C1-INH may 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) a Fab fragment, which is a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH domain and the CH1 domain; (iv) an Fv fragment consisting of the VL domain and the VH domain of one arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR) that retains functionality. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant techniques with a synthetic linker that allows them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule known as a single-chain Fv (scFv). See, e.g., U.S. Patent Nos. 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 may be obtained using any suitable technique, including conventional techniques known to those skilled in the art.

[0045] Any of the antibodies that bind to C1-INH as described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population, while a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the manner in which it is made.

[0046] Antibodies that bind to C1-INH may be produced by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, antibodies specific to C1-INH (e.g., human C1-INH) may be produced by conventional hybridoma technology. In other embodiments, antibodies specific to C1-INH may be isolated from an antibody library according to conventional antibody library screening techniques.

[0047] 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 mouse monoclonal antibody that specifically binds to human C1-INH, such as antibody clone MM06 or MM03 (also designated 10995-MM06 and 10995-MM03, respectively; 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 clone RP01 or RP02 (also designated 10995-RP01 and 10995-RP02, respectively; Sino Biological Inc.). In some embodiments, the antibody is RP02.

[0048] (c) Docking-Capture agent One of the fC1-INH binding substance and the C1-INH binding substance is conjugated with a docking agent that can bind to a capture substance also used in the LFA method and device as disclosed herein. In one embodiment, the docking agent is conjugated to the fC1-INH binding substance (e.g., FXIIa). In another embodiment, the docking agent may be conjugated to C1-INH (e.g., an antibody that binds to C1-INH).

[0049] The docking agent and the capture agent are members of a receptor-ligand pair, which refers to any pair of molecules that can bind 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 polystreptavidin.

[0050] (d) a detectable label One of the fC1-INH binding substance, C1-INH binding substance, and capture substance for use in the LFA method and device disclosed herein may be conjugated to a detectable label. In some embodiments, the fC1-INH binding substance is conjugated to a detectable label. In other embodiments, the C1-INH binding substance is conjugated to a detectable label. Alternatively, the capture substance is conjugated to a detectable label.

[0051] As used herein, a "detectable label" refers to any molecule that can directly or indirectly emit a detectable signal. In some embodiments, a detectable label may be a fluorophore (e.g., fluorescein). As used herein, the term "fluorophore" (also referred to as a "fluorescent label" or "fluorochrome") refers to a moiety that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength.

[0052] 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, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives (e.g., pyridyloxazole, 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., porphine, phthalocyanine, and bilirubin), or fluorescent proteins (e.g., green fluorescent protein).

[0053] In some embodiments, the detectable label is phycoerythrin.

[0054] In some embodiments, the detectable label is a chromophore (e.g., anthracene). In some embodiments, the detectable label is a semiconductor particle (e.g., a quantum dot). In some embodiments, the detectable label is linked to a semiconductor particle (e.g., a 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 linked to a gold particle. In some embodiments, the detectable label is linked to red gold particles. In some embodiments, the detectable label is linked to a latex particle. In some embodiments, the C1-INH binding agent is antibody RP02 conjugated to europium. In some embodiments, the C1-INH binding agent is antibody RP02 conjugated to a gold particle. In some embodiments, the C1-INH binding agent is antibody RP02 conjugated to a red gold particle. In some embodiments, the C1-INH binding agent is antibody RP02 conjugated to a latex particle.

[0055] II.LFA device In some aspects, the present disclosure provides a lateral flow assay (LFA) device for measuring fCl-INH in a sample containing fCl-INH. Reference is now made to Figures 1-4, which illustrate various embodiments of exemplary LFA devices described herein.

[0056] As shown in FIG. 1, device 100, in some embodiments, includes a conjugate pad 200, a membrane 300, and optionally an absorbent pad 400 and a support member 500 to which the conjugate pad 200, the membrane 300, and optionally the absorbent pad 400 are attached.

[0057] The conjugate pad 200 is in communication with the membrane 300, either directly or through a linker. If the device includes an absorbent pad 400, the conjugate pad 200 and the absorbent pad 400 are separated by the membrane 300, which is in communication, either directly or indirectly, with the absorbent pad 400. In some embodiments, the conjugate pad 200 overlaps the membrane 300, for example, by 2 to 6 mm (such as 3 mm as shown in FIG. 2). Alternatively, or in addition, the membrane 300 overlaps the absorbent pad 400, for example, by 2 to 6 mm (such as 3 mm as shown in FIG. 2).

[0058] The specific features and dimensions of the conjugate pad 200, membrane 300, absorbent pad 400, and support member 500 may be modified as needed to achieve desired results. As shown in Figure 2, in some embodiments, the support membrane has a length of 80 mm, which is the sum of the lengths of the conjugate pad (42 mm), membrane (25 mm), and absorbent pad (19 mm) minus the overlap of the sample pad and absorbent pad over the membrane (3 mm, 3 mm).

[0059] 3A, which is a top view of device 100, the device may include various zones (210, 220, 230) that, in some embodiments, are useful for immobilizing fC1-INH binding agents, C1-INH binding agents, and capture agents, such as those described herein. In some embodiments, device 100 includes first zone 210 and second zone 220, which may be on conjugate pad 200, and third zone 230, which may be on membrane 300.

[0060] Each of the fC1-INH binding agent, C1-INH binding agent, and capture agent may be immobilized on one of zones 210, 220, and 230 (which may be in any order). Any of these agents may be immobilized using any means known in the art. The agents may be directly or indirectly immobilized or bound to the surface of conjugate pad 200 and / or membrane 300. In some embodiments, the binding agent is immobilized to the surface by a covalent bond. In some embodiments, the binding agent is immobilized to the surface by a non-covalent bond. In some embodiments, the binding agent is immobilized to the surface by 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.

[0061] In some embodiments, an fC1-INH binding substance, such as FXIIa, is immobilized in the first zone 210. The fC1-INH binding substance may be conjugated to a docking substance, such as biotin. A C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the second zone 220. The C1-INH binding substance may be conjugated to a detectable label, such as those disclosed herein. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the third zone 230.

[0062] In some embodiments, an fC1-INH binding substance, such as FXIIa, is immobilized in the first zone 210. The fC1-INH binding substance may be conjugated to a detectable label, such as those described herein. A C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the second zone 220. The C1-INH binding substance may be conjugated to a docking substance, such as biotin. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the third zone 230.

[0063] In some embodiments, a C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the first zone 210. The C1-INH binding substance may be conjugated to a docking substance, such as biotin. A fC1-INH binding substance, such as FXIIa, may be immobilized in the second zone 220. The fC1-INH binding substance may be conjugated to a detectable label, such as those described herein. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the third zone 230.

[0064] In some embodiments, a C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the first zone 210. The C1-INH binding substance may be conjugated to a detectable label, such as those described herein. A fC1-INH binding substance, such as FXIIa, may be immobilized in the second zone 220. The fC1-INH binding substance may be conjugated to a docking substance, such as biotin. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the third zone 230.

[0065] In some embodiments, an fC1-INH binding substance, such as FXIIa, is immobilized in the first zone 210. The fC1-INH binding substance may be conjugated to a docking substance, such as biotin. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the second zone 220. The capture substance may be conjugated to a detectable label, such as those disclosed herein. A C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the third zone 230.

[0066] In some embodiments, an fC1-INH binding substance, such as FXIIa, is immobilized in the first zone 210. The fC1-INH binding substance may be conjugated to a detectable label, such as those described herein. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the second zone 220. A C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the third zone 230. The C1-INH binding substance may be conjugated to a docking substance, such as biotin.

[0067] In some embodiments, a C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the first zone 210. The C1-INH binding substance may be conjugated to a docking substance, such as biotin. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the second zone 220. The capture substance may be conjugated to a detectable label, such as those disclosed herein. A C1-INH binding substance, such as FXIIa, may be immobilized in the third zone 230.

[0068] In some embodiments, a C1-INH binding substance, such as an antibody that binds to C1-INH, may be immobilized in the first zone 210. The C1-INH binding substance may be conjugated to a detectable label, such as those described herein. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the second zone 220. An fC1-INH binding substance, such as FXIIa, may be immobilized in the third zone 230. The fC1-INH binding substance may be conjugated to a docking substance, such as biotin.

[0069] Any of the LFA devices disclosed herein may further include a fourth zone 240, which may be for depositing a sample such as those described herein, and optionally a fifth zone 250, which may be for depositing a buffer solution.

[0070] The fifth zone 250 may be located at one end of the device such that when a buffer solution is placed in the fifth zone 250, the buffer solution can flow through the device from the first zone 210 to the third zone 230. In some examples, the fifth zone 250 may overlap with the first zone 210. See FIG. 3B. In this case, a C1-INH binding substance, such as an antibody that binds C1-INH, may be immobilized in the first zone 210, which overlaps with the fifth zone 250. The C1-INH binding substance may be conjugated to a detectable label.

[0071] Alternatively, or in addition, the fourth zone 240 may be disposed between the fifth zone 250 and the second zone 220. In some instances, the fourth zone 240 and the second zone 220 may overlap. See Figure 3B. An fCl-INH binding agent, such as FXIIa, which may be conjugated to a docking agent, such as biotin, may be immobilized in the second zone 220.

[0072] As shown in FIG. 4, device 100 may further include a housing 600, which may be removable in some embodiments. An image of a device as described herein within the housing is shown in FIG. 5. Housing 600 may be configured to expose at least a portion of conjugate pad 200 and membrane 300 of device 100. In some embodiments, housing 600 includes a first opening for forming a buffer port 610, which may align with first zone 210. Housing 600 may further include a second opening for forming a sample port 620, which may align with second zone 220. Additionally, housing 600 may include a third opening for forming a test window 630, which may align with third zone 230.

[0073] In some embodiments, a C1-INH binding substance, such as an antibody that binds to C1-INH, is immobilized in the first zone 210, which is aligned with the buffer port 610. (FIG. 4). The C1-INH binding substance may be conjugated to a detectable label, such as those described herein. A fC1-INH binding substance, such as FXIIa, may be immobilized in the second zone 220, which may be aligned with the sample port 620. The fC1-INH binding substance may be conjugated to a docking substance, such as biotin. A capture substance, such as avidin (e.g., streptavidin), may be immobilized in the third zone 230, which may be aligned with the test window 630.

[0074] In some examples, a sample may be placed in the sample port 620, allowing binding of fC1-INH in the sample to the FXIIa-biotin conjugate. A buffer solution may be placed in the buffer port 610, allowing the C1-INH-binding substance in the first zone 210 to migrate with the buffer solution to the second zone 220. When the C1-INH-binding substance contacts the fC1-INH-FXIIa complex in the second zone 220, a C1-INH-binding substance / fC1-INH / FXIIa-biotin complex is formed. This complex will migrate with the buffer solution to the third zone 230 and be captured there by the interaction between biotin and streptavidin in the third zone 230. The signal emitted from the detectable label conjugated to the C1-INH binding substance in the third zone 230 aligned with the test window 630 may be measured, indicating the presence or level of fC1-INH in the sample.

[0075] Alternatively, or in addition, housing 600 may be transparent to facilitate visualization of sample port 610 and / or buffer port 620 and / or test window 630. In one embodiment, a portion of the housing is transparent. In some embodiments, the entire housing 600 is transparent.

[0076] Housing 600, in some embodiments, includes a label to facilitate identification of the sample or the result. In some embodiments, housing 600 includes one or more labels to facilitate identification of the result in test window 630. In some embodiments, the one or more labels identify the sample result.

[0077] It should be understood that various embodiments of the device, including the multiple components in the device as described herein (e.g., conjugate pad, membrane, absorbent pad, and support member), may be formed using any suitable materials, for example, using any suitable conjugate pad, using any suitable membrane, using any suitable absorbent pad, using any suitable support member, using any suitable binding material, and using any suitable combination thereof.

[0078] For example, the membrane 300 in an LFA device as disclosed herein can be any suitable membrane, including, but not limited to, a nitrocellulose membrane, a nylon membrane, a cellulose membrane, a polyvinylidine fluoride membrane, a polycarbonate membrane, a polypropylene membrane, a polyethylene membrane, a polytetrafluoroethylene membrane, and a polyparaphenylene terephthalamide membrane. In some embodiments, the membrane is a nitrocellulose membrane.

[0079] Any suitable support member may be used in the devices described herein. In some embodiments, the support member comprises a metal. In some embodiments, the support member comprises a plastic. In some embodiments, the support member comprises a plastic selected from the group consisting of styrene, polycarbonate, polypropylene, polyethylene, and polyvinyl chloride.

[0080] Any suitable pad may be used as the conjugate pad in the devices described herein. In some embodiments, the conjugate pad comprises cellulose or glass fiber. In some embodiments, the absorbent pad comprises cellulose or glass fiber.

[0081] The devices provided herein may further comprise a sample pad, hi some embodiments, the sample pad comprises cellulose or glass fiber.

[0082] It should be understood that various embodiments of the present invention may be formed with one or more of the above-described features. The above-described aspects and features of the present invention may be used in any suitable combination, as the present invention is not limited in this respect. It should also be understood that the drawings illustrate various components and features that may be incorporated into various embodiments of the present invention. For simplicity, some drawings may illustrate two or more optional features or components. However, the present invention is not limited to the specific embodiments disclosed in the drawings. It should be recognized that the present invention may include embodiments that include only some of the components illustrated in any one drawing and / or may include embodiments that combine components illustrated in different drawings.

[0083] III. Measurement of functional C1-INH Also provided herein are methods for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample. The assay methods disclosed herein all involve the use of the fC1-INH binding substance, C1-INH binding substance, and capture substance disclosed herein. One of the fC1-INH binding substance and the C1-INH acting substance is conjugated to a docking substance that binds to the capture substance. One of the fC1-INH binding substance, C1-INH acting substance, and capture substance is conjugated to a detectable label. The detectable label and the docking substance are conjugated to separate agents.

[0084] To carry out the assay methods disclosed herein, a sample suspected of containing fC1-INH can be contacted with the fC1-INH binding agent, the C1-INH binding agent, and the capture agent under conditions that allow for the formation of a complex comprising fC1-INH, the fC1-INH binding agent, the C1-INH binding agent, and the capture agent (through interaction with a docking agent conjugated to either the fC1-INH binding agent or the C1-INH binding agent). The presence or level of fC1-INH in the sample can be detected and / or quantified by measuring the signal emitted from a detectable label that can be conjugated to any one of the fC1-INH binding agent, the C1-INH binding agent, and the capture agent.

[0085] In some examples, a sample and an fC1-INH binding agent (e.g., FXIIa) may first be incubated for a suitable period of time (e.g., at least 5 minutes (e.g., 5 to 10 minutes)) to allow the formation of an fC1-INH / FXIIa complex. This complex may then be incubated with a C1-INH binding agent (e.g., an antibody that binds to C1-INH) to form a ternary complex, which may then be contacted with a capture agent that binds to either the fC1-INH binding agent or a docking agent conjugated to the C1-INH binding agent. The signal emitted from a detectable label conjugated to one of the components in the final complex may be measured to determine the presence / absence and / or level of fC1-INH in the sample.

[0086] In some embodiments, the methods for detecting and / or quantifying fC1-INH provided herein include: (i) contacting a sample with an fC1-INH binding agent, a C1-INH binding agent, and a capture agent to form a complex, wherein one of the fC1-INH binding agent and the C1-INH agent is conjugated to a docking agent that binds to the capture agent, and one of the fC1-INH binding agent, the C1-INH agent, and the capture agent is conjugated to a detectable label, and the detectable label and the docking agent are conjugated to separate agents; and (ii) detecting a signal emitted from the detectable label in the complex, wherein the presence of a signal emitted from the detectable label in the complex indicates the presence of fC1-INH in the sample.

[0087] 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 multi-well 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, for example, via a linker.

[0088] In some embodiments, the assay methods disclosed herein may be performed using any of the LFA devices disclosed herein. For example, a sample may be placed in sample port 620 (FIG. 4), and a buffer solution may be placed in buffer port 610. Sample port 620 may be aligned with second zone 220. Buffer port 610 may be aligned with first zone 210. The buffer solution will flow, for example, from first zone 210 to second zone 220 and third zone 230, along with the sample and the fC1-INH binding substance, C1-INH binding substance, and / or capture substance immobilized in first zone 210 and second zone 220. This allows the sample to contact the fC1-INH binding substance, C1-INH binding substance, and capture substance as the buffer solution passes through first zone 210, second zone 220, and third zone 230, resulting in the formation of a complex containing fC1-INH, fC1-INH binding substance, C1-INH binding substance, and capture substance in the sample. The presence or level of fC1-INH in the sample can be determined by measuring a signal emitted from a detectable label conjugated to one of the components in the complex.

[0089] In one example, a method for detecting and / or quantifying fC1-INH using an LFA device (e.g., a device configured as shown in FIG. 4 ) containing FXIIa-biotin and an anti-C1-INH antibody conjugated to europium particles is described for illustrative purposes only. In this example, the anti-C1-INH antibody conjugated to europium particles serves as a C1-INH binding substance conjugated to a detectable label, and the anti-C1-INH antibody conjugate is immobilized in the first zone 210. FXIIa conjugated to biotin serves as an fC1-INH binding substance 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 in the second zone 220 on the conjugate pad 200 via the sample port 620. When the sample contacts FXIIa-biotin in the second zone 220, fC1-INH in the sample binds to FXIIa, thereby forming a FXIIa-biotin:fC1-INH complex.

[0090] As used herein, the term "contacting" refers to exposing a sample to one or more binding substances for a suitable period of time sufficient to form a complex with fC1-INH and / or C1-INH in the sample, if present. In some embodiments, the sample and / or buffer contacts the one or more binding substances via capillary action, which migrates the sample and / or buffer across a conjugate pad or membrane.

[0091] A buffer may be placed in the first zone 210 on the conjugate pad 200 through the buffer port 610. The buffer may be placed in the first zone 210 any length of time after the sample is placed in the second zone 220; for example, the buffer may be placed at least 5 minutes after the sample is placed on the device. The buffer solubilizes the anti-C1-INH antibody conjugate and migrates 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 complexed with FXIIa-biotin, thereby forming a "sandwich." In this example, the fC1-INH sandwich therefore comprises FXIIa conjugated to biotin bound to fC1-INH, which is bound by anti-C1-INH antibodies conjugated to europium particles.

[0092] The buffer containing the fC1-INH sandwich continues to migrate up the conjugate pad 200 to the membrane 300, where streptavidin is immobilized in the third zone 230 (e.g., the test line). In this example, streptavidin functions as a capture agent that binds to the docking agent (specifically, biotin). When the buffer contacts the streptavidin in the third zone 230, the biotin in the fC1-INH sandwich binds to the streptavidin in the third zone 230, thereby capturing the fC1-INH sandwich. The presence of fC1-INH in the sample is then detected through the test window 630 based on the presence of a signal from the europium particles in the third zone 230. Detection of the fC1-INH sandwich is not limited to detection via europium particles. For example, the presence of fC1-INH may be detected via a detectable change in color or pH. If no fCl-INH is present in the sample, no fCl-INH sandwich is formed and no signal is detected in the third zone 230 .

[0093] After migrating into the third zone 230, the sample continues to migrate up the membrane 300 into the absorbent pad 400, which acts as a wick to draw the sample upward, thus removing background material from the third zone 230.

[0094] The methods provided herein encompass detecting and / or quantifying fC1-INH, or the lack thereof, 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 or at risk for a disorder mediated by a defect in fC1-INH (e.g., HAE).

[0095] 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 plasma. In some embodiments, the blood sample may be collected from a blood vessel (e.g., capillaries, veins, and arteries). In some embodiments, the blood sample may be obtained by a fingerstick method, which produces a drop(s) of blood. In some embodiments, after obtaining the blood sample, the blood sample is handled at 2-8°C. In the case of plasma or serum preparation, the plasma or serum may be prepared by centrifugation after blood collection. Plasma and serum samples may be stored at -80°C prior to analysis.

[0096] In some embodiments, the methods and / or devices described herein may further include a control line, which, as will be appreciated by those skilled in the art, may be used to verify that the method and / or device is functioning as intended (e.g., detecting fCl-INH).

[0097] IV. LFA Method and Equipment Applications The methods and devices described herein can be applied to disease assessment (e.g., disease diagnosis or prognosis). Assessment can include identifying a subject as being at risk for or having a disease as described herein (e.g., a disorder mediated by defects in fC1-INH). Assessment can also include monitoring disease treatment, such as evaluating the effectiveness of treatment for a disorder mediated by defects in fC1-INH. Examples of disorders mediated by defects in fC1-INH include, but are not limited to, hereditary angioedema (e.g., type I HAE and / or type II HAE), acquired angioedema (e.g., type I AAE and / or type II AAE), immune diseases associated with defects in C1-INH (e.g., systemic lupus erythematosus (SLE)), and cancers associated with defects in C1-INH (e.g., lymphoma).

[0098] In some embodiments, the method and device used herein is used to assess whether a subject has hereditary angioedema or whether the subject is at risk of hereditary angioedema.Generally, there are different types of hereditary angioedema, which show similar inflammatory reaction but have different etiologies.For example, type I HAE is associated with functional but low level C1-INH, while type II HAE is associated with non-functional C1-INH that exists at normal concentration.

[0099] A. Diagnosis In some embodiments, the methods and devices described herein are used to determine the level of fC1-INH in a biological sample (e.g., a serum sample, plasma sample, or blood sample) collected from a subject (e.g., a human patient suspected of having a disorder mediated by a defect in fC1-INH, such as HAE). The level of fC1-INH is then compared to a reference value to determine whether the subject has a disorder mediated by a defect in fC1-INH or whether the subject is at risk for a disorder mediated by a defect in fC1-INH. The reference value may be a control level of fC1-INH capable of binding to an fC1-INH binding agent (e.g., FXIIa) as described herein. In some embodiments, the control level is the level of fC1-INH in a control sample capable of binding to the fC1-INH binding agent. 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 does not have an apparent disorder mediated by a deficiency in fC1-INH at the time the level of fC1-INH is measured, or has no history of such a disease.

[0100] The control level may also be a predetermined level. Such a predetermined level may represent the level of fC1-INH in a population of subjects who do not have or are not at risk for a disorder mediated by a defect in fC1-INH. The predetermined level may take various forms. For example, it may be a single cutoff value, such as a median or mean. In some embodiments, such a predetermined level may be established based on a comparison of groups, such as when one defined group is known to have the target disease and another defined group is known not to have the target disease. Alternatively, the predetermined level may be a range, e.g., a range representing the level of fC1-INH in a control population within a predetermined percentile.

[0101] The control level as described herein may be determined by various methods. In some embodiments, the control level may be obtained by performing a known method. In some embodiments, the control level may be obtained by performing the same assay used to determine the level of fC1-INH in a sample from a subject. In some embodiments, the control level may be obtained by performing a method described herein. In some embodiments, the control level may be obtained using an apparatus described herein. In some embodiments, the control level may be obtained from members of a control population, and the results may be analyzed, for example, by a computational program, to obtain a control level (predetermined level) representing the level of fC1-INH in the control population.

[0102] By comparing the level of fC1-INH capable of binding to the fC1-INH binding substance in a sample obtained from a subject with a reference value as described herein, it is possible to determine whether the subject has a disease mediated by a defect in fC1-INH (e.g., HAE) or whether the subject is at risk for a disease mediated by a defect in fC1-INH (e.g., HAE). For example, if the level of fC1-INH bound to the fC1-INH binding substance in the subject deviates from the reference value (e.g., is reduced compared to the reference value), the candidate subject may be identified as having or at risk for a disease mediated by a defect in fC1-INH (e.g., HAE). The assays disclosed herein may be used to predetermine a cutoff value representing fC1-INH in normal subjects. Such a cutoff value may be used to determine whether the subject has a disease mediated by a defect in fC1-INH (e.g., HAE) or whether the subject is at risk for a disease mediated by a defect in fC1-INH (e.g., HAE). In some instances, a level of fC1-INH in a subject below a cutoff value may indicate risk or occurrence of disease.

[0103] As used herein, "reduced levels or levels below a reference value" means that the level of fC1-INH binding to the fC1-INH binding substance is lower than a reference value (such as a predetermined threshold or the level of fC1-INH binding to the fC1-INH binding substance in a control sample).

[0104] Decreased levels of fC1-INH binding to the fC1-INH binding agent include, for example, levels of fC1-INH that are 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more lower than baseline. Decreased levels of fC1-INH binding to the fC1-INH binding agent also include reducing the phenomenon from a non-zero state (e.g., some or detectable fC1-INH bound to the fC1-INH binding agent in the sample) to a zero state (e.g., no or only undetectable fC1-INH bound to the fC1-INH binding agent in the sample).

[0105] In some embodiments, the subject is a human patient with symptoms of a disease mediated by a defect in fC1-INH (e.g., those disclosed herein, such as HAE). For example, the subject has edema, swelling that is entirely or mainly peripheral; hives; redness, pain, and swelling without evidence of infection; non-histamine-mediated edema, recurrent bouts of swelling, or a combination thereof. In some embodiments, the subject does not have symptoms of a disease mediated by a defect in fC1-INH at the time the sample is collected, or does not have a history of symptoms of a disease mediated by a defect in fC1-INH, or does not have a history of a disease mediated by a defect in fC1-INH, such as HAE. In some embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0106] Examples of diseases mediated by defects in fC1-INH include non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, ventricular assist device or stent-associated thrombosis, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina, edema, eye disease, gout, intestinal bowel disease. disease), oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis - degenerative spinal disease, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral 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, protein misfolding-related diseases, angiogenesis-related diseases, hypertensive and diabetic nephropathy, allergic and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis), and tissue damage (e.g., burns or chemical injuries).

[0107] B. Evaluation of Treatment Efficacy The methods and devices described herein may also be applied to assess the effectiveness of treatment for diseases mediated by deficiencies in fC1-INH (e.g., HAE). For example, multiple biological samples (e.g., serum samples, plasma samples, or blood samples) may be collected from a subject receiving treatment before and after the treatment, or during the course of the treatment. The level of fC1-INH may be measured by any of the methods described herein. If the level of fC1-INH increases, remains the same, or increases after treatment or over the course of treatment (the level of fC1-INH in later samples compared to the level in earlier samples), it indicates that the treatment is effective.

[0108] If a subject is identified as not responding to treatment, a higher dose and / or frequency of administration of the therapeutic agent is administered to the identified subject. In some embodiments, the dosage or frequency of administration of the therapeutic agent is maintained, reduced, or discontinued in subjects who are identified as responding to treatment or who do not require further treatment. Alternatively, a different treatment may be administered to subjects who are found not to respond to the initial treatment.

[0109] Therapeutic agents include, but are not limited to, kallikrein binding agents, bradykinin B2 receptor antagonists, C1-INH replacement agents, DX-2930, and DX88 (see, e.g., International Publication No. WO2014 / 113701, which is incorporated by reference in its entirety). [Example]

[0110] In order to provide a more complete understanding of the devices and methods described herein, the following examples are presented. The examples described in this application are provided to illustrate the methods and compositions provided herein and should not be construed in any way as limiting the scope thereof.

[0111] Example 1: Preparation of a Lateral Flow Assay (LFA) Device for Detecting and / or Quantifying Functional C1-Esterase Inhibitor (fC1-INH) Membrane Striping The membrane was striped using FRONTLINE HR™ (BioDot). The front lines were washed with 10 cycles of wash buffer (0.05% BIO-TERGE® (Stepan Company) in diH2O) 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 polystreptavidin in 10 mM phosphate, pH 7.3, 0.5% sucrose. A polystreptavidin test line was striped onto the membrane (see, for example, the third zone 230 on membrane 300 in Figure 3A). The membrane was then labeled and dried at 40°C for 30 minutes. After striping, the front lines were washed with 10 cycles of wash buffer. The components for striping the membrane are listed in Table 1.

[0112] [Table 1]

[0113] Conjugate Pad Striping Anti-C1-INH Eu particle conjugate and FXIIa-biotin were striped onto a conjugation pad (Ahlstrom) using a FRONTLINE HR™ (BioDot). Prior to 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. The FRONTLINE was then washed with 10 cycles of wash buffer (0.05% BIO-TERGE® (Stepan Company) in diH2O). The front line was aligned so that the anti-C1-INH Eu particle conjugate was striped 15 mm from the bottom of the conjugate pad, and the FXIIa-biotin conjugate was striped 8 mm from the top of the conjugate pad, at rates of 10 μL / cm and 2.5 μL / cm, respectively. The positions of the anti-C1-INH Eu particle conjugate and the FXIIa-biotin conjugate aligned with the buffer and sample ports, respectively, of the custom SLA housing cassettes.

[0114] The front line was emptied and primed with either conjugate. The conjugate was striped onto a conjugate pad, which was then labeled and dried at 40°C for 30 minutes. The conjugate pad was sealed and allowed to dry. After striping, the front line was washed with 10 cycles of wash buffer. The components for striping the conjugate pad are listed in Table 2. The components for the Eu latex diluent and FXIIa-biotin diluent are listed in Tables 3 and 4, respectively.

[0115] [Table 2]

[0116] [Table 3]

[0117] [Table 4]

[0118] Preparation of anti-C1-INH Eu particle conjugate To prepare the anti-C1-INH Eu particle conjugate, 0.1 mg of anti-C1-INH antibody was exchanged into 50 mM borate, pH 8, using a ZEBA™ spin column (Thermo Fisher) using the manufacturer's protocol. The concentration of the antibody was determined by absorbance (A 280 , 1 mm, 1 OD = 1.4 mg / mL). Stock latex was rotated for 10 minutes and then sonicated for 10-15 seconds using a microtip sonicator (setting 25). The stock latex solution (10%) was diluted to 1% with 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, with a buffer volume equal to the initial volume of latex. The resulting solution was sonicated, microcentrifuged, and resuspended as previously described.

[0119] To prepare 15 mg / mL EDC in 0.1 M MES buffer, EDC was equilibrated to room temperature and weighed. The EDC solution was prepared within 10 minutes of use in preparing the anti-C1-INH Eu particle conjugate. For long-term storage, EDC stock powder was dried and frozen at -20°C.

[0120] To prepare 50 mg / mL sulfo-NHS in 0.1 M MES buffer, sulfo-NHS was equilibrated to room temperature and weighed. The sulfo-NHS solution was prepared within 10 minutes of use in preparing the anti-C1-INH Eu particle conjugate. The sulfo-NHS solution was activated by incubating it on a shaker at 1000 rpm for 30 minutes. This 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 initial volume of the latex solution (600 μL). This solution was then microcentrifuged, resuspended in borate buffer (300 μL), vortexed, and sonicated as previously described. Activated particles were dispensed at 50 μL per tube, and an appropriate amount of buffer and protein (e.g., 20 μg of protein (i.e., antibody)) was added to achieve a particle-to-protein mass ratio of 20:1. Tubes were vortexed immediately after buffer and protein addition.

[0121] The tubes were incubated on a shaker at 1,000 rpm for 2 hours at room temperature. After incubation, 10 μL / mL of 1 M ethanolamine was added, and the tubes were incubated on a shaker at 1,000 rpm for 30 minutes at room temperature. The tubes were microcentrifuged at 17,000 g for 10 minutes, and the pellets were resuspended in 7-day cured 1% casein and incubated overnight with shaking. After overnight incubation, the tubes were microcentrifuged, and the pellets were resuspended in 7-day cured 1% casein, vortexed, and sonicated. The particle conjugates were then striped onto conjugate pads as previously described. The components for the anti-C1-INH Eu particle conjugate are shown in Table 5.

[0122] [Table 5]

[0123] Lamination of components onto a backing card To stack the components onto an 80 mm long backing card (DCN), a straight razor blade was used to cut the backing sticker 39 mm from the bottom of the card. The backing sticker was removed from the top side of the card at this 39 mm position. A membrane was adhered to the card 39 mm from the bottom. An absorbent pad (Ahlstrom) was adhered to the top of the card, overlapping the top of the membrane by 3 mm. The remaining backing sticker was removed, and a conjugate pad was adhered to the bottom of the card, overlapping the bottom of the membrane by 3 mm. The components and component order are shown in Tables 6 and 7, respectively.

[0124] [Table 6]

[0125] [Table 7]

[0126] Preparation of test strips The card was placed in a Kinematic Cutter (Kinematic), which cut the card into 5.0 mm wide strips. Strips shorter than 5.0 mm or marked with a pen during striping were discarded. The cut strips were placed in foil bags with desiccant. The foil bags were sealed and stored in a dry box until use.

[0127] Example 2: Use of the LFA device to detect functional C1-esterase inhibitor (fC1-INH) in plasma samples from patients To perform a lateral flow assay (LFA) to determine the concentration of fC1-INH, test strips prepared as described in Example 1 above were placed in a custom stereolithography (SLA) cassette (see, e.g., Figure 4). Reference and quality controls were prepared by spiking C1-INH standards (Lot No. TCP103, Shire / Takeda) into C1-INH-depleted human K3-EDTA plasma (prepared by Shire / Takeda). A calibration curve was generated using control samples containing purified C1-INH at concentrations ranging from 0 mU / mL to 800 mU / mL. The control samples were diluted 1:20 with C1-INH-depleted medium. The sample was added to the sample port and incubated for 5 minutes. 30 μL of run buffer was added to the sample port, allowing the sample to flow onto the membrane and into the test window. 150 μL of run buffer was then 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). An image of the test strip in the cassette is shown in Figure 5. The measured intensity of the test line region was plotted against the concentration of C1-INH to generate the calibration curve shown in Figure 6, with an R of 0.97. 2 obtained.

[0128] Fifty normal K3-EDTA plasma samples were commercially obtained, and 50 HAE plasma samples were used with patient consent from SAHARA, a phase III, randomized, double-blind, placebo-controlled, two-period, three-sequence, partial crossover study evaluating the efficacy and safety of subcutaneous administration of 2000 IU of injectable C1 esterase inhibitor [human] liquid for the prevention of angioedema attacks in adolescents and adults with HAE.

[0129] The calibration curve in Figure 6 was then used to determine the concentration of fC1-INH in plasma samples from control subjects and subjects with hereditary angioedema (HAE). Briefly, plasma samples from subjects were diluted 1:20 with C1-INH-depleted plasma. The diluted plasma sample (20 μL) was added to the sample port and incubated for 5 minutes. The concentrations of fC1-INH determined using the test strips were within 20% of the concentration values ​​determined by ELISA.

[0130] As shown in Figure 8, by both methods, C1-INH levels were lower in subjects with HAE compared to healthy controls. The mean C1-INH concentrations measured were 1345 mU / mL and 1089 mU / mL in healthy controls and 275 mU / mL and 163 mU / mL in subjects with HAE by the chromogenic and LFA methods, respectively (Table 8). The SEM and 95% confidence intervals for the measurements are presented in Table 8. The C1-INH data for all HAE subjects obtained by the two methods, including the two HAE subjects who had C1-INH concentrations within the range of healthy controls, yielded an R of 0.86. 2 The mean ratios between normal controls and HAE subjects measuring fC1-INH were 4.9 and 6.7 for the chromogenic and LFA methods, respectively.

[0131] The receiver operating curve (ROC) for diagnostic performance based on samples from control subjects and subjects with HAE was 0.98, indicating that the C1-INH concentrations determined by LFA accurately distinguished between control subjects and subjects with HAE (Figure 7). The ROC curve showed that a C1-INH cut point of 496 mU / mL resulted in a sensitivity (true positive rate) of 94% and a specificity (false positive rate) of 96% (Figure 7). False negatives and false positives are listed in Table 9.

[0132] As shown in Table 8 and Figures 8-9, the results obtained using the LFA were compared with those obtained using a chromogenic ELISA assay. Briefly, the chromogenic method directly measures fC1-INH levels, involving C1s cleavage of a synthetic substrate to form a colored compound; a decrease in color intensity indicates inhibition of C1s enzymatic activity. The chromogenic assay was qualified with respect to precision, accuracy, linearity, and upper and lower limits of quantitation. Three quality controls and a standard curve with 10 standard points ranging from 1000 to 1.95 mU / mL were prepared using C1-INH protein (2000 IU Injectable C1 Esterase Inhibitor [Human] Solution, Shire / Takeda Pharmaceuticals). The highest and lowest points of the standard curve were used as anchor points. Briefly, K3-EDTA plasma samples and reference proteins were preincubated 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 thiobenzyl ester group, M-1300, Bachem) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) #D-8000, Biosynth). The reaction was incubated at room temperature for 40 min. The absorbance was recorded at 405 nm using a SpectraMax M5 plate reader with SoftMax Pro software.

[0133] [Table 8]

[0134] [Table 9]

[0135] Taken together, these results demonstrate that similar fC1-INH concentrations were detected in patient plasma samples by the LFA and ELISA (referred to as a chromogenic assay). Therefore, the LFA and test strips described herein may be effective tools for identifying patients with HAE based on the level of fC1-INH in plasma samples from patients. Results obtained using the LFA described herein correlated with the results of the chromogenic assay for assessing fC1-INH.

[0136] The rapid and sensitive LFA method and device disclosed herein can be implemented in physicians' offices for the rapid diagnosis of HAE (e.g., types I and II) based on fC1INH levels. Such methods and devices may offer low-cost consumables reimbursed by health insurance, a high level of confidence in quantitative results, ease of data interpretation by physicians, and / or a low level of need for confirmatory analysis. Such a rapid assay for diagnosing type I or type II HAE in the clinic could expand screening for HAE and more quickly identify new HAE patients. Currently, the global diagnostic rate for HAE is only 40%, and therefore, undiagnosed patients represent a high unmet need. The availability of a rapid test on a common device platform could expand HAE awareness. Furthermore, the rapid test for fC1INH disclosed herein may be useful for timely monitoring of HAE disease progression or response to treatment in clinical settings.

[0137] References 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 2. Aabom, A. et al. (2017) Complement factor C4 activation in patients with hereditary angioedema. Clinical Biochemistry 50 (15), 816-821. 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. 4. Csuka, D. et al. (2017) The role of the complement system in hereditary angioedema. Mol Immunol 89, 59-68. 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. 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. 7. Rosenstein, R.W. and Bloomster, T.G. (1989). Solid phase assay employing capillary flow. U.S. Patent No. 4,855,240. 8. May, K., Prior, ME, and Richards, I. (1997). Capillary immunoassay and device therefore comprising mobilizable particulate labeled reagents. US Patent No. 5,622,871. 9. O'Farrell, B. (2009). Evolution in Lateral Flow-Based Immunoassay Systems. In: Wong, RC and Tse, HY (eds.). Lateral Flow Immunoassay . Humana Press New York (NY). 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

[0138] Example 3: Competitive binding assay demonstrates the specificity of the lateral flow assay (LFA) device for detecting functional C1-esterase inhibitor (fC1-INH). The specificity of the lateral flow assay (LFA) device described herein for determining C1-INH concentrations 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 control samples. The intensity of the test line region was measured, and the percent signal reduction from the control reaction signal was calculated for each sample. For samples containing competing proteins, the intensity of the test line (TL) was reduced by 40% to 60% compared to the control sample (Table 10). The intensity of the TL was reduced by 55% with the addition of biotin-labeled BSA, demonstrating that unrelated proteins were not detected in the assay (Table 10). For unlabeled FXIIa and unlabeled antibody, the intensity of the TL was reduced by 61% and 48%, respectively, demonstrating the specificity of FXIIa and antibody for C1-INH signal generation (Table 10).

[0139] [Table 10]

[0140] The specificity of detection was further tested using heat-denatured C1-INH protein: the intensity of TL was not reduced by heating at 40°C, but by heating at 53°C, the intensity of TL was reduced to the intensity of the background signal (Table 11).

[0141] [Table 11]

[0142] Taken together, these results demonstrate that the LFA and test strips described herein are specific for the detection of fC1-INH in plasma samples.

[0143] Example 4: Use of blood samples from patients to detect functional C1-esterase inhibitor (fC1-INH) in an LFA device Briefly, a whole blood sample is collected by performing a fingerstick technique according to laboratory practices that will be apparent to those skilled in the art. After wiping away the first drop of blood, a second drop of blood forms on the finger. A sample loop is used to contact the second drop of blood, filling the loop with blood (FIG. 10A). The loop is then placed into a container such as a SampleTainer® bottle (FIG. 10B). Once the loop touches the bottom of the bottle, the bottle is snapped and twisted, breaking off the bottom of the shaft and leaving it inside the bottle. Finally, the bottle is capped and shaken to mix. The whole blood sample may be added to any device for analysis.

[0144] Example 5: Reagent Selection Reagents were selected for use in the methods and / or devices described herein. Two initial detection agents were developed: one utilizing europium nanoparticles conjugated to anti-fC1-INH Fab (FIG. 11A) and one using red gold nanoparticles conjugated to anti-fC1-INH Fab (FIG. 11B).

[0145] A sharp decrease in signal at the low end of the dynamic range was observed, suggesting that the system had reached its maximum signal. To reduce the slope of the relationship, various amounts of different reagents were evaluated. Two reagents were evaluated as test lines: streptavidin printed at a concentration of 0.5 mg / mL and a polymeric version of streptavidin (Polystreptavidin®). Streptavidin was found to have a higher signal than polystreptavidin, while polystreptavidin was found to have more 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 C1-INH / Sinlyse® 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.

[0146] Different agents were also evaluated for the test lines. Briefly, three different test lines were generated: human biotinylated FXIIa (B-HFXIIa) mixed with streptavidin; B-HFXIIa mixed with polystreptavidin; and HFXIIa (non-biotinylated) alone. The use of HFXIIa resulted in a positive, albeit low, signal, whereas B-HFXIIa with streptavidin and B-HFXIIa with polystreptavidin did not have an appreciable positive signal (Figure 13).

[0147] Additionally, anti-C1-INH antibodies were also evaluated for use in the methods described herein. Four europium conjugates (anti-C1-INH Fab and anti-C1-INH 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) (FIG. 14). The signals obtained with each of the antibodies RP01, RP02, MM03, and MM06 were enhanced compared to the Fab conjugates. Polyclonal antibody RP02 was selected for further analysis.

[0148] We also evaluated buffer conditions. For example, buffers containing inorganic buffering agents (IBA) and organic buffering agents (OBA) were prepared at various pH values ​​from 7.0 to 9.5. The IBA-containing buffer performed better (e.g., higher signal) at the higher end of the pH range (pH 9.5), while the OBA-containing buffer performed better (e.g., higher signal) in the physiological pH range (approximately 7 to 7.5) (Figure 15).

[0149] Finally, detectable substances conjugated to anti-C1-INH binding agents were also evaluated. In particular, antibody RP02 was evaluated using a europium conjugate or a red gold conjugate and compared to anti-C1-INH Fab. The Fab conjugate did not produce a positive signal, whereas the RP02 antibody with the red gold conjugate produced a positive signal (Figure 16).

[0150] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless otherwise specified, each disclosed feature is only one example of a generic series of equivalent or similar features. From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure and can make various changes and modifications to the present disclosure to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the following claims.

[0151] Equivalents and Scope 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 disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0152] In the claims, articles such as "a," "an," and "the" can mean one or more unless indicated otherwise or clear from the context. A claim or description including "or" between one or more members of a group is deemed to stand if one, more than one, or all of the group members are present in, used in, or associated with a given product or process, unless indicated otherwise or clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, used in, or associated with a given product or process. The present disclosure includes embodiments in which two or more, or all of the group members are present in, used in, or associated with a given product or process.

[0153] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends from the same base claim. Where elements are presented as a list (e.g., in Markush group format), each subgroup of elements is also disclosed, and any element(s) may be deleted from the group. In general, when the present disclosure or aspects of the present disclosure are referred to as comprising certain elements and / or features, it should be understood that certain embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features. For the sake of brevity, those embodiments have not been specifically described in those terms herein. It should also be noted that the terms "comprising" and "containing" are intended to be open-ended, permitting the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges may, in various embodiments of the present disclosure, assume any specific value or subrange within the stated range, to the nearest tenth of the lower limit of the range, unless the context clearly dictates otherwise.

[0154] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Furthermore, any specific embodiments of the present disclosure that fall within the prior art may be expressly excluded from one or more of the claims. Such embodiments may be excluded even if the exclusion is not explicitly set forth herein because they are deemed to be known to those of ordinary skill in the art. Any specific embodiments of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0155] Those skilled in the art will recognize, or be able to ascertain using no more than 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 to this description may be made without departing from the spirit or scope of the present disclosure as defined in the following claims.

Claims

1. 1. A device for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH), comprising: The device comprises: (i) a conjugate pad comprising a first zone and a second zone in which a first agent and a second agent are immobilized, respectively; (ii) a membrane in communication with the conjugate pad; (iii) a control line; and Including, the membrane includes a third zone in which a third agent is immobilized; the first agent is a functional C1-inhibitor (fC1-INH) binding agent or a C1-inhibitor (C1-INH) binding agent, and the second agent is a fC1-INH binding agent or a C1-INH binding agent; the first agent and the second agent are different from each other; the third agent is a capture agent capable of binding to a docking agent; one of the fC1-INH binding agent and the C1-INH binding agent is conjugated to a detectable label, one of the fC1-INH binding agent and the C1-INH binding agent is conjugated to the docking agent, and the detectable label and the docking agent are conjugated to separate binding agents; the conjugate pad further comprises a fourth zone for disposing a biological sample and a fifth zone for disposing a buffer, the buffer flowing through the device in the order of the first zone, the second zone, and the third zone; Device.

2. the first agent, the second agent, and the third agent are the C1-INH binding agent, the fC1-INH binding agent, and the capture agent, respectively; or the first agent, the second agent, and the third agent are the fC1-INH binding agent, the C1-INH binding agent, and the capture agent, respectively; 10. The apparatus of claim 1.

3. the first agent is conjugated to the detectable label and the second agent is conjugated to the docking agent; or the first agent is conjugated to the docking agent and the second agent is conjugated to the detectable label; 3. The device according to claim 1 or 2.

4. The device of any one of claims 1 to 3, wherein the fC1-INH binding substance is the activated form of factor XII (FXIIa).

5. The device according to any one of claims 1 to 4, wherein the C1-INH binding substance is an antibody that binds to C1-INH.

6. the docking agent and the capture agent are members of a receptor-ligand pair; The device according to any one of claims 1 to 5.

7. 7. The device of claim 6, wherein the receptor-ligand pair comprises biotin and avidin.

8. The device of claim 7 , wherein the docking agent is biotin and the capture agent is avidin.

9. The device of claim 8 , wherein the avidin is streptavidin or polystreptavidin.

10. 10. The device of claim 1, 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 device of claim 10 , wherein the detectable label is europium.

12. The device of claim 10 , wherein the detectable label is colloidal gold.

13. The device of any one of claims 1 to 12, wherein the detectable label is linked to a latex particle.

14. The apparatus of any one of claims 1 to 13, wherein the fourth zone overlaps the second zone.

15. 15. The device of claim 1, wherein the first agent is a C1-INH binding agent disposed in the first zone, the second agent is a fC1-INH binding agent disposed in the second zone, and the third agent is a capture agent disposed in the third zone.

16. 16. The device of claim 15, wherein the C1-INH binding agent is an antibody that binds to C1-INH and is conjugated to the detectable label, the fC1-INH binding agent is FXIIa conjugated to a docking agent that is biotin, and the capture agent is avidin, which may be streptavidin or polystreptavidin.

17. an absorbent pad in communication with the membrane, the absorbent pad and the conjugate pad being separated by the membrane; The apparatus of any one of claims 1 to 16, further comprising:

18. a support member to which the conjugate pad, the membrane, and / or the absorbent pad are attached; 20. The apparatus of claim 17, further comprising:

19. The device of any one of claims 1 to 18, further comprising a housing.

20. 20. The device of claim 19, wherein the housing includes a first opening to form a buffer port, a second opening to form a sample port, and a third opening to form a test window.

21. 21. The device of claim 20, wherein the sample port is disposed between the buffer port and the test window.

22. 22. The device of claim 20 or 21, wherein the buffer port is aligned with the first zone in which the C1-INH binding substance is located.

23. 23. The device of any one of claims 20 to 22, wherein the sample port is aligned with the second zone in which the fCl-INH binding substance is located.

24. The device of any one of claims 20 to 23, wherein the test window is aligned with the third zone in which the capture substance is located.

25. 1. A method for detecting and / or quantifying functional C1-esterase inhibitor (fC1-INH) in a sample, comprising: (i) placing a sample in a sample port of an apparatus according to any one of claims 20 to 24; (ii) placing a buffer in a buffer port of the device, the buffer flowing in a direction from the first zone to the third zone; (iii) detecting a signal in a test window of the device; (iv) determining the presence of fCl-INH in the sample or measuring the level of fCl-INH in the sample based on the presence or intensity of a signal in the test window; A method comprising:

26. 26. The method of claim 25, wherein step (ii) is carried out at least 5 minutes after step (i).

27. The method of any one of claims 25 to 26, wherein the sample is a biological sample obtained from a subject.

28. 28. The method of claim 27, wherein the biological sample is a serum sample, a plasma sample, or a blood sample.

29. 29. The method of claim 27 or 28, wherein the subject is a human patient suspected of having or at risk for a disorder mediated by a defect in fC1-INH.

30. 30. The method of claim 29, wherein the disorder mediated by defective fC1-INH is selected from the group consisting of hereditary angioedema (HAE), acquired angioedema (AAE), and C1-INH-associated immune disease.

31. 31. The method of claim 30, wherein the subject has symptoms of HAE.

32. 32. The method of claim 30 or 31, wherein the HAE is type I HAE or type II HAE.

33. 31. The method of claim 30, wherein the subject has no symptoms of HAE, no history of symptoms of HAE, or no history of HAE.

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